Structural determinants of voltage sensitivity in hyperpolarization-activated ion channels and their persistence in the evolutionary scale

Structural determinants of voltage sensitivity in hyperpolarization-activated ion channels and their persistence in the evolutionary scale | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Structural determinants of voltage sensitivity in hyperpolarization-activated ion channels and their persistence in the evolutionary scale Karla G Alvarez-Villagómez, Daniel Balleza This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8117147/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted 9 You are reading this latest preprint version Abstract HCN channels have a reverse electromechanical coupling mechanism, where hyperpolarized membrane potentials facilitate pore opening through an inward displacement of the S4 segment of the voltage-sensing domain (VSD). This voltage dependence is finely regulated by the binding of cAMP to an intracellular domain (CNBD). Of the four widely studied isoforms of human HCN channels, the HCN3 channel is practically insensitive to cAMP or is even inhibited by it, but the structural determinants underlying this unexpected behavior are still unclear. Here, we evaluated the possible role of flexibility in very specific regions of the VSD that could be determinants for this behavior. Hence, part of the S2-S3L linker is significantly rigid in HCN3, which correlates with low atomic mobility for this region in proximity to the C-linker subdomain of the opposite subunit. We built structural models using AlphaFold 3 and Swiss-Model and thus reconstructed the disordered regions that connect the transmembrane segments of the VSD and that in some of the structures deposited in the PDB have not been resolved. Besides, in an attempt to reveal the evolutionary trends that this transmembrane domain may have undergone, we conducted a comparative study with phylogenetically distant HCN channels and found an interesting tendency to lose sensitivity to cAMP as VSD flexibility is lost. Our analysis confirms a large body of published experimental findings. Finally, we found that in metazoans, two types of HCN channels clearly diverge: (1) those that are highly sensitive to cAMP with moderate flexibility profiles in protostomes, and (2) those that show less marked sensitivity to this ligand in deuterostomes. We also propose a possible evolutionary scenario for the appearance of cAMP-modulated HCN channels in the last eukaryotic common ancestor (LECA). Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Please have a look at courier new font provided for text in article. In the superfamily of voltage-gated ion channels (VGIC), hyperpolarization-activated and cyclic nucleotide-gated channels (HCN) occupy a prominent place. These channels are essentially activated when the cell membrane hyperpolarizes and the intracellular voltage becomes even more negative than its resting potential (Sartiari et al. 2017). These properties enable the cells that express them to regulate the frequency of action potentials and thus act as pacemaker currents (Baruscotti and DiFrancesco 2004). Although this activity responds to the transmembrane potential, the voltage dependence for the opening activity of these channels is reduced in the presence of a specific ligand, cyclic AMP. In this way, cAMP exerts a modulatory role on these channels, facilitating the action of voltage and increasing the channel open probability (Saponaro et al. 2021a). The electromechanical coupling mechanism that facilitates the opening of HCN channels has been extensively studied (Lee and Mackinnon 2019; Saponaro et al. 2021b; Burtscher et al. 2024 ). Intracellular cAMP, in parallel, facilitates the activation of these channels by shifting the voltage dependence toward more depolarizing potentials (Cuttle et al. 2001 ). This allows channel activation to occur with less hyperpolarization. However, the details of the allosteric modulation that allows the voltage sensor domain (VSD) to communicate with the cytosolic cyclic nucleotide-binding domain (CNBD) are less well known. Some studies have demonstrated autoinhibition mechanisms where cAMP facilitates tetramerization of a more energetically stable form of the HCN4 channel, which is compatible with the activated state (Akimoto et al. 2014 ; vanSchouwen et al. 2015 ). Four isoforms (HCN1-4) with characteristic activation kinetics have been described in mammalian cells (Stieber et al. 2005 ). These paralogs exhibit 80–90% sequence identity between the start of the first transmembrane segment, S1, to the end of the cytosolic CNBD, although in terms of sequence length they are different (Jackson et al. 2007 ). However, the role of the nucleotide ligand clearly exerts different modulatory effects on each isoform (Wainger et al. 2001 ; Stieber et al. 2003 ). Typically, the biophysical parameter that allows to estimate the voltage dependence between different isoforms of the same channel, or to evaluate the effect of point mutations on the gating machinery of ion channels, is the half-activation voltage ΔV 1/2 , which allows to quantify the membrane potential at which a voltage-activated ion channel reaches 50% of its maximum ionic current. Thus, it has been determined that of the four known isoforms, HCN2 and HCN4 exhibit the highest sensitivity to cAMP in terms of the V 1/2 shift (HCN2: 12–14 mV; HCN4: 11–23 mV), while the HCN1 channel is poorly modulated by this ligand (~ 2 mV) (Kaupp and Seifert 2001 ). The HCN3 isoform, on the contrary, is insensitive to cAMP. Indeed, the activation curve in the presence of ligand shifts to more hyperpolarized potentials (Místrik et al. 2005; Stieber et al. 2005 ). This observation has been interpreted in terms of an anomalous autoinhibition mechanism, which depends on a structural change in the domains that communicate cAMP binding with the channel activation machinery (Stieber 2005). The search for HCN orthologous channels and the modulatory role of cyclic nucleotides has led to a better understanding of the nature of these proteins and their role in cell physiology. Thus, cAMP-modulated HCN-like channels have been described in urochordates and lower vertebrates (Gauss et al. 1998 ; Shin et al. 2004 ; Baker et al. 2015 ; Wobig et al. 2020 ) as well as in arthropods (Gisselmann et al. 2003 , 2005a,b). Hyperpolarization-activated channels have even been found in plants, although these do not appear to be regulated by cyclic monophosphate nucleotides, as their CNBDs have been found to lack specific residues for binding to these ligands (Lu et al. 2022 ). However, HCN-like sequences show considerable identity at the amino acid residue level but significant differences from their mammalian counterparts ( Suppl. Figure 1 ), providing key clues for deciphering the evolutionary relationships between HCN channels. Eight bacterial homologues with significant sequence similarity to eukaryotic HCN channels have been described. These include weakly voltage dependent channels found in bacteria, and so-called as AqK (from Aquifex aeolicus ), SthK (from Spirochaeta thermophila ), AmaK (from Arthrospira maxima ), TerK (from Trichodesmium erythraeum ), LpcK (from Lyngbya sp. PCC 8106), LbiK (from Leptospira biflexa ), and LliK (from Leptospira licerasiae ) (Brams et al. 2014 ; James et al. 2017 ; Romero-Romero et al. 2019 ). Of all these channels, only SthK has been extensively studied by the group of C. Nimigean, who describe it as a channel activated by cyclic nucleotides and modulated by depolarizing voltages, which places it functionally closer to cyclic nucleotide–gated (CNG) channels in comparison with the HCN ones (Schmidpeter et al. 2018; Gao et al. 2022 ). In 2003, the first discovery of a hyperpolarization-activated prokaryotic channel, MVP, was reported in Methanococcus jannaschii , an extremely thermophilic methane-producing archaebacterium (Sesti et al. 2003 ). This channel is activated after the downward displacement of S4 segments in the VSD, similar to the helical motion also described for the sea urchin HCN channel and the human HCN1 channel (Dai et al. 2019 ; Burtscher et al. 2024 ). On the other hand, some studies have shed light on the evolution of these proteins, suggesting that gene duplication phenomena may have occurred independently in different lineages of the animal kingdom. These studies have identified regions that are important for the specific functions of each isoform, as well as for those specific to each species (Jackson et al. 2007 ). To our knowledge, cyclic nucleotide-dependent ion channels have not been described in the Archaea domain. The structural basis of the allosteric coupling of cAMP binding to the voltage-sensitive transmembrane domain to modulate the voltage sensitivity in HCN channels remains unknown. However, several electromechanical models have been proposed to reveal the cytoplasmic-to-transmembrane-domain movements as result of the cAMP binding event (Lee and Mackinnon 2017 ; Akimoto et al. 2018; Gross et al. 2018 ; Wang et al. 2020 ; Saponaro et al. 2021a). In this study, we take advantage on the structural information available in the Protein Data Bank, as well as the extensive electrophysiological studies that have been reported by various research groups to understand the activation and modulation by cAMP of these ion channels (reviewed, for example, in Sartiani et al. 2017 and Saponaro et al. 2021b), to compare structure information with functional data in terms of their voltage dependence and their degree of sensitivity to cAMP. We also wonder whether the compositional variations between each isoform, as well as the functional differences found in the paralogous proteins studied, could be explained in terms of the intrinsic flexibility of the VSD throughout the evolutionary scale. The findings described in this study point to a relationship in which HCN channels that are more sensitive to the modulating effect of cAMP exhibit significantly more flexible voltage sensors than those whose amino acid composition is intrinsically more rigid. We also discovered that sensitivity to cAMP as a modulator of voltage dependence in these channels is poor in early animals, which is linked to low flexibility profiles in the VSDs in those channels. On the contrary, sensitivity to this ligand is significantly higher in protostomes than in deuterostomes. These relationships are also significant when considering the MVP channel of the Archaea lineage, which is the hyperpolarization-activated channel that exhibits the greatest intrinsic rigidity, consistent with a hyperthermophilic origin for this class of proteins in a thermoadaptive scenario close to the root of the tree of life. Methods Data extraction and Structural Dataset The sequences and structural data known for the four isoforms of human HCN channels, whose functional characterization is widely reported in the literature, are deposited in the GenBank and the Protein Data Bank, with the following accessions: 5U6O_1 (hHCN1), NP_001185.3 (hHCN2), NP_065948.1 (hHCN3), NP_005468.1 (hHCN4). We also include four paralogous sequences found in Xenopus laevis , with high similarity but not yet functionally characterized: XP_031750156.1 (XtHCN1), XP_002939815. 3 (XtHCN2), XP_031747278.1 (XtHCN3), XP_017947882.2 (XtHCN4). It was particularly interesting to include HCN-like channels whose functional characterization has already been reported in terms of the change in voltage activation midpoint dependent on cyclic AMP. These channels include the following isoforms: QKM75727.1 (DrHCNL1) and XP_009302941.1 (DrHCNL2) from zebrafish ( Danio rerio ) (Wobig et al. 2020 ); NP_999729.1 (spHCN, SpIh) from sea urchin ( Strongylocentrotus purpuratus ) (Shin et al. 2004 ; Fynn et al. 2007); ALO50060.1 (NvHCN1) and ALO50059.1 (NvHCN2) from the starlet sea anemone ( Nematostella vectensis ) (Baker et al. 2015 ); AAQ16311.1 (PaIh) from spiny lobster ( Panulirus argus ) (Gisselmann et al. 2005b); AAX78396.1 (DmHCN, Ih) from fruit fly ( Drosophila melanogaster ) (Gisselmann et al. 2005a); NP_001011568.1 (AmIh) from honey bee ( Apis mellifera ) (Gisselmann et al. 2003 ) and the archaeal MVP channel of Methanocaldococcus jannaschii (WP_010869632.1) (Sesti et al. 2003 ). Likewise, the following sequences were included for the reconstruction of phylogenetic relationships: AAC07678.1 (AqK) from Aquifex aeolicus , WP_131007350.1 (MaVP) from the archaeon Methanofervidicoccus abyssi , Q38998.2 (AKT1) and NP_199436.1 (KAT1) from Arabidopsis thaliana , and ABF71886.1 (hERG1) from human. Molecular phylogenetic analysis For molecular phylogenetic analysis, HCN and HCN-like sequences were aligned using the Clustal W program (Thompson et al., 1994 ) and a neighbor-joining tree was constructed using the online version of MAFFT (Katoh et al., 2019 ). We included sequences from two hyperpolarization-activated plant channels, AKT1 and KAT1, the bacterial channel AqK with the CNBD domain, and the hERG channel, which exhibits an “intrinsic ligand” that mimics a cyclic nucleotide within a homologous CNBD (Codding and Trudeau 2019 ). Protein flexibility estimation The estimation of intrinsic flexibility for primary protein sequences has been previously described in our research group using the FlexiProt predictor (Balleza 2023 ; García-Morales and Balleza 2023 ). In brief, this algorithm allows us to predict the local flexibility of each of the 20 side chains by correlating the amino acid composition based on the two neighbors for each position. With this normalized B factor (nBf), we assign a relative flexibility associated with the structural characteristics and conformational freedom of each side chain in any typical sequence of any protein. In this study, we also calculated the mean B factor (mBf) corresponding to the transmembrane domain of the voltage sensor (S1-S4) in each protein evaluated. This parameter is the result of the average of each nBf based on the number of amino acids for each protein segment, expressed as the inverse of the absolute value for this weighted average: Structure Modelling The structures of human HCN1-4 channels have been deposited in the PDB with varying degrees of atomic resolution, and include structures obtained by Cryo-EM, except for the spHCN channel, which was obtained by integrative structure determination. Except for the HCN2 channel, whose structures are known only for the cytoplasmic regions and not the transmembrane region of interest to us, the deposited structures include the voltage-sensing domains. However, these structures generally lack information on the linker regions between segments S1 to S4, which could be related to a high mobility rate for these regions. This led us to generate high-quality three-dimensional models using AlphaFold3 (Abramson et al. 2024 ) or using the SWISS-MODEL homology modeling server (Waterhouse et al. 2018 ) with specific PDB files as follow: HCN1 (5u6o, 3.50 Å); HCN2 (6gyn, 3.40 Å) (however, the 5u6o structure was also evaluated for this specific model); HCN3 (8inz, 2.72 Å); HCN4 (6gyn) and spHCN (8zzw). Thereby, we generated high-quality structures that modeled the missing regions in each protein. Each model was validated using both the MolProbity tool ( http://molprobity.biochem.duke.edu/index.php ) and the Procheck server ( https://saves.mbi.ucla.edu/ ), mainly evaluating stereochemical parameters such as the presence of unfavored rotamers and C β deviations > 0.25Å. Hence, the best-rated models were chosen for further analysis. Three visualizers were used to analyze each structure: PyMOL (The PyMOL Molecular Graphics System, version 3.0 Schrödinger, LLC.), Chimera X (Goddard et al. 2018 ), and DSV (Biovia 2019 ) Molecular Dynamics Simulations and Vibrational Entropy Estimation Dynamic simulations were executed using the CABSflex 2.0 server ( http://biocomp.chem.uw.edu.pl/CABSflex2/submit ), using the SS1 mode with restraints for residues structured in regular secondary regions (C-linker). This type of simulations allows the prediction of protein flexibility by determining the root-mean-square fluctuation (RMSF) (Kurcinski et al., 2018). CABSflex is based on the CABS (C-Alpha, Beta, and Side-chains) model of polypeptide chains, which is a medium-resolution coarse-grain model in which the main chain of a polypeptide is replaced by two pseudoatoms per residue. The CABS uses Monte Carlo dynamics of a single protein or a set of chains to reproduce local movements. This reproduces very well the flexibility of proteins and their long-term dynamic patterns (Kolinski 2004 ). To evaluate the possible restrictions of each flexibility profile that we calculated previously (Fig. 2 ), in terms of the stability of the VSD and the intercalated C-linker, we followed a coarse-grained Normal Mode Analysis (NMA) strategy that considers the physicochemical nature of each amino acid and thus allows to calculate significant changes in vibrational entropy in harmonic motions of the protein structure. This was done using the DynaMut predictor (Rodrigues et al., 2018 ), a consensus predictor of protein stability based on the vibrational entropy changes predicted by an Elastic Network Contact model (ENCoM) (Frappier et al., 2017 ). Results and Discussion Phylogenetic characterization of Hyperpolarization activated HCN channels The HCN channel subfamily is represented by four isoforms, HCN1-4 in mammals, and is closely related to the CNG subfamily of cyclic nucleotide-gated ion channels. These two groups of ion channels, along with the KCNH (Kv 10-12 ) subfamily channels, form one of the largest clades of the Voltage-Gated Ion Channel, VGIC, superfamily (Yu and Catterall 2004). However, unlike CNG channels, HCN channels are highly voltage-sensitive, with the HCN1 isoform being the most sensitive in terms of the activation time constant (t −100 mV = 67 16 ms) (Stieber et al. 2005). A previous study determined that HCN1-4 isoforms form a paralogous group that was established before the origin of the vertebrate clade, having been present in the common ancestor of fish and tetrapods (Jackson et al. 2007). This proposal was strengthened when the first report of a functional isoform, spHCN, present in sea urchin sperm was made (Gauss et al. 1988). Decades later, the discovery of functional HCN-like isoforms in early metazoans and some arthropods was also reported (Marx et al. 1999; Gisselmann et al. 2003, 2005; Baker et al. 2015). Thus, the evolutionary history of the HCN family is becoming increasingly complete, strongly suggesting that it probably evolved through independent gene duplications, as has been proposed elsewhere (Jackson et al. 2007). To confirm the nature of these evolutionary relationships, a phylogenetic analysis was performed using the neighborhood-joining method ( Fig. 1 ), and the estimated distances for each of the clades among the species were calculated. It should be noted that in this analysis an important criterion was to select ion channels that have been functionally characterized by electrophysiological techniques. Thus, the phylogenetic analysis revealed that the four isoforms present in mammals formed a monophyletic group, which is closely related to the clade of HCN channels described in arthropods but distant from both the spHCN channel (Echinodermata) and one of the two isoforms described as HCN in Nematostella vectensis (Cnidaria), NvHCN1 (Baker et al. 2015). Interestingly, the second isoform described by that research group, NvHCN2, is consistently positioned close to the HCN1-4 clade present in mammals and amphibians. In this phylogenetic analysis, we also included two HCN-like channels discovered in zebrafish, HCNL-1 and HCNL-2, which, unlike typical HCN channels, show low permeability to Family IA metal cations (Na + , K + ) and preferentially conduct protons (Wobig et al. 2020). In this sequence analysis, we also included the hERG channel, whose intrinsic ligand facilitates its activation in the absence of cAMP (Codding et al. 2020). Consistent with these differences, this channel clearly occupies an outgroup with the hyperpolarization-activated plant AKT1 and KAT1 channels, slightly closer to the clade of HCN and HCN-like channels described above. Finally, we included three prokaryotic sequences for hyperpolarization-activated channels, MVP and probably MaVP, present in Methanocaldococcus jannaschii and Methanofervidicoccus abyssi , respectively (Sesti et al. 2007), as well as the AqK channel described by us, which carries a cyclic nucleotide-binding domain (CNBD) connected by a C-linker to the VSD (Romero et al. 2019). This substructure is typical of channels of the HCN family, as well as CNG and KCNH (Codding et al. 2020). The intrinsic flexibility of the voltage sensor correlates with the ligand-induced change in the activation V 1/2 of HCN channels Once we defined the phylogenetic relationships among all the HCN channels that have been previously functionally characterized, we decided to try to find some correlation between their composition, their structure and some of their activation parameters. With this objective in mind, we defined a more detailed study of the VSD, since it is well known that the downward movement of one of its four transmembrane (TM) segments, S4, is atypical within the VGIC superfamily, and that this displacement is probably conserved throughout the evolutionary scale (Randich et al. 2014, Dai et al. 2019, Wu et al. 2023). We also decided to focus on the midpoint activation voltage parameter, V 1/2 , in response to the presence of cAMP, since this ligand interacts specifically with each isoform, positively or negatively modulating the voltage dependence for the activation of these channels. We reasoned that it would be possible to reveal subtle differences encoded in the VSD sequence for each isoform or paralogous protein, which would reveal differences in the modulatory effect of each channel. For example, it is widely known that HCN1, 2, and 4 channels respond to cAMP by shifting the activation curve toward more depolarized voltages, i.e. by making the channel more sensitive in terms of the activation voltage. However, the HCN3 channel responds in the opposite way, shifting the voltage curve toward more hyperpolarized potentials in presence of cAMP, which is interpreted as an antagonistic effect of this ligand by desensitizing the protein in terms of its activation voltage (Stieber et al. 2005). Hence, it was very tempting to ask the reason behind the apparent lack of sensitivity of the human HCN3 channel in response to this ligand. We wondered whether the shift towards more hyperpolarized voltages described in the HCN3 channel in response to the presence of the cyclic nucleotide could be the result of some structural determinant in the VSD. Thus, we calculated the intrinsic flexibility (1/mBf) for each of the TM segments that integrate the VSD, which have around 141 to 143 amino acid residues for the isoforms described in human cells ( Table 1 ). Fig. 2 shows the intrinsic flexibilities calculated for each of the TM segments (S1-S4) that integrate the voltage sensor module in five of the best-characterized HCN channels studied to date. It is noteworthy that of the four isoforms present in human cells, the three that respond positively to cAMP by shifting the activation curve toward less polarized voltages (i.e., HCN1, 2, and 4) are relatively more flexible than the HCN3 isoform, whose response to this nucleotide is opposite and therefore not modulated by intracellular cAMP (Stieber et al. 2005). Likewise, we also note that the estimated flexibility index for the voltage sensor present in the spHCN channel is even lower, making it more compositionally rigid than the HCN3 channel. This also correlates with an apparent insensitivity to the modulatory effect of cAMP in this protein (Shin et al. 2004). It is also important to note that the divergence time between echinoderms and mammals is more than 210 million years (dos Reis et al. 2015). We also analyzed in detail each of the VSD sequences in the four human HCN1-4 isoforms, finding that the most divergent of these is precisely the HCN3 isoform ( Fig. 2 ). Then we tried to find the subregion within the VSD where this evolutionary divergence is most noticeable, since in general these sequences are very similar in terms of amino acid residue identity ( Suppl. Table 1 ) and conservative substitutions ( data not shown ). Thus, we found a small fragment of 15 residues very close to the only intracellular linker of the VSD (S2-S3L). This fragment extends from position D202 to N216 (HCN1); D271 to K285 (HCN2); E153 to R167 (HCN3) and D322 to K336 (HCN4) and is decisive for estimating the flexibility profile of the entire VSD. Fig. 2a shows this part of the sequence in black, and it is clear that only in the case of the HCN3 channel, the flexibility profile for this region is the lowest of the four proteins, which determines that the flexibility of the entire VSD in this protein is the lowest one ( Fig. 2b ). In this analysis, we also included a comparison with four other channels of interest: spHCN, CNGA1, hERG1, and Kv7.2. It should be noted here that spHCN does not show a particularly rigid analogous fragment, but it does have a less flexible overall VSD profile than the human orthologous channels, while in the case of Kv7.2, the VSD flexibility profile was the lowest of all the analyzed sequences. On the other hand, as expected, the flexibility profile of the VSD-like belonging to the CNGA1 channel is the highest of all, consistent with previous reports (Romero-Romero et al. 2019), while in the case of the human ether-a-go-go related K + channel 1 (hERG1), its profile was slightly higher than that found for HCN3. However, as mentioned previously, this channel includes an intrinsic ligand in its cytoplasmic structure, which prevents it from the direct binding of cAMP (Codding et al. 2019). Table 1. Changes in the activation midpoints depending on cAMP binding and intrinsic flexibility of the voltage sensor Channel VSD Num. of residues Positions 1/mBf V 1/2 shift (mV) Reference hHCN1 141 S139-H279 1.786 +6.0 ± 1.3 Wainger et al. (2001) Stieber et al. (2005) hHCN2 141 S208-H348 1.809 +18.7 ± 2.3 Wainger et al. (2001) Mistrik et al. (2005) Stieber et al. (2005) Peters et al. (2024) hHCN3 143 S90-H232 1.730 −3.95 ± 2.9 Mistrik et al. (2005) Stieber et al. (2005) hHCN4 141 S259-H399 1.784 +15.8 ± 2.3 Stieber et al. (2005) Schweizer et al. (2010) Peters et al. (2024) Wu et al. (2025) NvHCN1 142 S141-M282 1.702 0.0 Baker et al. (2015) NvHCN2 141 S218-V358 1.710 +6.1 ± 1.0 Baker et al. (2015) spHCN 148 S212-F359 1.674 −3.1 ± 1.8 Shin et al. (2004) Flynn et al. (2007) DmHCN (Ih) 144 R748-Y891 1.697 +21 Gisselmann et al. (2005a) AmHCN 140 F89-Y228 1.691 +18.9 Gisselmann et al. (2003) PaHCN 140 S94-Y235 1.748 +41 Gisselmann et al. (2005b) zHCNL1 159 P44-E202 1.702 +4.7 Wobig et al. (2020) zHCNL2 157 P56-V212 1.784 ND Wobig et al. (2020) MVP 113 R7-K119 1.657 0.0 Sesti et al. (2003) CNGA1 130 S31-T160 1.845 ND Xue et al. (2021) Based on these observations, we then conducted a compositional analysis for three specific ion channels: (1) the CNGA1 channel, whose activation depends exclusively on the binding of cyclic nucleotides and which has a highly flexible S4-S5 linker as a requirement for transitioning to the open state (Mazzolini et al. 2018); (2) the hERG1, which has a conserved stretch of amino acids in the cyclic nucleotide-binding homology (CNBH) domain that simulates the presence of a cyclic nucleotide and whose presence makes these channels insensitive to the modulatory effects of such ligands (Brelidze et al. 2009); and finally (3) the Kv7.2 channel, whose activation is independent of cAMP, although a certain regulatory effect, still poorly understood, mediated by cAMP has also been reported (van der Horst et al. 2020) but which clearly has a compositional profile rich in rigid amino acids, as we recently reported (Balleza et al. 2019). The results of this part of the analysis clearly suggest that flexible voltage sensors are highly sensitive to activation by cyclic nucleotides or are more prone to their modulatory effects, while those with more rigid profiles are typically insensitive to the effects of this class of intracellular ligands. Therefore, to demonstrate the possible correlation between the modulating effect of cAMP on HCN and HCN-like channels throughout the evolutionary scale, we compared data reported in the literature corresponding to the shifts in the activation curves for each channel (DV 1/2 ), as a function of the intrinsic flexibility of each corresponding VSD. With this rationale, we found that these displacements clearly fall into two categories: HCN channels present in protostome organisms (Arthropoda) and those encoded in the genome of deuterostome organisms, including mammals, fish, echinoderms, and cnidarians. The activation midpoint (V 1/2 ) for each of these channels is a key functional parameter which measure the voltage at which each channel reaches 50% of its total activation. Fig. 3 describes those data, and it should be noted that, in an attempt to reveal possible evolutionary trends, we included the archaeobacterial MVP channel in this analysis, which, to our surprise, has a highly rigid voltage sensor, in fact the most rigid of all the channels studied here. This correlates very well with the habitat of these thermophilic microorganisms, echoing some theories of protein thermoadaptation that postulate a preference for rigid structures in environments where the temperature is significantly high (Radestock and Gohlke 2011; Lecocq et al. 2021). Overall, these results indicate that the HCN channels described in arthropods are significantly more sensitive to the modulatory effects of cAMP, while those described in cnidarians, echinoderms, and chordates (Deuterostomata) are significantly less likely to exhibit the modulatory effect of this ligand in terms of the shift of the activation curves. However, as anticipated, there is a clear relationship between compositionally flexible voltage sensors and greater sensitivity to cAMP, both in protostome and deuterostome channels. Besides, it can also be noted that, at least in the case of deuterostome animals, voltage sensors are comparatively more rigid in animals that diverged early in the evolution of the tree of life (Cnidaria, Echinodermata, Actinopterygii), compared to those described in mammals (HCN1-4). Even more noteworthy is that among the isoforms described in mammals, the VSD present in the HCN3 channel shows the lowest flexibility profile, which is consistent with its insensitivity to cAMP, as described above. The intrinsic flexibility of extramembrane linkers is key in the conformational freedom of the voltage sensor in HCN channels. To gain further insight into the structural changes related to the variation in cAMP sensitivity, we evaluated the tertiary structure in each voltage sensor in terms of their relative intrinsic flexibility. To do this, we initiated a search in the Protein Data Bank, reviewing all available structural information corresponding to the four human HCN isoforms (HCN1-4), finding only three partially complete structures for HCN1 (5u6o), HCN3 (8inz), and HCN4 (6gyn). In this part of the analysis, we chose structures in the absence of the ligand (cAMP) precisely to focus on the inherent flexibility of each amino acid sequence in those proteins. Fig. 4 depicts a structural analysis for these three HCN channels using a scan of the B factor in each tetramer as a comparative criterion. Since the B factor is a measure for quantifying atomic vibrational movements (Sun et al. 2019), our analysis clearly indicates that HCN3 shows reduced vibrational variations within the VSD, i.e. it exhibits significant rigidity in relation to HCN4 and HCN1. Thus, the VSDs of these proteins can be ordered according to their increasing flexibility as follows: HCN3>HCN4>HCN1. These results are consistent with both the evolutionary distances in the cladogram shown above ( Fig. 1 ) and the cAMP sensitivity previously mentioned ( Fig. 3 ). It is tempting to suggest that the low sensitivity of the HCN1 channel to cAMP could be related to greater rigidity of the C-linker and part of the CNBD in this protein, even though the flexibility of the VSD itself is comparable to that shown by HCN4 (compare structural flexibility profile in Fig. 4 with cAMP sensitivity data included in Fig. 3 ). This analysis also suggests that the HCN domain (HCND), which occupies a strategic position in the architecture of HCN channels, forming a continuous mechanical interface between the VSD and the C-linker/CNBD (Porro et al. 2019, 2020) ( Fig. 4 ), is also key in coupling the allosteric changes of the CNBD and transmitting them to the voltage sensor in response to cAMP binding. Thus, in rigid voltage sensors such as those present in HCN3, the associated rigidity of the HCND in this channel prevents it from being positively modulated with cAMP, while in channels with more flexible voltage sensors, with less rigid HCNDs such as those present in HCN1 and HCN4, the greater degree of flexibility of the HCND allows these proteins to more effectively transmit the conformational changes derived from the binding to this ligand. However, the lack of a complete structure for the HCN2 channel made it impossible to perform a comprehensive comparison of the four known isoforms in the same phylogenetic clade. In addition, since the structures available in the PDB are incomplete, particularly with regard to the S2-S3L and S3-S4L linkers, we decide to obtain high-quality structural models using templates with high compositional similarity for each of the four proteins. Thus, this technical limitation, typical of cryogenic electron microscopy (Lučič et al. 2013), as well as the probable difficulty of obtaining a complete structure for the HCN2 channel due to its high intrinsic flexibility ( Fig. 2b ), we decided to generate homology models based on specific templates with high similarity using the SWISS-MODEL server (Waterhouse et al. 2018). In this part of the analysis, we also included a refined model of the spHCN channel from the sea urchin ( Fig. 5 ), a widely studied channel belonging to a phylogenetic clade significantly more distant from their orthologs present in mammals. With these models, we identify small structural differences that could explain the contribution of each linker in terms of the intrinsic flexibility of each voltage sensor. Thus, once we generated the five tetramerized models for each channel and these were validated using Ramachandran conformer analysis, we detected some residues of interest in dynamic terms, as they are located in unfavorable areas of these plots, specifically in the S2-S3L or the CNBD ( Suppl. Fig. 2 ). Therefore, we evaluated the contribution of missing sequences in the available cryo-EM structures, with special attention to the S2-S3L and S3-S4L linkers, whose overall contribution to the flexibility of the voltage sensor is decisive ( Fig. 2a ). Table 2. Relative flexibilities (1/mBf) for the three linkers within the VSD in HCN channels S1-S2L Position S2-S3L Position S3-S4L Position hHCN1 2.565 F166-T173 2.812 R195-D209 2.392 G242-T250 hHCN2 3.124 F235-A242 2.256 R264-P279 2.490 I312-A320 hHCN3 4.714 F117-P124 1.914 R146-A163 2.049 E191-V210 hHCN4 3.777 F286-T292 2.141 T316-D329 2.600 D364-A371 spHCN 2.196 F239-T247 2.436 R269-Q287 2.143 G315-R326 We thus discovered significant structural differences in terms of the intrinsic flexibilities of the three linkers that could explain why the HCN3 channel and spHCN are practically insensitive to the modulatory effects of cAMP. These results are shown in Table 2 and clearly indicate that the HCN3 isoform has the two most rigid linkers in this group of proteins; these are the S2-S3L (dark blue) and S3-S4L (green) linkers ( Fig. 5B ). Therefore, HCN3 is the only one of the four mammalian isoforms capable of organizing itself into a partial 3 10 helix outside the cell. This substructure is promoted by a Pro residue at position 194, which is only present in this isoform. In turn, inside the cell, the S2-S3L linker of HCN3 is the only one that consistently folds into an antiparallel beta sheet. This substructure was never obtained for any of the other homology models, including spHCN, which is intrinsically more rigid than HCN3. This substructure was never obtained for any of the other homology models, including spHCN, which is intrinsically more rigid than HCN3. In contrast, in the case of HCN1, 2, and 4, this linker is configured in a more disordered manner, with varying degrees of flexibility between them. With regard to the structure of the spHCN channel, to our surprise, the S2-S3L linker is not organized in beta configurations, but is much more flexible, with the exception of the corresponding linker in HCN1 ( Table 2 ). However, we found that this channel has the most rigid S1-S2L linker (cyan) ( Fig. 5B , arrowheads) of these five channels. This rigidity manifests in the form of a more compact and less extended loop compared to those present in mammalian orthologs, as it includes two key residues in the stabilization of helical substructures: K243 and M245. This finding is consistent with the fact that both residues are particularly prone to stabilizing a-helical structures (Blaber et al. 1993; Monera et al. 1995), which stiffens this part of the protein. Regarding the structure of the spHCN channel, to our surprise, the S2-S3L linker is not organized in b-configurations, but is much more flexible (mBf = 2.436), with the exception of the corresponding linker in HCN1 (mBf = 2.812) ( Table 2 ). However, we found that this channel has the most rigid S1-S2L linker (mBf = 2.196) of these five channels ( Fig. 5B , asterisk). This rigidity manifests in the form of a more compact two-turn w-loop compared to those present in mammalian orthologs, as it includes two key residues in the stabilization of helical substructures: D244 and S246. In the first case, D244 forms a double salt bridge with K335 (S4), while position S246 forms a double H-bond with K243, which in turn forms another H-bond with residue F240. This substructure gives this w-loop great rigidity, which is consistent with previous reports establishing that some omega loops can form turns that show a preference for specific amino acids, such as aspartate, serine, and asparagine (Papaleo et al. 2016), which is present at position 242. Thus, S1-S2L acquires a w-loop with a very ordered configuration, while the RHFLE motif in the extracellular S3-S4L forms a highly rigid and stable a-helical lid. Taken together, these structural traits are consistent with the high degree of rigidity of the entire VSD in the spHCN channel and its low sensitivity to the modulatory effect of cAMP. This is also consistent with considerable experimental evidence indicating that both w-loops and the presence of helical substructures in extracellular linkers are typically located in allosterically regulated regions of proteins (Papaleo et al. 2016). Common hydrogen bond interactions in the voltage sensor domain of HCN channels Once we had identified the main differences in tertiary structure that could explain the different sensitivities to voltage and the modulating effects of cAMP in the five HCN channels studied, we decided to compare the number of H-bonds that stabilize each VSD, based on the relative contribution of the S2-S3L and S3-S4L linkers, which are the most rigid in each structure and, in turn, are most closely associated with the propagation machinery that couples the allosteric effects of cAMP when binding to the CNBD of each channel. Table 3. Number of H-bonds and relevant van der Waals interactions in selected HCN channel VSDs HCND-VSD Position S2-S3L Relevant interactions S3-S4L Relevant interactions hHCN1 182 M94-Y289 10 (5.5%) R126-E205 ( 2)(HCND) T198-I206 ( b ) N200-S203 ( b ) I135-G197 (HCND) 6 (3.3%) D244-E246 S245-Y248 V247-R252 K249-R252 hHCN2 209 M163-Y358 7 (3.3%) R195-I275 ( 2)(HCND) I267-I276 ( 2)( b ) N272-E274 4 (1.9%) E309-D313 D313-E315 E315-R318 ( 3 10 ) T319-R321 hHCN3 171 G50-Y242 10 (5.8%) V151-G154 ( b ) V151-A155 ( 2)( b ) R162-T166 A163-T166 A163-R167 R146-W84 (HCND) R146-I86 (HCND) 11 (6.4%) E193-L196 ( 3 10 ) D197-E199 D197-V200 Y201-R205 A204-R208 V210-T213 hHCN4 212 M214-Y409 8 (3.8%) R246-I326 ( 2)(HCND) I318-I327 ( 2)( b ) N323-E325 E325-R332 6 (2.8%) E360-R372 ( 2) E366-K369 ( 3 10 ) spHCN 229 S212-F359 24 (10.5%) K195-E279 ( 2)(HCND) E196-Q280 (HCND) R199-Q280 ( 2)(HCND) V205-R269 L206-R269 ( 2) R215-T270 I272-V281 ( 2)( b ) N274-K276 16 (7.0%) G315-Q317 ( 2) Q317-F321 ( a ) N318-L322 ( a ) R319-E323 ( a ) H320-V324 ( a ) Hence, the most rigid VSDs that are insensitive to cAMP (spHCN, HCN3) have a greater number of H-bonds in the S2-S3L and S3-S4L loops compared to flexible VSDs that are highly sensitive to this cyclic nucleotide. Indeed, the effect of rigidity in these linkers could also explain the relatively scarce modulatory effect exhibited by cAMP in terms of the displacement of the mean activation voltage for the HCN1 channel ( Table 3 ). Furthermore, the presence of a-helical or extracellular 3 10 substructures in the five VSDs studied, except for HCN1, could in turn be related to their low sensitivity to cAMP. On the other hand, although the extracellular 3 10 element present in the S3-S4L of HCN3 was modeled as a helix, as in the cAMP-sensitive HCN2 and HCN4 channels, this region of just 3 residues (one turn) which exhibits a [PRL] motif at positions 194-196 in HCN3 but modeled only for the D subunit of the tetramer. This could indicate that such region is particularly dynamic for this protein. In that context, the presence of the b-turn at the S2-S3L inside the cell and in close contact with the C-linker should considerably restrict its sensitivity to cAMP ( Fig. 6 ). Table 3 shows the H-bonds that support this hypothesis, i.e., the relative rigidity of the linkers, in terms of the number of H-bonds in those regions at the VSD, determines the modulatory effects of cAMP in HCN channels. Molecular dynamics simulations To confirm the position of the intracellular S2-S3L linker in each of the five HCN structures studied, and with the aim of evaluating their conformational freedom, we performed molecular dynamics (MD) CABS Monte Carlo simulations (Nithin et al. 2024) using homology models generated with SWISS-MODEL (Waterhouse et al. 2018) ( see Methods). In each case, cAMP-free (apo) structures were used, and since a structure for the HCN2 channel has not yet been obtained, we decided to use the HCN4 structure as a proper template. This is because in terms of HCN4 responsiveness to the modulatory effects of cAMP, it is more similar to the HCN2 channel (Stieber et al. 2005), although phylogenetically the HCN1 channel is closer to HCN2 ( Fig. 1 ). Besides, these five proteins were selected because they have been the most studied using electrophysiological and patch-clamp fluorometry experiments. This approach facilitates a better comparison of the compositional/structural analysis we performed, contrasting it with the reported information regarding the voltage-dependent activation and the modulating effects of cAMP in each of these channels. As shown in Fig. 7a , we calculated the fluctuation profiles of atomic positions for each residue in the HCND-VSD domain present in the five proteins studied using the CABS-flex computational tool (Jamroz et al. 2014; Kuriata et al. 2018). Furthermore, using Normal Mode Analysis with DynaMut (Rodrigues et al. 2018) and the Anisotropic Network Model (ANM) FF, we were able to estimate the magnitude of the deformations/fluctuations by representing substructures in the form of tubes with low (blue), moderate (white), and high (red) mobility, including regions where each of them experiences deformations from thin to thick ( Fig. 7b ). In this analysis, we used the SS1 mode for the estimation of the root mean square fluctuation (rmsf) in CABS-flex, whose algorithm establishes steric restrictions between residues, at least one of which belongs to a secondary structure element; such is the case of the “elbow” formed by the C-linker of subunit S with the disordered S2-S3L linker present in subunit S + 2 . Our results indicate that the distance between these two structural elements is maximum for the HCN3 channel (~10 Å), which could hinder the propagation of the allosteric wave and make it insensitive to the modulatory effect of cAMP. In the case of the HCN2 channel, which is highly sensitive to the modulatory effect of cAMP, the distance is reduced to 6.5 Å. Besides, we believe that the presence of the antiparallel beta element present in HCN3 significantly restricts the conformational freedom of the disordered part of the b-turn that runs from residue V151 to G154 in that channel. Hence, the absence of the b-substructure in HCN2, on the other hand, manifests in the form of a high conformational freedom for the S2-S3L linker, which could be consistent with the poor packing of the D271 residue in a ‘forbidden’ region of the Ramachandran map of HCN2 or the Q280 residue in the same linker of spHCN, which, on the contrary, is found in a less prohibited area of the (ϕ-ψ) map) ( Suppl. Fig. 2 ), this in turn would be related to high dynamic flexibility for that region (Momen 2017). Therefore, a significant deformability of the S2-S3L could facilitate the propagation of allosteric perturbations triggered from the cAMP binding site in the distal CNBD. The fact that changes in the structural rigidity of the HCN2 channel propagate from the cAMP binding sites via the C-linker has been extensively documented elsewhere (Pfleger et al. 2021). On the other hand, although we found an even shorter distance, i.e., ~5 Å between the S2-S3L and the elbow of the C-linker in the spHCN channel, the high intrinsic rigidity of the VSD of this protein ( Fig. 3 ) must be severely limiting the conformational freedom of the entire transmembrane region, which is consistent with the low cAMP sensitivity reported for this protein. In conclusion, the swapped architecture of the S2-S3L with respect to the C-linker between subunits, which involves possible specific interactions between the S and S + 2 subunits, must be crucial to understanding the cooperativity established between the cAMP binding sites in the CNBD and the VSD, which in turn would allosterically modulate the opening of the HCN channel pore. A relevant interaction could be between residues T273 ( S )/K491 ( S + 2 ) in HCN2 and T324 ( S )/K542 ( S + 2 ) in HCN4, which in the studied models are about 6.5 Å apart in both cases, while in HCN3 the pair of residues involved correspond to positions E153 ( S ) and K375 ( S + 2 ), which are almost twice as far apart, i.e., 12.5 Å. In the spHCN channel, residues S277 ( S ) and K492 ( S + 2 ) are closer, i.e., 9.3 Å, and given the high deformability of the intracellular elbow in spHCN ( Fig. 7b ), it is conceivable that this motif could contact the S2-S3L even though it is considerably rigid. Furthermore, the substitution of a Thr residue for a Ser residue at position 277 of this protein could also be decisive in explaining the shift in the activation V 1/2 in presence of cAMP, as this is a less polarizable residue than Thr (Swart et al. 2004). With the ultimate goal of confirming the relative flexibilities in the HCND-VSD domains of these four proteins, we generated Dynamical Cross-Correlation Maps (DCCM) using the ANM FF in DynaMut. This FF is encoded to optimally predict residue fluctuations in regular secondary structural elements. To do this, a uniform force constant is adopted for all springs in coarse-grained simulations; nodes are identified by the positions of Ca atoms, and the total potential of the system is a sum of harmonic potentials (Atilgan et al. 2001). With this strategy, the DCCMs obtained reveal the dynamic nature of each domain in proteins that are close but not identical. Thus, the role of flexibility/rigidity in the configuration of conformational landscapes reveals that the HCND-VSD domain of the HCN3 channel does indeed exhibit the least flexible conformational landscape of mammalian channels and that the same domain in the spHCN channel also shows fewer areas of flexibility ( Suppl. Fig. 3 ). Taken together, these results are consistent with our general hypothesis and thus, HCN channels with flexible sensors (HCN2, HCN4) are more likely to respond to the allosteric propagation resulting from cAMP binding, while those with less flexible landscapes (HCN3, spHCN) respond with difficulty to this type of conformational stimulus. Finally, we performed the same analysis for the CNGA1 channel, a cyclic nucleotide-gated channel ( Suppl. Fig. 4 ). Consistent with our hypothesis, this channel exhibited the highest flexibility profile of those studied here ( Fig. 2b ), with a high deformability index in the S2-S3L/C-linker and a very high flexibility DCCM. This is consistent with a high dependence on cAMP for its activation, typical of channels in this subfamily, channels largely insensitive to membrane potential (James and Zagotta 2018). The persistence of HCN channels on the evolutionary scale In this study, we have made an effort to identify the structural determinants that could explain the distinctive modulatory effects exhibited by cAMP among the four isoforms of the HCN channel in mammals. Our results clearly suggest that the degree of conformational freedom exhibited by the S2-S3L linker and its possible interaction with the ‘elbow’ region in the C-linker could be decisive in the propagation of conformational changes triggered by cAMP binding, which allosterically couple the binding of this intracellular ligand to the transmembrane voltage sensor domain. When performing a compositional analysis of the VSD in the four mammalian HCN variants, we found a remarkable correlation between intrinsically very flexible voltage sensors (HCN2) and high sensitivity to cAMP in terms of the V 1/2 of activation. Thus, the intrinsically most rigid channel of the four isoforms (HCN3) is a channel that is practically insensitive to cAMP. Our study also reveals that the HCN3 channel is phylogenetically closest to HCN channels present in metazoans that diverged very early in evolution (Echinodermata, Cnidaria, Arthropoda). This phylogenetic proximity is remarkable and could indicate that the HCN3 clade represents the product of a very early duplication event in chordates (Jackson et al. 2007). Interestingly, the flexibility profiles for HCN and HCN-like channels in basal deuterostomes (Cnidaria, Echinodermata, Actinopterygii) are very low, as is the profile of the HCN3 channel, which also correlates with very low sensitivity to cAMP. However, those HCN channels described in arthropods have shown very high sensitivity to the modulatory effects of cAMP (Gisselmann et al. 2003, 2005a, 2005b) ( Fig. 3 ). This could reflect the very early divergence of protostomes during the early stages of animal evolution (Wheat and Wahlberg 2013). On the other hand, given that HCN channels have been found in the olfactory receptor neurons of A. mellifera and D. melanogaster (Marx et al., 1999), as well as being present in Heliothis virescens (Krieger et al. 1999) and P. argus (Gisselmann et al. 2005b), it has been postulated that the mechanisms of chemosensory transduction in mammals and arthropods may be profoundly different (Sato and Touhara 2008; Kaupp 2010). Thus, metabotropic signaling, dependent on G protein-coupled receptors, is typical in mammalian olfactory neurons, providing a wide range of positive and negative regulations in these animals in response to diverse environmental odors. In contrast, ionotropic signaling in insects is an adaptation that improves the speed of chemosensation in animals that fly rapidly and track sudden changes in the concentration and quality of multiple odors (Kaupp 2010). This is consistent with the fact that mice are able to discriminate between different odors in less than 250 ms (Abraham et al., 2004), but honeybees are able to distinguish between them in as little as 200 ms (Wright et al., 2009). From a physiological perspective, one final consideration is worth noting. Given these profound differences between HCN channels in protostome and deuterostome animals, is it possible to distinguish any attributes in the activation kinetics of these proteins that would be consistent with the metabotropic or ionotropic pathways of each type of organism? The kinetics of the so-called Ih current varies significantly due to evolutionary diversification in arthropods (Jackson et al. 2007). In that context, Ih activation kinetics in insects are inherently fast, closely resembling the rapid mammalian HCN1 isoform (Gisselmann et al. 2003). In crustaceans, the significant acceleration of the activation time constant that has been reported for the Ih current in the presence of cyclic nucleotides, closely resembles the rapid kinetics typical of the mammalian HCN1 channel (Gisselmann et al. 2005b). Besides, unlike what happens in mammals, in arthropods such as D. melanogaster , crab ( Carcinus maenas ), and cockroach ( Periplaneta americana ) typically only one HCN channel gene is expressed that retains the characteristics of the ancestral fast channel (Jackson et al. 2007). This would be consistent with the typical fast activation kinetics and strong voltage dependence exhibited by HCN channels in arthropods (Gisselmann et al. 2003, 2005a, 2005b) compared to their mammalian orthologs, which exhibit a wide range of voltage sensitivities on a scale from hundreds of milliseconds to several tens of seconds (Wahl-Schott and Biel 2009). Finally, the persistence of CNBD throughout the evolutionary scale remains a mystery of life (Berman et al. 2005). Its ancestry is enigmatic in the hyperthermophilic order Aquificales (García-Morales et al. 2019). However, to the best of our knowledge, it has not yet been possible to express this channel described in the bacterium Aquifex aeolicus (AqK) and recording the possible voltage dependence or sensitivity to the presence of cAMP in this channel. The archaeobacterial MVP channel, on the other hand, although it exemplifies a minimalist model of hyperpolarization-activated channels, does not have a CNBD, and consequently cAMP has no modulatory effect on its activation behavior. In addition, it is highly selective for potassium ( P Na / P K ≈ 0.01), while HCN channels are poorly selective for monovalent cations ( P Na / P K ≈ 0.2) and have a conductance close to ~ 1-2 pS (HCN2) compared to 37 pS for MVP (Sesti et al. 2003; Randich et al. 2014). These profound differences strongly suggest that very early in the evolution of life, a lineage of archaeobacterial cells may have contributed the reverse electromechanical coupling module, typical of HCN channels, but lacking sensitivity to cyclic nucleotides. On the other hand, an ancestral bacterial lineage provides the CNBD module that recognizes cAMP and was able to couple it to the transmembrane domain through a gene fusion event (Pasek et al. 2006; Poole and Penny 2007; Marsh and Teichmann 2010). Thus, the first eukaryotic cell appeared, already possessing both attributes in a single ancestral HCN protein ( Fig. 8 ). This protein diverges into two lineages with very different activation kinetics for protostomes and deuterostomes. A last conclusion Although HCN channels are principally operated by voltage, the modulatory effect of cAMP is enhanced by the degree of deformability acquired by specific structural elements in these proteins. Thus, we have described in some detail a probable intersubunit interaction that connects the S2-S3L linker to the elbow of the C-linker with the subunit in front. This interaction depends on the intrinsic flexibility of the former to facilitate the allosteric transmission that couples the cAMP-binding event with the displacement of the C-linker and, in turn, with a probable interaction through S2-S3L. This linker in turn directly contacts the TM region that detects changes in membrane voltage. Furthermore, there appears to be an evolutionary trend in which these channels have become more flexible in key regions of their architecture, and this goes hand in hand with a greater ability to become sensitized when interacting with cAMP through an intracellular module that has persisted for millions of years of biological evolution. Declarations Supplementary information The online version contains supplementary material available at https://link.springer.com/journal Acknowledgments The authors would like to thank Dr. E. Rudiño of the Institute of Biotechnology (UNAM) for his valuable contributions and comments on this work, as well as his helpful discussions. KGAV received a grant (CVU: 693618) from the Secretaría de Ciencia, Humanidades, Tecnología e Innovación (Secihti). Author contributions KGAV contributed to the acquisition and compilation of protein sequences and the analysis of structural data; DB conceived and designed this work, performed all flexibility and molecular dynamics analysis, and wrote and prepared the manuscript. Funding This project was funded in part by a grant from the National Technological Institute of Mexico: “Scientific Research Projects, Technological Development, and Innovation 2025—Federal Technological Institutes and Centers” to DB (grant reference number 21871.25-P). Data availability The modeling code, analysis, and protein models can be found in the following repository: https://github.com/DBalleza-Flexiprot/Voltage-Sensor-Domain Declarations The authors declare that they have no competing financial interests. References Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, Bodenstein SW, Evans DA, Hung CC, O'Neill M, Reiman D, Tunyasuvunakool K, Wu Z, Žemgulytė A, Arvaniti E, Beattie C, Bertolli O, Bridgland A, Cherepanov A, Congreve M, Cowen-Rivers AI, Cowie A, Figurnov M, Fuchs FB, Gladman H, Jain R, Khan YA, Low CMR, Perlin K, Potapenko A, Savy P, Singh S, Stecula A, Thillaisundaram A, Tong C, Yakneen S, Zhong ED, Zielinski M, Žídek A, Bapst V, Kohli P, Jaderberg M, Hassabis D, Jumper JM.Accurate structure prediction of biomolecular interactions with AlphaFold 3. 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Sci STKE. 2004. 2004(253):re15. doi: 10.1126/stke.2532004re15 Additional Declarations No competing interests reported. Supplementary Files Ramachandran.pdf Suppl.docx Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted Editorial decision: Revision requested 29 Dec, 2025 Reviews received at journal 29 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers agreed at journal 19 Nov, 2025 Reviewers invited by journal 19 Nov, 2025 Editor assigned by journal 18 Nov, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 14 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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14:13:05","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82301,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/4e2156502b5047a31dd1af30.png"},{"id":96849336,"identity":"772313d9-2c18-4ec0-a709-7e8db6f12869","added_by":"auto","created_at":"2025-11-26 17:06:53","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75859,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/a07acaee53598c91d2a7dac5.png"},{"id":96918953,"identity":"9e81293c-8990-42e8-9dcb-ff143083b77f","added_by":"auto","created_at":"2025-11-27 14:12:53","extension":"xml","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":235697,"visible":true,"origin":"","legend":"","description":"","filename":"743d071909a548d89ce188a5cf270a271structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/aad333f8fea0f3e524e0a531.xml"},{"id":96918112,"identity":"90a15388-7fb7-4c6a-9c8d-377d70d306a7","added_by":"auto","created_at":"2025-11-27 14:11:10","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":249671,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/a196084b374dffba620a1b1d.html"},{"id":96849300,"identity":"0f9579ba-3a48-4388-98d5-ec6ce7fef85e","added_by":"auto","created_at":"2025-11-26 17:06:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82830,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic tree of 22 proteins with high similarity to HCN channels. Three prokaryotic sequences were used as a coherent outgroup, including two archaeobacterial sequences lacking CNBD but activated by hyperpolarization (MVP, MaVP) and one bacterial sequence that includes a CNBD but for which there is no evidence of voltage-dependent activation (AqK). The sequences shown in red boxes represent those that exhibit comparatively flexible voltage sensors compared to those that are more rigid, shown in black boxes. These channels have been electrophysiologically characterized, and data are available on their relative sensitivity to cAMP.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/fe9d1b5f52e372db342245c8.png"},{"id":96919617,"identity":"8de7f9af-6402-4024-ae18-b8a2830b9484","added_by":"auto","created_at":"2025-11-27 14:14:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":157749,"visible":true,"origin":"","legend":"\u003cp\u003eEstimation of the intrinsic flexibility of the VSD. (A) Flexibility profiles for VSDs in five HCN channels; the cyclic nucleotide-dependent channel CNGA1; the hERG channel, which carries a cyclic nucleotide binding homology domain (CNBHD) but does not require binding to cAMP for activation; and the voltage-dependent channel Kv7.2 (KCNQ2). (B) Weighted flexibilities of VSDs for each representative sequence in terms of mean B-factor. The inset shows an overlap between three S2-S3L linkers, illustrating a tendency toward greater flexibility in channels that are increasingly dependent on cAMP binding.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/d539486219b2c26169b9ea0a.png"},{"id":96849301,"identity":"a47a841b-e07a-4613-9c0d-92d604fb0c50","added_by":"auto","created_at":"2025-11-26 17:06:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92491,"visible":true,"origin":"","legend":"\u003cp\u003eShift in the activation midpoint in HCN channels as a function of the intrinsic flexibility of the VSD. Two trends can be observed, whereby the intrinsically more flexible sequences are more likely to respond to the modulating effect of cAMP on the voltage dependence for those channels. Two distinct types of sensitivity are clearly observed: (1) flexible channels sensitive to cAMP in deuterostome animals (\u003cem\u003eblue\u003c/em\u003e) and (2) channels highly sensitive to cAMP but less flexible in protostome animals (Arthropoda, \u003cem\u003ered\u003c/em\u003e). The Archaean MVP channel was included, which has the lowest flexibility profile of all the voltage sensors studied and follows the trend of channels in deuterostome animals more harmoniously.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/5672ab79d5e0f238c395ab97.png"},{"id":96918292,"identity":"9d009f4c-06e7-44bb-9818-9450e2dab22c","added_by":"auto","created_at":"2025-11-27 14:11:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":364639,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the B-factors for three human HCN channels deposited in the PDB. The B-factors are shown in the respective structures for HCN1 (5u6o), HCN3 (8inz), and HCN4 (6gyn). The voltage sensor with the most rigid profile corresponds to HCN3, consistent with the estimate of its intrinsic flexibility presented in Fig. 2b. The relative positions of the CNBD, the intercalated C-linker, and the HCND present in each protein are also shown.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/4169c4e687b63c1a5ea454f2.png"},{"id":96919162,"identity":"693d3929-ce5a-40f9-b3ed-7c6dc0216d09","added_by":"auto","created_at":"2025-11-27 14:13:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":398582,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Comparison of model structures generated with Swiss-Model showing the typical regions of HCN channels: HCN domain (HCND, salmon); voltage sensor domain (VSD, teal); pore domain (PD, olive); C-linker (orange); cyclic nucleotide binding domain (CNBD, blue jay). The region of greatest rigidity in each protein is highlighted in black, according to the FlexiProt 3.0 predictor. (\u003cstrong\u003eB\u003c/strong\u003e) Tertiary structure predicted by Swiss-Model for the HCND-VSD domain of spHCN and HCN1-4 channels in ascending order of intrinsic flexibility: spHCN \u0026lt; HCN3 \u0026lt; HCN1 = HCN4 \u0026lt; HCN2. The S2-S3L linker is highlighted in deep blue, with a b-turn configuration in HCN3. Also highlighted are ordered substructures in S3-S4L in spHCN (a-helix) and HCN2-4 (3\u003csub\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/sub\u003e), as well as preordered zones in S1-S2L of spHCN (arrowheads and w-loops) and disordered zones within the S3 segment in HCN2 and HCN4 that provide flexibility to the VSD in those proteins (asterisks).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/38861b031338f5059e6e2c8a.png"},{"id":96849312,"identity":"5f9dbeb3-6736-45c0-bfca-d519416e989b","added_by":"auto","created_at":"2025-11-26 17:06:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":240988,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Tertiary structure of the transmembrane region (TM) showing the spatial arrangement of the four TM segments of the voltage sensor (VSD) and the HSND of one of the four subunits that form the HCN3 channel. Highly flexible and semi-disordered regions are shown as loops in the hydrophobic core of the sensor (brown), and more ordered regions are shown as cylinders. Both the S2-S3L linker and the HCND face the aqueous interior of the cell and expose a highly hydrophilic surface (blue/white). The VSD was visualized using BioVia Discovery Studio. (\u003cstrong\u003eB\u003c/strong\u003e) Network of hydrogen bonds that stabilize the extracellular S3-S4L linker and form a 3\u003csub\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/sub\u003e helix turn. (\u003cstrong\u003eC\u003c/strong\u003e) Network of H-bonds stabilizing the b-turn in the intracellular linker S2-S3L. H-bonds are displayed in dotted sky blue lines, and the images were visualized using Chimera X (\u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003eC\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/3b766cc7126e817b911dc705.png"},{"id":96849308,"identity":"ae5a37ce-5006-4cbe-9803-307959df1870","added_by":"auto","created_at":"2025-11-26 17:06:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":275488,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Root-mean-square fluctuation (rmsf) per residue of the HCND-VSD domain generated with CABS-Flex. This MD parameter is a measure of the average deviation of atomic positions from their mean positions over time. (\u003cstrong\u003eB\u003c/strong\u003e) DynaMut structural stability prediction and deformation/fluctuation associated energy in the structural context of two opposing subunits for the HCN2, HCN3, and spHCN channels. The magnitude of atomic fluctuation is represented as tubes of varying thickness, colored red (high), white (moderate), and blue (low).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/bebe26629659e5a585bce159.png"},{"id":96919636,"identity":"179dc1ce-0e55-4b17-94a3-a2b4a26c1449","added_by":"auto","created_at":"2025-11-27 14:14:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":142204,"visible":true,"origin":"","legend":"\u003cp\u003ePossible evolutionary scenario for the acquisition of cyclic nucleotide-gated channel domains in the last eukaryotic common ancestor (LECA). Each domain is depicted by color, where red (CNBD), blue (VSD), and teal (PD) represent each of the cell lineages where channels associated with the CNBD (Bacteria) and the hyperpolarization-activated VSD (Archaea/Eukaria) have been found. Through a possible ancestral gene fusion event, the cAMP recognition region is integrated and selected into a new hybrid channel, resulting in a channel that can be activated by hyperpolarization of the membrane potential and regulated by binding to intracellular cAMP.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/755b3cb08e7ae35fa93f7d28.png"},{"id":104250706,"identity":"f0b0caa3-a13d-43ca-b20b-a3b0bb3a3e3d","added_by":"auto","created_at":"2026-03-09 16:06:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3140293,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/447c55b8-08ed-4126-835e-de6ba98f802c.pdf"},{"id":96918930,"identity":"b4ff0a87-0571-4006-be3c-4cfcb0ed69de","added_by":"auto","created_at":"2025-11-27 14:12:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10420115,"visible":true,"origin":"","legend":"","description":"","filename":"Ramachandran.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/97df4fb99c55cdb41f3466d1.pdf"},{"id":96849307,"identity":"8edb4113-2b44-47f4-ba20-295ef66f606a","added_by":"auto","created_at":"2025-11-26 17:06:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":909998,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.docx","url":"https://assets-eu.researchsquare.com/files/rs-8117147/v1/cdad291f3df69acf9374c4cc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural determinants of voltage sensitivity in hyperpolarization-activated ion channels and their persistence in the evolutionary scale","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cspan name=\"Emphasis\"\u003ePlease have a look at courier new font provided for text in article.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eIn the superfamily of voltage-gated ion channels (VGIC), hyperpolarization-activated and cyclic nucleotide-gated channels (HCN) occupy a prominent place. These channels are essentially activated when the cell membrane hyperpolarizes and the intracellular voltage becomes even more negative than its resting potential (Sartiari et al. 2017). These properties enable the cells that express them to regulate the frequency of action potentials and thus act as pacemaker currents (Baruscotti and DiFrancesco 2004). Although this activity responds to the transmembrane potential, the voltage dependence for the opening activity of these channels is reduced in the presence of a specific ligand, cyclic AMP. In this way, cAMP exerts a modulatory role on these channels, facilitating the action of voltage and increasing the channel open probability (Saponaro et al. 2021a). The electromechanical coupling mechanism that facilitates the opening of HCN channels has been extensively studied (Lee and Mackinnon 2019; Saponaro et al. 2021b; Burtscher et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Intracellular cAMP, in parallel, facilitates the activation of these channels by shifting the voltage dependence toward more depolarizing potentials (Cuttle et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). This allows channel activation to occur with less hyperpolarization. However, the details of the allosteric modulation that allows the voltage sensor domain (VSD) to communicate with the cytosolic cyclic nucleotide-binding domain (CNBD) are less well known. Some studies have demonstrated autoinhibition mechanisms where cAMP facilitates tetramerization of a more energetically stable form of the HCN4 channel, which is compatible with the activated state (Akimoto et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; vanSchouwen et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFour isoforms (HCN1-4) with characteristic activation kinetics have been described in mammalian cells (Stieber et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). These paralogs exhibit 80\u0026ndash;90% sequence identity between the start of the first transmembrane segment, S1, to the end of the cytosolic CNBD, although in terms of sequence length they are different (Jackson et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, the role of the nucleotide ligand clearly exerts different modulatory effects on each isoform (Wainger et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e; Stieber et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). Typically, the biophysical parameter that allows to estimate the voltage dependence between different isoforms of the same channel, or to evaluate the effect of point mutations on the gating machinery of ion channels, is the half-activation voltage \u0026Delta;V\u003csub\u003e\u003cstrong\u003e1/2\u003c/strong\u003e\u003c/sub\u003e, which allows to quantify the membrane potential at which a voltage-activated ion channel reaches 50% of its maximum ionic current. Thus, it has been determined that of the four known isoforms, HCN2 and HCN4 exhibit the highest sensitivity to cAMP in terms of the V\u003csub\u003e\u003cstrong\u003e1/2\u003c/strong\u003e\u003c/sub\u003e shift (HCN2: 12\u0026ndash;14 mV; HCN4: 11\u0026ndash;23 mV), while the HCN1 channel is poorly modulated by this ligand (~\u0026thinsp;2 mV) (Kaupp and Seifert \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). The HCN3 isoform, on the contrary, is insensitive to cAMP. Indeed, the activation curve in the presence of ligand shifts to more hyperpolarized potentials (M\u0026iacute;strik et al. 2005; Stieber et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). This observation has been interpreted in terms of an anomalous autoinhibition mechanism, which depends on a structural change in the domains that communicate cAMP binding with the channel activation machinery (Stieber 2005).\u003c/p\u003e\n\u003cp\u003eThe search for HCN orthologous channels and the modulatory role of cyclic nucleotides has led to a better understanding of the nature of these proteins and their role in cell physiology. Thus, cAMP-modulated HCN-like channels have been described in urochordates and lower vertebrates (Gauss et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Shin et al. \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Baker et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wobig et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) as well as in arthropods (Gisselmann et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e, 2005a,b). Hyperpolarization-activated channels have even been found in plants, although these do not appear to be regulated by cyclic monophosphate nucleotides, as their CNBDs have been found to lack specific residues for binding to these ligands (Lu et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, HCN-like sequences show considerable identity at the amino acid residue level but significant differences from their mammalian counterparts (\u003cstrong\u003eSuppl. Figure\u0026nbsp;1\u003c/strong\u003e), providing key clues for deciphering the evolutionary relationships between HCN channels.\u003c/p\u003e\n\u003cp\u003eEight bacterial homologues with significant sequence similarity to eukaryotic HCN channels have been described. These include weakly voltage dependent channels found in bacteria, and so-called as AqK (from \u003cem\u003eAquifex aeolicus\u003c/em\u003e), SthK (from \u003cem\u003eSpirochaeta thermophila\u003c/em\u003e), AmaK (from \u003cem\u003eArthrospira maxima\u003c/em\u003e), TerK (from \u003cem\u003eTrichodesmium erythraeum\u003c/em\u003e), LpcK (from \u003cem\u003eLyngbya\u003c/em\u003e sp. PCC 8106), LbiK (from \u003cem\u003eLeptospira biflexa\u003c/em\u003e), and LliK (from \u003cem\u003eLeptospira licerasiae\u003c/em\u003e) (Brams et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; James et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Romero-Romero et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Of all these channels, only SthK has been extensively studied by the group of C. Nimigean, who describe it as a channel activated by cyclic nucleotides and modulated by depolarizing voltages, which places it functionally closer to cyclic nucleotide\u0026ndash;gated (CNG) channels in comparison with the HCN ones (Schmidpeter et al. 2018; Gao et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). In 2003, the first discovery of a hyperpolarization-activated prokaryotic channel, MVP, was reported in \u003cem\u003eMethanococcus jannaschii\u003c/em\u003e, an extremely thermophilic methane-producing archaebacterium (Sesti et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). This channel is activated after the downward displacement of S4 segments in the VSD, similar to the helical motion also described for the sea urchin HCN channel and the human HCN1 channel (Dai et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Burtscher et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the other hand, some studies have shed light on the evolution of these proteins, suggesting that gene duplication phenomena may have occurred independently in different lineages of the animal kingdom. These studies have identified regions that are important for the specific functions of each isoform, as well as for those specific to each species (Jackson et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). To our knowledge, cyclic nucleotide-dependent ion channels have not been described in the Archaea domain.\u003c/p\u003e\n\u003cp\u003eThe structural basis of the allosteric coupling of cAMP binding to the voltage-sensitive transmembrane domain to modulate the voltage sensitivity in HCN channels remains unknown. However, several electromechanical models have been proposed to reveal the cytoplasmic-to-transmembrane-domain movements as result of the cAMP binding event (Lee and Mackinnon \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Akimoto et al. 2018; Gross et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Saponaro et al. 2021a). In this study, we take advantage on the structural information available in the Protein Data Bank, as well as the extensive electrophysiological studies that have been reported by various research groups to understand the activation and modulation by cAMP of these ion channels (reviewed, for example, in Sartiani et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e and Saponaro et al. 2021b), to compare structure information with functional data in terms of their voltage dependence and their degree of sensitivity to cAMP. We also wonder whether the compositional variations between each isoform, as well as the functional differences found in the paralogous proteins studied, could be explained in terms of the intrinsic flexibility of the VSD throughout the evolutionary scale. The findings described in this study point to a relationship in which HCN channels that are more sensitive to the modulating effect of cAMP exhibit significantly more flexible voltage sensors than those whose amino acid composition is intrinsically more rigid. We also discovered that sensitivity to cAMP as a modulator of voltage dependence in these channels is poor in early animals, which is linked to low flexibility profiles in the VSDs in those channels. On the contrary, sensitivity to this ligand is significantly higher in protostomes than in deuterostomes. These relationships are also significant when considering the MVP channel of the Archaea lineage, which is the hyperpolarization-activated channel that exhibits the greatest intrinsic rigidity, consistent with a hyperthermophilic origin for this class of proteins in a thermoadaptive scenario close to the root of the tree of life.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData extraction and Structural Dataset\u003c/h2\u003e\u003cp\u003eThe sequences and structural data known for the four isoforms of human HCN channels, whose functional characterization is widely reported in the literature, are deposited in the GenBank and the Protein Data Bank, with the following accessions: 5U6O_1 (hHCN1), NP_001185.3 (hHCN2), NP_065948.1 (hHCN3), NP_005468.1 (hHCN4). We also include four paralogous sequences found in \u003cem\u003eXenopus laevis\u003c/em\u003e, with high similarity but not yet functionally characterized: XP_031750156.1 (XtHCN1), XP_002939815. 3 (XtHCN2), XP_031747278.1 (XtHCN3), XP_017947882.2 (XtHCN4). It was particularly interesting to include HCN-like channels whose functional characterization has already been reported in terms of the change in voltage activation midpoint dependent on cyclic AMP. These channels include the following isoforms: QKM75727.1 (DrHCNL1) and XP_009302941.1 (DrHCNL2) from zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) (Wobig et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); NP_999729.1 (spHCN, SpIh) from sea urchin (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e) (Shin et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Fynn et al. 2007); ALO50060.1 (NvHCN1) and ALO50059.1 (NvHCN2) from the starlet sea anemone (\u003cem\u003eNematostella vectensis\u003c/em\u003e) (Baker et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); AAQ16311.1 (PaIh) from spiny lobster (\u003cem\u003ePanulirus argus\u003c/em\u003e) (Gisselmann et al. 2005b); AAX78396.1 (DmHCN, Ih) from fruit fly (\u003cem\u003eDrosophila melanogaster\u003c/em\u003e) (Gisselmann et al. 2005a); NP_001011568.1 (AmIh) from honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e) (Gisselmann et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and the archaeal MVP channel of \u003cem\u003eMethanocaldococcus jannaschii\u003c/em\u003e (WP_010869632.1) (Sesti et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Likewise, the following sequences were included for the reconstruction of phylogenetic relationships: AAC07678.1 (AqK) from \u003cem\u003eAquifex aeolicus\u003c/em\u003e, WP_131007350.1 (MaVP) from the archaeon \u003cem\u003eMethanofervidicoccus abyssi\u003c/em\u003e, Q38998.2 (AKT1) and NP_199436.1 (KAT1) from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and ABF71886.1 (hERG1) from human.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMolecular phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eFor molecular phylogenetic analysis, HCN and HCN-like sequences were aligned using the Clustal W program (Thompson et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and a neighbor-joining tree was constructed using the online version of MAFFT (Katoh et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We included sequences from two hyperpolarization-activated plant channels, AKT1 and KAT1, the bacterial channel AqK with the CNBD domain, and the hERG channel, which exhibits an \u0026ldquo;intrinsic ligand\u0026rdquo; that mimics a cyclic nucleotide within a homologous CNBD (Codding and Trudeau \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eProtein flexibility estimation\u003c/h3\u003e\n\u003cp\u003eThe estimation of intrinsic flexibility for primary protein sequences has been previously described in our research group using the FlexiProt predictor (Balleza \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Garc\u0026iacute;a-Morales and Balleza \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In brief, this algorithm allows us to predict the local flexibility of each of the 20 side chains by correlating the amino acid composition based on the two neighbors for each position. With this normalized B factor (nBf), we assign a relative flexibility associated with the structural characteristics and conformational freedom of each side chain in any typical sequence of any protein. In this study, we also calculated the mean B factor (mBf) corresponding to the transmembrane domain of the voltage sensor (S1-S4) in each protein evaluated. This parameter is the result of the average of each nBf based on the number of amino acids for each protein segment, expressed as the inverse of the absolute value for this weighted average:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"196\" height=\"100\"\u003e\u003c/p\u003e\n\u003ch3\u003eStructure Modelling\u003c/h3\u003e\n\u003cp\u003eThe structures of human HCN1-4 channels have been deposited in the PDB with varying degrees of atomic resolution, and include structures obtained by Cryo-EM, except for the spHCN channel, which was obtained by integrative structure determination. Except for the HCN2 channel, whose structures are known only for the cytoplasmic regions and not the transmembrane region of interest to us, the deposited structures include the voltage-sensing domains. However, these structures generally lack information on the linker regions between segments S1 to S4, which could be related to a high mobility rate for these regions. This led us to generate high-quality three-dimensional models using AlphaFold3 (Abramson et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) or using the SWISS-MODEL homology modeling server (Waterhouse et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with specific PDB files as follow: HCN1 (5u6o, 3.50 \u0026Aring;); HCN2 (6gyn, 3.40 \u0026Aring;) (however, the 5u6o structure was also evaluated for this specific model); HCN3 (8inz, 2.72 \u0026Aring;); HCN4 (6gyn) and spHCN (8zzw). Thereby, we generated high-quality structures that modeled the missing regions in each protein. Each model was validated using both the MolProbity tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://molprobity.biochem.duke.edu/index.php\u003c/span\u003e\u003cspan address=\"http://molprobity.biochem.duke.edu/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the Procheck server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://saves.mbi.ucla.edu/\u003c/span\u003e\u003cspan address=\"https://saves.mbi.ucla.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), mainly evaluating stereochemical parameters such as the presence of unfavored rotamers and C\u003csub\u003e\u003cb\u003eβ\u003c/b\u003e\u003c/sub\u003e deviations\u0026thinsp;\u0026gt;\u0026thinsp;0.25\u0026Aring;. Hence, the best-rated models were chosen for further analysis. Three visualizers were used to analyze each structure: PyMOL (The PyMOL Molecular Graphics System, version 3.0 Schr\u0026ouml;dinger, LLC.), Chimera X (Goddard et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and DSV (Biovia \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eMolecular Dynamics Simulations and Vibrational Entropy Estimation\u003c/h3\u003e\n\u003cp\u003eDynamic simulations were executed using the CABSflex 2.0 server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biocomp.chem.uw.edu.pl/CABSflex2/submit\u003c/span\u003e\u003cspan address=\"http://biocomp.chem.uw.edu.pl/CABSflex2/submit\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using the SS1 mode with restraints for residues structured in regular secondary regions (C-linker). This type of simulations allows the prediction of protein flexibility by determining the root-mean-square fluctuation (RMSF) (Kurcinski et al., 2018). CABSflex is based on the CABS (C-Alpha, Beta, and Side-chains) model of polypeptide chains, which is a medium-resolution coarse-grain model in which the main chain of a polypeptide is replaced by two pseudoatoms per residue. The CABS uses Monte Carlo dynamics of a single protein or a set of chains to reproduce local movements. This reproduces very well the flexibility of proteins and their long-term dynamic patterns (Kolinski \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo evaluate the possible restrictions of each flexibility profile that we calculated previously (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), in terms of the stability of the VSD and the intercalated C-linker, we followed a coarse-grained Normal Mode Analysis (NMA) strategy that considers the physicochemical nature of each amino acid and thus allows to calculate significant changes in vibrational entropy in harmonic motions of the protein structure. This was done using the DynaMut predictor (Rodrigues et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), a consensus predictor of protein stability based on the vibrational entropy changes predicted by an Elastic Network Contact model (ENCoM) (Frappier et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic characterization of Hyperpolarization activated HCN channels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HCN channel subfamily is represented by four isoforms, HCN1-4 in mammals, and is closely related to the CNG subfamily of cyclic nucleotide-gated ion channels. These two groups of ion channels, along with the KCNH (Kv\u003cstrong\u003e\u003csub\u003e10-12\u003c/sub\u003e\u003c/strong\u003e) subfamily channels, form one of the largest clades of the Voltage-Gated Ion Channel, VGIC, superfamily (Yu and Catterall 2004). However, unlike CNG channels, HCN channels are highly voltage-sensitive, with the HCN1 isoform being the most sensitive in terms of the activation time constant (t\u003cstrong\u003e\u003csub\u003e\u0026minus;100 mV\u003c/sub\u003e\u003c/strong\u003e = 67\u0026nbsp;\u003cbr\u003e\u003cimg width=\"12\" height=\"20\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABIAAAAeBAMAAADTIlMfAAAAAXNSR0IArs4c6QAAABtQTFRFAAAAAAAAAAA6ADqQkDoAkNv/2////7Zm///bxPh3hAAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAJ0lEQVQoU2NgoD7gcE6AGko2i10QDIQCQAaRbQqKXgx/othBTCgAAN/1CvlL7CmfAAAAAElFTkSuQmCC\" alt=\"image\"\u003e\u0026nbsp;16 ms) (Stieber et al. 2005). A previous study determined that HCN1-4 isoforms form a paralogous group that was established before the origin of the vertebrate clade, having been present in the common ancestor of fish and tetrapods (Jackson et al. 2007). This proposal was strengthened when the first report of a functional isoform, spHCN, present in sea urchin sperm was made (Gauss et al. 1988). Decades later, the discovery of functional HCN-like isoforms in early metazoans and some arthropods was also reported (Marx et al. 1999; Gisselmann et al. 2003, 2005; Baker et al. 2015). Thus, the evolutionary history of the HCN family is becoming increasingly complete, strongly suggesting that it probably evolved through independent gene duplications, as has been proposed elsewhere (Jackson et al. 2007).\u003c/p\u003e\n\u003cp\u003eTo confirm the nature of these evolutionary relationships, a phylogenetic analysis was performed using the neighborhood-joining method (\u003cstrong\u003eFig. 1\u003c/strong\u003e), and the estimated distances for each of the clades among the species were calculated. It should be noted that in this analysis an important criterion was to select ion channels that have been functionally characterized by electrophysiological techniques. Thus, the phylogenetic analysis revealed that the four isoforms present in mammals formed a monophyletic group, which is closely related to the clade of HCN channels described in arthropods but distant from both the spHCN channel (Echinodermata) and one of the two isoforms described as HCN in \u003cem\u003eNematostella vectensis\u003c/em\u003e (Cnidaria), NvHCN1 (Baker et al. 2015). Interestingly, the second isoform described by that research group, NvHCN2, is consistently positioned close to the HCN1-4 clade present in mammals and amphibians. In this phylogenetic analysis, we also included two HCN-like channels discovered in zebrafish, HCNL-1 and HCNL-2, which, unlike typical HCN channels, show low permeability to Family IA metal cations (Na\u003cstrong\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e, K\u003cstrong\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e) and preferentially conduct protons (Wobig et al. 2020). In this sequence analysis, we also included the hERG channel, whose intrinsic ligand facilitates its activation in the absence of cAMP (Codding et al. 2020). Consistent with these differences, this channel clearly occupies an outgroup with the hyperpolarization-activated plant AKT1 and KAT1 channels, slightly closer to the clade of HCN and HCN-like channels described above. Finally, we included three prokaryotic sequences for hyperpolarization-activated channels, MVP and probably MaVP, present in \u003cem\u003eMethanocaldococcus jannaschii\u003c/em\u003e and \u003cem\u003eMethanofervidicoccus abyssi\u003c/em\u003e, respectively (Sesti et al. 2007), as well as the AqK channel described by us, which carries a cyclic nucleotide-binding domain (CNBD) connected by a C-linker to the VSD (Romero et al. 2019). This substructure is typical of channels of the HCN family, as well as CNG and KCNH (Codding et al. 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe intrinsic flexibility of the voltage sensor correlates with the ligand-induced change in the activation V\u003csub\u003e1/2\u003c/sub\u003e of HCN channels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce we defined the phylogenetic relationships among all the HCN channels that have been previously functionally characterized, we decided to try to find some correlation between their composition, their structure and some of their activation parameters. With this objective in mind, we defined a more detailed study of the VSD, since it is well known that the downward movement of one of its four transmembrane (TM) segments, S4, is atypical within the VGIC superfamily, and that this displacement is probably conserved throughout the evolutionary scale (Randich et al. 2014, Dai et al. 2019, Wu et al. 2023).\u003c/p\u003e\n\u003cp\u003eWe also decided to focus on the midpoint activation voltage parameter, V\u003cstrong\u003e\u003csub\u003e1/2\u003c/sub\u003e\u003c/strong\u003e, in response to the presence of cAMP, since this ligand interacts specifically with each isoform, positively or negatively modulating the voltage dependence for the activation of these channels. We reasoned that it would be possible to reveal subtle differences encoded in the VSD sequence for each isoform or paralogous protein, which would reveal differences in the modulatory effect of each channel. For example, it is widely known that HCN1, 2, and 4 channels respond to cAMP by shifting the activation curve toward more depolarized voltages, i.e. by making the channel more sensitive in terms of the activation voltage. However, the HCN3 channel responds in the opposite way, shifting the voltage curve toward more hyperpolarized potentials in presence of cAMP, which is interpreted as an antagonistic effect of this ligand by desensitizing the protein in terms of its activation voltage (Stieber et al. 2005). Hence, it was very tempting to ask the reason behind the apparent lack of sensitivity of the human HCN3 channel in response to this ligand. We wondered whether the shift towards more hyperpolarized voltages described in the HCN3 channel in response to the presence of the cyclic nucleotide could be the result of some structural determinant in the VSD. Thus, we calculated the intrinsic flexibility (1/mBf) for each of the TM segments that integrate the VSD, which have around 141 to 143 amino acid residues for the isoforms described in human cells (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2\u003c/strong\u003e shows the intrinsic flexibilities calculated for each of the TM segments (S1-S4) that integrate the voltage sensor module in five of the best-characterized HCN channels studied to date. It is noteworthy that of the four isoforms present in human cells, the three that respond positively to cAMP by shifting the activation curve toward less polarized voltages (i.e., HCN1, 2, and 4) are relatively more flexible than the HCN3 isoform, whose response to this nucleotide is opposite and therefore not modulated by intracellular cAMP (Stieber et al. 2005). Likewise, we also note that the estimated flexibility index for the voltage sensor present in the spHCN channel is even lower, making it more compositionally rigid than the HCN3 channel. This also correlates with an apparent insensitivity to the modulatory effect of cAMP in this protein (Shin et al. 2004). It is also important to note that the divergence time between echinoderms and mammals is more than 210 million years (dos Reis et al. 2015).\u003c/p\u003e\n\u003cp\u003eWe also analyzed in detail each of the VSD sequences in the four human HCN1-4 isoforms, finding that the most divergent of these is precisely the HCN3 isoform (\u003cstrong\u003eFig. 2\u003c/strong\u003e). Then we tried to find the subregion within the VSD where this evolutionary divergence is most noticeable, since in general these sequences are very similar in terms of amino acid residue identity (\u003cstrong\u003eSuppl.\u003c/strong\u003e \u003cstrong\u003eTable 1\u003c/strong\u003e) and conservative substitutions (\u003cem\u003edata not shown\u003c/em\u003e). Thus, we found a small fragment of 15 residues very close to the only intracellular linker of the VSD (S2-S3L). This fragment extends from position D202 to N216 (HCN1); D271 to K285 (HCN2); E153 to R167 (HCN3) and D322 to K336 (HCN4) and is decisive for estimating the flexibility profile of the entire VSD. \u003cstrong\u003eFig. 2a\u003c/strong\u003e shows this part of the sequence in black, and it is clear that only in the case of the HCN3 channel, the flexibility profile for this region is the lowest of the four proteins, which determines that the flexibility of the entire VSD in this protein is the lowest one (\u003cstrong\u003eFig. 2b\u003c/strong\u003e). In this analysis, we also included a comparison with four other channels of interest: spHCN, CNGA1, hERG1, and Kv7.2. It should be noted here that spHCN does not show a particularly rigid analogous fragment, but it does have a less flexible overall VSD profile than the human orthologous channels, while in the case of Kv7.2, the VSD flexibility profile was the lowest of all the analyzed sequences. On the other hand, as expected, the flexibility profile of the VSD-like belonging to the CNGA1 channel is the highest of all, consistent with previous reports (Romero-Romero et al. 2019), while in the case of the human ether-a-go-go related K\u003cstrong\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e channel 1 (hERG1), its profile was slightly higher than that found for HCN3. However, as mentioned previously, this channel includes an intrinsic ligand in its cytoplasmic structure, which prevents it from the direct binding of cAMP (Codding et al. 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eChanges in the activation midpoints depending on cAMP binding and intrinsic flexibility of the voltage sensor\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eChannel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVSD Num. of residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePositions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e1/mBf\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eV\u003csub\u003e1/2\u003c/sub\u003e shift\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mV)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eS139-H279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e+6.0 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eWainger et al. (2001)\u003c/p\u003e\n \u003cp\u003eStieber et al. (2005)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eS208-H348\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e+18.7 \u0026plusmn; 2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eWainger et al. (2001)\u003c/p\u003e\n \u003cp\u003eMistrik et al. (2005)\u003c/p\u003e\n \u003cp\u003eStieber et al. (2005)\u003c/p\u003e\n \u003cp\u003ePeters et al. (2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eS90-H232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026minus;3.95 \u0026plusmn; 2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eMistrik et al. (2005)\u003c/p\u003e\n \u003cp\u003eStieber et al. (2005)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eS259-H399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e+15.8 \u0026plusmn; 2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eStieber et al. (2005)\u003c/p\u003e\n \u003cp\u003eSchweizer et al. (2010)\u003c/p\u003e\n \u003cp\u003ePeters et al. (2024)\u003c/p\u003e\n \u003cp\u003eWu et al. (2025)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNvHCN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eS141-M282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.702\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eBaker et al. (2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNvHCN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eS218-V358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+6.1 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eBaker et al. (2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003espHCN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eS212-F359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026minus;3.1 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eShin et al. (2004)\u003c/p\u003e\n \u003cp\u003eFlynn et al. (2007)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDmHCN (Ih)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eR748-Y891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.697\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eGisselmann et al. (2005a)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmHCN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eF89-Y228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.691\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+18.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eGisselmann et al. (2003)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePaHCN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eS94-Y235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eGisselmann et al. (2005b)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ezHCNL1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eP44-E202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.702\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eWobig et al. (2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ezHCNL2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eP56-V212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eWobig et al. (2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMVP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eR7-K119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eSesti et al. (2003)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCNGA1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eS31-T160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eXue et al. (2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBased on these observations, we then conducted a compositional analysis for three specific ion channels: (1) the CNGA1 channel, whose activation depends exclusively on the binding of cyclic nucleotides and which has a highly flexible S4-S5 linker as a requirement for transitioning to the open state (Mazzolini et al. 2018); (2) the hERG1, which has a conserved stretch of amino acids in the cyclic nucleotide-binding homology (CNBH) domain that simulates the presence of a cyclic nucleotide and whose presence makes these channels insensitive to the modulatory effects of such ligands (Brelidze et al. 2009); and finally (3) the Kv7.2 channel, whose activation is independent of cAMP, although a certain regulatory effect, still poorly understood, mediated by cAMP has also been reported \u0026nbsp;(van der Horst et al. 2020) but which clearly has a compositional profile rich in rigid amino acids, as we recently reported (Balleza et al. 2019). The results of this part of the analysis clearly suggest that flexible voltage sensors are highly sensitive to activation by cyclic nucleotides or are more prone to their modulatory effects, while those with more rigid profiles are typically insensitive to the effects of this class of intracellular ligands.\u003c/p\u003e\n\u003cp\u003eTherefore, to demonstrate the possible correlation between the modulating effect of cAMP on HCN and HCN-like channels throughout the evolutionary scale, we compared data reported in the literature corresponding to the shifts in the activation curves for each channel (DV\u003cstrong\u003e\u003csub\u003e1/2\u003c/sub\u003e\u003c/strong\u003e), as a function of the intrinsic flexibility of each corresponding VSD. With this rationale, we found that these displacements clearly fall into two categories: HCN channels present in protostome organisms (Arthropoda) and those encoded in the genome of deuterostome organisms, including mammals, fish, echinoderms, and cnidarians. The activation midpoint (V\u003cstrong\u003e\u003csub\u003e1/2\u003c/sub\u003e\u003c/strong\u003e) for each of these channels is a key functional parameter which measure the voltage at which each channel reaches 50% of its total activation. \u003cstrong\u003eFig. 3\u003c/strong\u003e describes those data, and it should be noted that, in an attempt to reveal possible evolutionary trends, we included the archaeobacterial MVP channel in this analysis, which, to our surprise, has a highly rigid voltage sensor, in fact the most rigid of all the channels studied here. This correlates very well with the habitat of these thermophilic microorganisms, echoing some theories of protein thermoadaptation that postulate a preference for rigid structures in environments where the temperature is significantly high (Radestock and Gohlke 2011; Lecocq et al. 2021).\u003c/p\u003e\n\u003cp\u003eOverall, these results indicate that the HCN channels described in arthropods are significantly more sensitive to the modulatory effects of cAMP, while those described in cnidarians, echinoderms, and chordates (Deuterostomata) are significantly less likely to exhibit the modulatory effect of this ligand in terms of the shift of the activation curves. However, as anticipated, there is a clear relationship between compositionally flexible voltage sensors and greater sensitivity to cAMP, both in protostome and deuterostome channels. Besides, it can also be noted that, at least in the case of deuterostome animals, voltage sensors are comparatively more rigid in animals that diverged early in the evolution of the tree of life (Cnidaria, Echinodermata, Actinopterygii), compared to those described in mammals (HCN1-4). Even more noteworthy is that among the isoforms described in mammals, the VSD present in the HCN3 channel shows the lowest flexibility profile, which is consistent with its insensitivity to cAMP, as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe intrinsic flexibility of extramembrane linkers is key in the conformational freedom of the voltage sensor in HCN channels.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain further insight into the structural changes related to the variation in cAMP sensitivity, we evaluated the tertiary structure in each voltage sensor in terms of their relative intrinsic flexibility. To do this, we initiated a search in the Protein Data Bank, reviewing all available structural information corresponding to the four human HCN isoforms (HCN1-4), finding only three partially complete structures for HCN1 (5u6o), HCN3 (8inz), and HCN4 (6gyn). In this part of the analysis, we chose structures in the absence of the ligand (cAMP) precisely to focus on the inherent flexibility of each amino acid sequence in those proteins. \u003cstrong\u003eFig. 4\u003c/strong\u003e depicts a structural analysis for these three HCN channels using a scan of the B factor in each tetramer as a comparative criterion. Since the B factor is a measure for quantifying atomic vibrational movements (Sun et al. 2019), our analysis clearly indicates that HCN3 shows reduced vibrational variations within the VSD, i.e. it exhibits significant rigidity in relation to HCN4 and HCN1. Thus, the VSDs of these proteins can be ordered according to their increasing flexibility as follows: HCN3\u0026gt;HCN4\u0026gt;HCN1. These results are consistent with both the evolutionary distances in the cladogram shown above (\u003cstrong\u003eFig. 1\u003c/strong\u003e) and the cAMP sensitivity previously mentioned (\u003cstrong\u003eFig. 3\u003c/strong\u003e). It is tempting to suggest that the low sensitivity of the HCN1 channel to cAMP could be related to greater rigidity of the C-linker and part of the CNBD in this protein, even though the flexibility of the VSD itself is comparable to that shown by HCN4 (compare structural flexibility profile in \u003cstrong\u003eFig. 4\u003c/strong\u003e with cAMP sensitivity data included in \u003cstrong\u003eFig. 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThis analysis also suggests that the HCN domain (HCND), which occupies a strategic position in the architecture of HCN channels, forming a continuous mechanical interface between the VSD and the C-linker/CNBD (Porro et al. 2019, 2020) (\u003cstrong\u003eFig. 4\u003c/strong\u003e), is also key in coupling the allosteric changes of the CNBD and transmitting them to the voltage sensor in response to cAMP binding. Thus, in rigid voltage sensors such as those present in HCN3, the associated rigidity of the HCND in this channel prevents it from being positively modulated with cAMP, while in channels with more flexible voltage sensors, with less rigid HCNDs such as those present in HCN1 and HCN4, the greater degree of flexibility of the HCND allows these proteins to more effectively transmit the conformational changes derived from the binding to this ligand.\u003c/p\u003e\n\u003cp\u003eHowever, the lack of a complete structure for the HCN2 channel made it impossible to perform a comprehensive comparison of the four known isoforms in the same phylogenetic clade. In addition, since the structures available in the PDB are incomplete, particularly with regard to the S2-S3L and S3-S4L linkers, we decide to obtain high-quality structural models using templates with high compositional similarity for each of the four proteins. Thus, this technical limitation, typical of cryogenic electron microscopy (Lučič et al. 2013), as well as the probable difficulty of obtaining a complete structure for the HCN2 channel due to its high intrinsic flexibility (\u003cstrong\u003eFig. 2b\u003c/strong\u003e), we decided to generate homology models based on specific templates with high similarity using the SWISS-MODEL server (Waterhouse et al. 2018). In this part of the analysis, we also included a refined model of the spHCN channel from the sea urchin (\u003cstrong\u003eFig. 5\u003c/strong\u003e), a widely studied channel belonging to a phylogenetic clade significantly more distant from their orthologs present in mammals. With these models, we identify small structural differences that could explain the contribution of each linker in terms of the intrinsic flexibility of each voltage sensor.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThus, once we generated the five tetramerized models for each channel and these were validated using Ramachandran conformer analysis, we detected some residues of interest in dynamic terms, as they are located in unfavorable areas of these plots, specifically in the S2-S3L or the CNBD (\u003cstrong\u003eSuppl. Fig. 2\u003c/strong\u003e). Therefore, we evaluated the contribution of missing sequences in the available cryo-EM structures, with special attention to the S2-S3L and S3-S4L linkers, whose overall contribution to the flexibility of the voltage sensor is decisive (\u003cstrong\u003eFig. 2a\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eRelative flexibilities (1/mBf) for the three linkers within the VSD in HCN channels\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS1-S2L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS2-S3L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS3-S4L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.565\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eF166-T173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2.812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eR195-D209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eG242-T250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e3.124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eF235-A242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2.256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eR264-P279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eI312-A320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e4.714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eF117-P124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.914\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eR146-A163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eE191-V210\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e3.777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eF286-T292\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2.141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eT316-D329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eD364-A371\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003espHCN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eF239-T247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2.436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eR269-Q287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eG315-R326\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eWe thus discovered significant structural differences in terms of the intrinsic flexibilities of the three linkers that could explain why the HCN3 channel and spHCN are practically insensitive to the modulatory effects of cAMP. These results are shown in \u003cstrong\u003eTable 2\u003c/strong\u003e and clearly indicate that the HCN3 isoform has the two most rigid linkers in this group of proteins; these are the S2-S3L (dark blue) and S3-S4L (green) linkers (\u003cstrong\u003eFig. 5B\u003c/strong\u003e). Therefore, HCN3 is the only one of the four mammalian isoforms capable of organizing itself into a partial 3\u003cstrong\u003e\u003csub\u003e10\u003c/sub\u003e\u003c/strong\u003e helix outside the cell. This substructure is promoted by a Pro residue at position 194, which is only present in this isoform. In turn, inside the cell, the S2-S3L linker of HCN3 is the only one that consistently folds into an antiparallel beta sheet. This substructure was never obtained for any of the other homology models, including spHCN, which is intrinsically more rigid than HCN3. This substructure was never obtained for any of the other homology models, including spHCN, which is intrinsically more rigid than HCN3. In contrast, in the case of HCN1, 2, and 4, this linker is configured in a more disordered manner, with varying degrees of flexibility between them.\u003c/p\u003e\n\u003cp\u003eWith regard to the structure of the spHCN channel, to our surprise, the S2-S3L linker is not organized in beta configurations, but is much more flexible, with the exception of the corresponding linker in HCN1 (\u003cstrong\u003eTable 2\u003c/strong\u003e). However, we found that this channel has the most rigid S1-S2L linker (cyan) (\u003cstrong\u003eFig. 5B\u003c/strong\u003e, arrowheads) of these five channels. This rigidity manifests in the form of a more compact and less extended loop compared to those present in mammalian orthologs, as it includes two key residues in the stabilization of helical substructures: K243 and M245. This finding is consistent with the fact that both residues are particularly prone to stabilizing a-helical structures (Blaber et al. 1993; Monera et al. 1995), which stiffens this part of the protein.\u003c/p\u003e\n\u003cp\u003eRegarding the structure of the spHCN channel, to our surprise, the S2-S3L linker is not organized in\u0026nbsp;b-configurations, but is much more flexible (mBf = 2.436), with the exception of the corresponding linker in HCN1 (mBf = 2.812) (\u003cstrong\u003eTable 2\u003c/strong\u003e). However, we found that this channel has the most rigid S1-S2L linker (mBf = 2.196) of these five channels (\u003cstrong\u003eFig. 5B\u003c/strong\u003e, asterisk). This rigidity manifests in the form of a more compact two-turn w-loop compared to those present in mammalian orthologs, as it includes two key residues in the stabilization of helical substructures: D244 and S246. In the first case, D244 forms a double salt bridge with K335 (S4), while position S246 forms a double H-bond with K243, which in turn forms another H-bond with residue F240. This substructure gives this w-loop great rigidity, which is consistent with previous reports establishing that some omega loops can form turns that show a preference for specific amino acids, such as aspartate, serine, and asparagine (Papaleo et al. 2016), which is present at position 242. Thus, S1-S2L acquires a w-loop with a very ordered configuration, while the RHFLE motif in the extracellular S3-S4L forms a highly rigid and stable a-helical lid. Taken together, these structural traits are consistent with the high degree of rigidity of the entire VSD in the spHCN channel and its low sensitivity to the modulatory effect of cAMP. This is also consistent with considerable experimental evidence indicating that both w-loops and the presence of helical substructures in extracellular linkers are typically located in allosterically regulated regions of proteins (Papaleo et al. 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCommon hydrogen bond interactions in the voltage sensor domain of HCN channels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce we had identified the main differences in tertiary structure that could explain the different sensitivities to voltage and the modulating effects of cAMP in the five HCN channels studied, we decided to compare the number of H-bonds that stabilize each VSD, based on the relative contribution of the S2-S3L and S3-S4L linkers, which are the most rigid in each structure and, in turn, are most closely associated with the propagation machinery that couples the allosteric effects of cAMP when binding to the CNBD of each channel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eNumber of H-bonds and relevant van der Waals interactions in selected HCN channel VSDs\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHCND-VSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS2-S3L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelevant interactions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS3-S4L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelevant\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003einteractions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eM94-Y289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e(5.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eR126-E205\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(HCND)\u003c/p\u003e\n \u003cp\u003eT198-I206\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eN200-S203\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eI135-G197\u003c/p\u003e\n \u003cp\u003e(HCND)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e(3.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eD244-E246\u003c/p\u003e\n \u003cp\u003eS245-Y248\u003c/p\u003e\n \u003cp\u003eV247-R252\u003c/p\u003e\n \u003cp\u003eK249-R252\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eM163-Y358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003cp\u003e(3.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eR195-I275\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(HCND)\u003c/p\u003e\n \u003cp\u003eI267-I276\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eN272-E274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e(1.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eE309-D313\u003c/p\u003e\n \u003cp\u003eD313-E315\u003c/p\u003e\n \u003cp\u003eE315-R318\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003e3\u003csub\u003e10\u003c/sub\u003e\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eT319-R321\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eG50-Y242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e(5.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eV151-G154\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eV151-A155\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eR162-T166\u003c/p\u003e\n \u003cp\u003eA163-T166\u003c/p\u003e\n \u003cp\u003eA163-R167\u003c/p\u003e\n \u003cp\u003eR146-W84\u003c/p\u003e\n \u003cp\u003e(HCND)\u003c/p\u003e\n \u003cp\u003eR146-I86\u003c/p\u003e\n \u003cp\u003e(HCND)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e(6.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eE193-L196\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003e3\u003csub\u003e10\u003c/sub\u003e\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eD197-E199\u003c/p\u003e\n \u003cp\u003eD197-V200\u003c/p\u003e\n \u003cp\u003eY201-R205\u003c/p\u003e\n \u003cp\u003eA204-R208\u003c/p\u003e\n \u003cp\u003eV210-T213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehHCN4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eM214-Y409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e(3.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eR246-I326\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(HCND)\u003c/p\u003e\n \u003cp\u003eI318-I327\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eN323-E325\u003c/p\u003e\n \u003cp\u003eE325-R332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e(2.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eE360-R372\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)\u003c/p\u003e\n \u003cp\u003eE366-K369\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003e3\u003csub\u003e10\u003c/sub\u003e\u003c/strong\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003espHCN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eS212-F359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003cp\u003e(10.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eK195-E279\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(HCND)\u003c/p\u003e\n \u003cp\u003eE196-Q280\u003c/p\u003e\n \u003cp\u003e(HCND)\u003c/p\u003e\n \u003cp\u003eR199-Q280\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(HCND)\u003c/p\u003e\n \u003cp\u003eV205-R269\u003c/p\u003e\n \u003cp\u003eL206-R269\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)\u003c/p\u003e\n \u003cp\u003eR215-T270\u003c/p\u003e\n \u003cp\u003eI272-V281\u003c/p\u003e\n \u003cp\u003e(\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)(\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eN274-K276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003cp\u003e(7.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eG315-Q317\u003c/p\u003e\n \u003cp\u003e(\u003cbr\u003e\u003cbr\u003e\u003cimg width=\"11\" height=\"19\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABAAAAAcBAMAAACaHyIpAAAAAXNSR0IArs4c6QAAACpQTFRFAAAAAAAAAAA6OpC2OpDbZrbbZrb/kDo6kNv/tmYAtmY625A625Bm///b97lStQAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAVklEQVQYV2NgoDrYAjSRdxmQWOjAwHBTDcjgEmHgnZgAsmihw00xsIVcwhMDIDYvFIW6YKIIhMEpPBGoEQgmOnCC5bhEgdpBqhcCCbD+oyArKqD6yKYAy6IOoPs7qsEAAAAASUVORK5CYII=\" alt=\"image\"\u003e2)\u003c/p\u003e\n \u003cp\u003eQ317-F321\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eN318-L322\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eR319-E323\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eH320-V324\u003c/p\u003e\n \u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eHence, the most rigid VSDs that are insensitive to cAMP (spHCN, HCN3) have a greater number of H-bonds in the S2-S3L and S3-S4L loops compared to flexible VSDs that are highly sensitive to this cyclic nucleotide. Indeed, the effect of rigidity in these linkers could also explain the relatively scarce modulatory effect exhibited by cAMP in terms of the displacement of the mean activation voltage for the HCN1 channel (\u003cstrong\u003eTable 3\u003c/strong\u003e). Furthermore, the presence of\u0026nbsp;a-helical or extracellular 3\u003cstrong\u003e\u003csub\u003e10\u003c/sub\u003e\u003c/strong\u003e substructures in the five VSDs studied, except for HCN1, could in turn be related to their low sensitivity to cAMP. On the other hand, although the extracellular 3\u003cstrong\u003e\u003csub\u003e10\u003c/sub\u003e\u003c/strong\u003e element present in the S3-S4L of HCN3 was modeled as a helix, as in the cAMP-sensitive HCN2 and HCN4 channels, this region of just 3 residues (one turn) which exhibits a [PRL] motif at positions 194-196 in HCN3 but modeled only for the D subunit of the tetramer. This could indicate that such region is particularly dynamic for this protein. In that context, the presence of the b-turn at the S2-S3L inside the cell and in close contact with the C-linker should considerably restrict its sensitivity to cAMP (\u003cstrong\u003eFig. 6\u003c/strong\u003e). \u003cstrong\u003eTable 3\u003c/strong\u003e shows the H-bonds that support this hypothesis, i.e., the relative rigidity of the linkers, in terms of the number of H-bonds in those regions at the VSD, determines the modulatory effects of cAMP in HCN channels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular dynamics simulations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the position of the intracellular S2-S3L linker in each of the five HCN structures studied, and with the aim of evaluating their conformational freedom, we performed molecular dynamics (MD) CABS Monte Carlo simulations (Nithin et al. 2024) using homology models generated with SWISS-MODEL (Waterhouse et al. 2018) (\u003cem\u003esee\u003c/em\u003e Methods). In each case, cAMP-free (apo) structures were used, and since a structure for the HCN2 channel has not yet been obtained, we decided to use the HCN4 structure as a proper template. This is because in terms of HCN4 responsiveness to the modulatory effects of cAMP, it is more similar to the HCN2 channel (Stieber et al. 2005), although phylogenetically the HCN1 channel is closer to HCN2 (\u003cstrong\u003eFig. 1\u003c/strong\u003e). Besides, these five proteins were selected because they have been the most studied using electrophysiological and patch-clamp fluorometry experiments. This approach facilitates a better comparison of the compositional/structural analysis we performed, contrasting it with the reported information regarding the voltage-dependent activation and the modulating effects of cAMP in each of these channels.\u003c/p\u003e\n\u003cp\u003eAs shown in \u003cstrong\u003eFig. 7a\u003c/strong\u003e, we calculated the fluctuation profiles of atomic positions for each residue in the HCND-VSD domain present in the five proteins studied using the CABS-flex computational tool (Jamroz et al. 2014; Kuriata et al. 2018). Furthermore, using Normal Mode Analysis with DynaMut (Rodrigues et al. 2018) and the Anisotropic Network Model (ANM) FF, we were able to estimate the magnitude of the deformations/fluctuations by representing substructures in the form of tubes with low (blue), moderate (white), and high (red) mobility, including regions where each of them experiences deformations from thin to thick (\u003cstrong\u003eFig. 7b\u003c/strong\u003e). In this analysis, we used the SS1 mode for the estimation of the root mean square fluctuation (rmsf) in CABS-flex, whose algorithm establishes steric restrictions between residues, at least one of which belongs to a secondary structure element; such is the case of the \u0026ldquo;elbow\u0026rdquo; formed by the C-linker of subunit \u003cem\u003eS\u003c/em\u003e with the disordered S2-S3L linker present in subunit \u003cem\u003eS + 2\u003c/em\u003e. Our results indicate that the distance between these two structural elements is maximum for the HCN3 channel (~10 \u0026Aring;), which could hinder the propagation of the allosteric wave and make it insensitive to the modulatory effect of cAMP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the case of the HCN2 channel, which is highly sensitive to the modulatory effect of cAMP, the distance is reduced to 6.5 \u0026Aring;. Besides, we believe that the presence of the antiparallel beta element present in HCN3 significantly restricts the conformational freedom of the disordered part of the\u0026nbsp;b-turn that runs from residue V151 to G154 in that channel. Hence, the absence of the\u0026nbsp;b-substructure in HCN2, on the other hand, manifests in the form of a high conformational freedom for the S2-S3L linker, which could be consistent with the poor packing of the D271 residue in a \u0026lsquo;forbidden\u0026rsquo; region of the Ramachandran map of HCN2 or the Q280 residue in the same linker of spHCN, which, on the contrary, is found in a less prohibited area of the (ϕ-\u0026psi;) map) (\u003cstrong\u003eSuppl. Fig. 2\u003c/strong\u003e), this in turn would be related to high dynamic flexibility for that region (Momen 2017). Therefore, a significant deformability of the S2-S3L could facilitate the propagation of allosteric perturbations triggered from the cAMP binding site in the distal CNBD. The fact that changes in the structural rigidity of the HCN2 channel propagate from the cAMP binding sites via the C-linker has been extensively documented elsewhere (Pfleger et al. 2021). On the other hand, although we found an even shorter distance, i.e., ~5 \u0026Aring; between the S2-S3L and the elbow of the C-linker in the spHCN channel, the high intrinsic rigidity of the VSD of this protein (\u003cstrong\u003eFig. 3\u003c/strong\u003e) must be severely limiting the conformational freedom of the entire transmembrane region, which is consistent with the low cAMP sensitivity reported for this protein.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the swapped architecture of the S2-S3L with respect to the C-linker between subunits, which involves possible specific interactions between the \u003cem\u003eS\u003c/em\u003e and \u003cem\u003eS + 2\u003c/em\u003e subunits, must be crucial to understanding the cooperativity established between the cAMP binding sites in the CNBD and the VSD, which in turn would allosterically modulate the opening of the HCN channel pore. A relevant interaction could be between residues T273 (\u003cem\u003eS\u003c/em\u003e)/K491 (\u003cem\u003eS + 2\u003c/em\u003e) in HCN2 and T324 (\u003cem\u003eS\u003c/em\u003e)/K542 (\u003cem\u003eS + 2\u003c/em\u003e) in HCN4, which in the studied models are about 6.5 \u0026Aring; apart in both cases, while in HCN3 the pair of residues involved correspond to positions E153 (\u003cem\u003eS\u003c/em\u003e) and K375 (\u003cem\u003eS + 2\u003c/em\u003e), which are almost twice as far apart, i.e., 12.5 \u0026Aring;. In the spHCN channel, residues S277 (\u003cem\u003eS\u003c/em\u003e) and K492 (\u003cem\u003eS + 2\u003c/em\u003e) are closer, i.e., 9.3 \u0026Aring;, and given the high deformability of the intracellular elbow in spHCN (\u003cstrong\u003eFig. 7b\u003c/strong\u003e), it is conceivable that this motif could contact the S2-S3L even though it is considerably rigid. Furthermore, the substitution of a Thr residue for a Ser residue at position 277 of this protein could also be decisive in explaining the shift in the activation V\u003cstrong\u003e\u003csub\u003e1/2\u003c/sub\u003e\u003c/strong\u003e in presence of cAMP, as this is a less polarizable residue than Thr (Swart et al. 2004).\u003c/p\u003e\n\u003cp\u003eWith the ultimate goal of confirming the relative flexibilities in the HCND-VSD domains of these four proteins, we generated Dynamical Cross-Correlation Maps (DCCM) using the ANM FF in DynaMut. This FF is encoded to optimally predict residue fluctuations in regular secondary structural elements. To do this, a uniform force constant is adopted for all springs in coarse-grained simulations; nodes are identified by the positions of Ca\u0026nbsp;atoms, and the total potential of the system is a sum of harmonic potentials (Atilgan et al. 2001). With this strategy, the DCCMs obtained reveal the dynamic nature of each domain in proteins that are close but not identical. Thus, the role of flexibility/rigidity in the configuration of conformational landscapes reveals that the HCND-VSD domain of the HCN3 channel does indeed exhibit the least flexible conformational landscape of mammalian channels and that the same domain in the spHCN channel also shows fewer areas of flexibility (\u003cstrong\u003eSuppl. Fig. 3\u003c/strong\u003e). Taken together, these results are consistent with our general hypothesis and thus, HCN channels with flexible sensors (HCN2, HCN4) are more likely to respond to the allosteric propagation resulting from cAMP binding, while those with less flexible landscapes (HCN3, spHCN) respond with difficulty to this type of conformational stimulus. Finally, we performed the same analysis for the CNGA1 channel, a cyclic nucleotide-gated channel (\u003cstrong\u003eSuppl. Fig. 4\u003c/strong\u003e). Consistent with our hypothesis, this channel exhibited the highest flexibility profile of those studied here (\u003cstrong\u003eFig. 2b\u003c/strong\u003e), with a high deformability index in the S2-S3L/C-linker and a very high flexibility DCCM. This is consistent with a high dependence on cAMP for its activation, typical of channels in this subfamily, channels largely insensitive to membrane potential (James and Zagotta 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe persistence of HCN channels on the evolutionary scale\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we have made an effort to identify the structural determinants that could explain the distinctive modulatory effects exhibited by cAMP among the four isoforms of the HCN channel in mammals. Our results clearly suggest that the degree of conformational freedom exhibited by the S2-S3L linker and its possible interaction with the \u0026lsquo;elbow\u0026rsquo; region in the C-linker could be decisive in the propagation of conformational changes triggered by cAMP binding, which allosterically couple the binding of this intracellular ligand to the transmembrane voltage sensor domain. When performing a compositional analysis of the VSD in the four mammalian HCN variants, we found a remarkable correlation between intrinsically very flexible voltage sensors (HCN2) and high sensitivity to cAMP in terms of the V\u003cstrong\u003e\u003csub\u003e1/2\u003c/sub\u003e\u003c/strong\u003e of activation. Thus, the intrinsically most rigid channel of the four isoforms (HCN3) is a channel that is practically insensitive to cAMP. Our study also reveals that the HCN3 channel is phylogenetically closest to HCN channels present in metazoans that diverged very early in evolution (Echinodermata, Cnidaria, Arthropoda). This phylogenetic proximity is remarkable and could indicate that the HCN3 clade represents the product of a very early duplication event in chordates (Jackson et al. 2007). Interestingly, the flexibility profiles for HCN and HCN-like channels in basal deuterostomes (Cnidaria, Echinodermata, Actinopterygii) are very low, as is the profile of the HCN3 channel, which also correlates with very low sensitivity to cAMP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, those HCN channels described in arthropods have shown very high sensitivity to the modulatory effects of cAMP (Gisselmann et al. 2003, 2005a, 2005b) (\u003cstrong\u003eFig. 3\u003c/strong\u003e). This could reflect the very early divergence of protostomes during the early stages of animal evolution (Wheat and Wahlberg 2013). On the other hand, given that HCN channels have been found in the olfactory receptor neurons of \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eD. melanogaster\u0026nbsp;\u003c/em\u003e(Marx et al., 1999), as well as being present in \u003cem\u003eHeliothis virescens\u003c/em\u003e (Krieger et al. 1999) and \u003cem\u003eP. argus\u003c/em\u003e (Gisselmann et al. 2005b), it has been postulated that the mechanisms of chemosensory transduction in mammals and arthropods may be profoundly different (Sato and Touhara 2008; Kaupp 2010). Thus, metabotropic signaling, dependent on G protein-coupled receptors, is typical in mammalian olfactory neurons, providing a wide range of positive and negative regulations in these animals in response to diverse environmental odors. In contrast, ionotropic signaling in insects is an adaptation that improves the speed of chemosensation in animals that fly rapidly and track sudden changes in the concentration and quality of multiple odors (Kaupp 2010). This is consistent with the fact that mice are able to discriminate between different odors in less than 250 ms (Abraham et al., 2004), but honeybees are able to distinguish between them in as little as 200 ms (Wright et al., 2009).\u003c/p\u003e\n\u003cp\u003eFrom a physiological perspective, one final consideration is worth noting. Given these profound differences between HCN channels in protostome and deuterostome animals, is it possible to distinguish any attributes in the activation kinetics of these proteins that would be consistent with the metabotropic or ionotropic pathways of each type of organism? The kinetics of the so-called Ih current varies significantly due to evolutionary diversification in arthropods (Jackson et al. 2007). In that context, Ih activation kinetics in insects are inherently fast, closely resembling the rapid mammalian HCN1 isoform (Gisselmann et al. 2003). In crustaceans, the significant acceleration of the activation time constant that has been reported for the Ih current in the presence of cyclic nucleotides, closely resembles the rapid kinetics typical of the mammalian HCN1 channel (Gisselmann et al. 2005b). Besides, unlike what happens in mammals, in arthropods such as \u003cem\u003eD. melanogaster\u003c/em\u003e, crab (\u003cem\u003eCarcinus maenas\u003c/em\u003e), and cockroach (\u003cem\u003ePeriplaneta americana\u003c/em\u003e) typically only one HCN channel gene is expressed that retains the characteristics of the ancestral fast channel (Jackson et al. 2007). This would be consistent with the typical fast activation kinetics and strong voltage dependence exhibited by HCN channels in arthropods (Gisselmann et al. 2003, 2005a, 2005b) compared to their mammalian orthologs, which exhibit a wide range of voltage sensitivities on a scale from hundreds of milliseconds to several tens of seconds (Wahl-Schott and Biel 2009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the persistence of CNBD throughout the evolutionary scale remains a mystery of life (Berman et al. 2005). Its ancestry is enigmatic in the hyperthermophilic order Aquificales (Garc\u0026iacute;a-Morales et al. 2019). However, to the best of our knowledge, it has not yet been possible to express this channel described in the bacterium \u003cem\u003eAquifex aeolicus\u003c/em\u003e (AqK) and recording the possible voltage dependence or sensitivity to the presence of cAMP in this channel. The archaeobacterial MVP channel, on the other hand, although it exemplifies a minimalist model of hyperpolarization-activated channels, does not have a CNBD, and consequently cAMP has no modulatory effect on its activation behavior. In addition, it is highly selective for potassium (\u003cem\u003eP\u003c/em\u003e\u003cstrong\u003e\u003csub\u003eNa\u003c/sub\u003e\u003c/strong\u003e/\u003cem\u003eP\u003c/em\u003e\u003cstrong\u003e\u003csub\u003eK\u003c/sub\u003e\u003c/strong\u003e \u0026asymp; 0.01), while HCN channels are poorly selective for monovalent cations (\u003cem\u003eP\u003c/em\u003e\u003cstrong\u003e\u003csub\u003eNa\u003c/sub\u003e\u003c/strong\u003e/\u003cem\u003eP\u003c/em\u003e\u003cstrong\u003e\u003csub\u003eK\u003c/sub\u003e\u003c/strong\u003e \u0026asymp; 0.2) and have a conductance close to ~ 1-2 pS (HCN2) compared to 37 pS for MVP (Sesti et al. 2003; Randich et al. 2014). These profound differences strongly suggest that very early in the evolution of life, a lineage of archaeobacterial cells may have contributed the reverse electromechanical coupling module, typical of HCN channels, but lacking sensitivity to cyclic nucleotides. On the other hand, an ancestral bacterial lineage provides the CNBD module that recognizes cAMP and was able to couple it to the transmembrane domain through a gene fusion event (Pasek et al. 2006; Poole and Penny 2007; Marsh and Teichmann 2010). Thus, the first eukaryotic cell appeared, already possessing both attributes in a single ancestral HCN protein (\u003cstrong\u003eFig. 8\u003c/strong\u003e). This protein diverges into two lineages with very different activation kinetics for protostomes and deuterostomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eA last conclusion\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough HCN channels are principally operated by voltage, the modulatory effect of cAMP is enhanced by the degree of deformability acquired by specific structural elements in these proteins. Thus, we have described in some detail a probable intersubunit interaction that connects the S2-S3L linker to the elbow of the C-linker with the subunit in front. This interaction depends on the intrinsic flexibility of the former to facilitate the allosteric transmission that couples the cAMP-binding event with the displacement of the C-linker and, in turn, with a probable interaction through S2-S3L. This linker in turn directly contacts the TM region that detects changes in membrane voltage. Furthermore, there appears to be an evolutionary trend in which these channels have become more flexible in key regions of their architecture, and this goes hand in hand with a greater ability to become sensitized when interacting with cAMP through an intracellular module that has persisted for millions of years of biological evolution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at https://link.springer.com/journal\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. E. Rudiño of the Institute of Biotechnology (UNAM) for his valuable contributions and comments on this work, as well as his helpful discussions. KGAV received a grant (CVU: 693618) from the Secretaría de Ciencia, Humanidades, Tecnología e Innovación (Secihti).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKGAV contributed to the acquisition and compilation of protein sequences and the analysis of structural data; DB conceived and designed this work, performed all flexibility and molecular dynamics analysis, and wrote and prepared the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded in part by a grant from the National Technological Institute of Mexico: “Scientific Research Projects, Technological Development, and Innovation 2025—Federal Technological Institutes and Centers” to DB (grant reference number 21871.25-P).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe modeling code, analysis, and protein models can be found in the following repository: https://github.com/DBalleza-Flexiprot/Voltage-Sensor-Domain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eAbramson \u003c/strong\u003eJ, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, Bodenstein SW, Evans DA, Hung CC, O\u0026apos;Neill M, Reiman D, Tunyasuvunakool K, Wu Z, Žemgulytė A, Arvaniti E, Beattie C, Bertolli O, Bridgland A, Cherepanov A, Congreve M, Cowen-Rivers AI, Cowie A, Figurnov M, Fuchs FB, Gladman H, Jain R, Khan YA, Low CMR, Perlin K, Potapenko A, Savy P, Singh S, Stecula A, Thillaisundaram A, Tong C, Yakneen S, Zhong ED, Zielinski M, Ž\u0026iacute;dek A, Bapst V, Kohli P, Jaderberg M, Hassabis D, Jumper JM.Accurate structure prediction of biomolecular interactions with AlphaFold 3. 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Neuron. 2021 Apr 21;109(8):1302-1313.e4. doi: 10.1016/j.neuron.2021.02.007\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eYu\u003c/strong\u003e FH, Catterall WA. The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. 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This voltage dependence is finely regulated by the binding of cAMP to an intracellular domain (CNBD). Of the four widely studied isoforms of human HCN channels, the HCN3 channel is practically insensitive to cAMP or is even inhibited by it, but the structural determinants underlying this unexpected behavior are still unclear. Here, we evaluated the possible role of flexibility in very specific regions of the VSD that could be determinants for this behavior. Hence, part of the S2-S3L linker is significantly rigid in HCN3, which correlates with low atomic mobility for this region in proximity to the C-linker subdomain of the opposite subunit. We built structural models using AlphaFold 3 and Swiss-Model and thus reconstructed the disordered regions that connect the transmembrane segments of the VSD and that in some of the structures deposited in the PDB have not been resolved. Besides, in an attempt to reveal the evolutionary trends that this transmembrane domain may have undergone, we conducted a comparative study with phylogenetically distant HCN channels and found an interesting tendency to lose sensitivity to cAMP as VSD flexibility is lost. Our analysis confirms a large body of published experimental findings. Finally, we found that in metazoans, two types of HCN channels clearly diverge: (1) those that are highly sensitive to cAMP with moderate flexibility profiles in protostomes, and (2) those that show less marked sensitivity to this ligand in deuterostomes. We also propose a possible evolutionary scenario for the appearance of cAMP-modulated HCN channels in the last eukaryotic common ancestor (LECA).\u003c/p\u003e","manuscriptTitle":"Structural determinants of voltage sensitivity in hyperpolarization-activated ion channels and their persistence in the evolutionary scale","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 17:06:47","doi":"10.21203/rs.3.rs-8117147/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-29T23:10:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-29T18:05:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T20:32:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175257846941122295064801369925016376458","date":"2025-11-21T15:49:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60487849941304098820010788224390533771","date":"2025-11-19T11:53:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-19T09:21:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-18T09:26:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T09:23:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pflügers Archiv - European Journal of Physiology","date":"2025-11-14T16:56:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a5ce57a8-8606-4701-a9d8-3a6b9f8a4e87","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:02:40+00:00","versionOfRecord":{"articleIdentity":"rs-8117147","link":"https://doi.org/10.1007/s00424-026-03153-4","journal":{"identity":"pflugers-archiv-european-journal-of-physiology","isVorOnly":false,"title":"Pflügers Archiv - European Journal of Physiology"},"publishedOn":"2026-03-07 15:57:49","publishedOnDateReadable":"March 7th, 2026"},"versionCreatedAt":"2025-11-26 17:06:47","video":"","vorDoi":"10.1007/s00424-026-03153-4","vorDoiUrl":"https://doi.org/10.1007/s00424-026-03153-4","workflowStages":[]},"version":"v1","identity":"rs-8117147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8117147","identity":"rs-8117147","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}