FGF12A Regulates Nav1.5 via CaM-regulated and CaM-independent Mechanisms

FGF12A Regulates Nav1.5 via CaM-regulated and CaM-independent Mechanisms | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results FGF12A Regulates Nav1.5 via CaM-regulated and CaM-independent Mechanisms View ORCID Profile Lucy Woodbury , View ORCID Profile Paweorn Angsutararux , View ORCID Profile Martina Marras , Emily Wagner , View ORCID Profile Carlota Abella , Anna Li , Jonathan R. Silva doi: https://doi.org/10.1101/2025.01.02.631125 Lucy Woodbury 1 Department of Biomedical Engineering, McKelvey School of Engineering, Washington University in St. Louis , St. Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lucy Woodbury Paweorn Angsutararux Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paweorn Angsutararux Martina Marras 1 Department of Biomedical Engineering, McKelvey School of Engineering, Washington University in St. Louis , St. Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martina Marras Emily Wagner 1 Department of Biomedical Engineering, McKelvey School of Engineering, Washington University in St. Louis , St. Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carlota Abella 3 Baylor College of Medicine , Houston, TX, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carlota Abella Anna Li 1 Department of Biomedical Engineering, McKelvey School of Engineering, Washington University in St. Louis , St. Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jonathan R. Silva 1 Department of Biomedical Engineering, McKelvey School of Engineering, Washington University in St. Louis , St. Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: jonsilva{at}wustl.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Opening of the cardiac voltage-gated Na+ channel (Nav1.5) is responsible for robust depolarization of the cardiac action potential, while inactivation, which rapidly follows, allows for repolarization. Regulation of both the voltage- and time-dependent kinetics of Nav1.5 inactivation can alter the ability of the heart to initiate and sustain a re-entrant arrhythmia. The C-terminal domain (CTD) of Nav1.5 has been shown to modulate fast inactivation of the channel, and multiple auxiliary proteins bind to the CTD, including calmodulin (CaM) and intracellular fibroblast growth factor 12A (FGF12A). Recently, a non-canonical CaM-binding site was also discovered on the N-terminal of A-splice variants of iFGFs. We performed cut-open Vaseline gap (COVG) voltage-clamp to test whether FGF12A with and without CaM regulates Nav1.5 gating. In WT Nav1.5 channels, FGF12A with and without CaM present had a minimal effect on the voltage dependence of both activation and inactivation. Conversely, when CaM is absent on the Nav1.5 CTD (IQ/AA), a dramatic shift in steady-state inactivation (SSI) occurred, regardless of whether CaM was present on FGF12A. These two distinct mechanisms are operative in Nav1.5 LQT3 mutations where FGF12A requires CaM to shift in the voltage-dependence of inactivation, but not to inhibit the persistent late current. We conclude that there are two distinct mechanisms by which FGF12A modulates the Nav1.5 channel: CaM-regulated alteration of the voltage dependence of inactivation and CaM-independent inhibition of persistent late current. Introduction Voltage-gated sodium (Nav) channels are responsible for the initiation of the action potential in excitable cells including neurons and myocytes. The Nav1.5 sodium channel, encoded by the SCN5A gene, is the most prevalent Nav channel in cardiomyocytes and is responsible for the rapid post-stimulus depolarization of the myocyte ( Catterall et al., 2005 ). Nav1.5 contains four homologous repeats (I-IV) each comprising six transmembrane helices (S1-S6) with the first four defining the voltage-sensing domain (VSD). The S4 helix carries multiple positive charges, which translocate in response to depolarization of the cell membrane to actuate channel gating ( Catterall, 2023 ; Catterall et al., 2005 ; D et al., 2020). Nav1.5 gating has been described in terms of closed (deactivated), open (activated), and inactivated states ( Ahern et al., 2016 ; Mangold et al., 2017 ) where channels transition from closed to open in response to depolarizing changes in membrane potential and then to inactivated as an elevated membrane potential is maintained. From the closed state, strong depolarization of the plasma membrane will activate the first three VSDs (I-III). Notably, VSD-IV is not required for channel-pore opening, however it is required for inactivation ( Capes et al., 2013 ; Chanda and Bezanilla, 2002 ). The activation of the VSD-IV allows a motif in the intracellular III-IV linker, isoleucine-phenylalanine-methionine (IFM), to bind to a hydrophobic pocket that is proximal to the intracellular side of the pore (D et al., 2020; Jiang et al., 2021 ). The IFM motif then allosterically alters the conformation of the S5-S6 helices to close the pore and produce a fast inactivation of the channel, allowing the cardiac action potential to eventually repolarize, returning the membrane to negative potentials ( Li et al., 2021 ). The C-terminal domain (CTD) of Nav1.5 has also been implicated in modulating activation and inactivation kinetics ( Deschênes et al., 2001 ). Recent structures of Nav1.5 channel in its open state have shown the channel CTD interacting with the III-IV linker, modulating fast inactivation ( Biswas et al., 2024 ; Shen et al., 2017 ; Wang et al., 2012 ). Numerous variants within SCN5A are known to predispose patients to cardiac arrhythmia. This pathology can arise when variants alter the peak and late components of the Na + current (I Na ) causing either a gain or loss of function. One of these, Long QT syndrome (LQTS), arises from the prolongation of ventricular repolarization that manifests as a prolonged ventricular myocyte action potential and corrected Q-T interval of an ECG. LQTS can be caused by gain-of-function mutations that typically affect the late or persistent Na + current (I Na-L ). Normally, I Na,L is relatively small compared to the peak I Na (0.1 – 1%). Variants that significantly increase I Na,L bring excess positive charges into the myocyte that prolong the action potential, leading to proarrhythmic triggered activity that includes early and delayed afterdepolarizations ( Han et al., 2018 ). Loss-of-function mutations within SCN5A are usually associated with Brudaga syndrome which has over 300 associated variants ( Mizusawa and Wilde, 2012 ), which can cause slowed conduction and dispersion of repolarization. Multiple subunits interact with Nav1.5 to regulate its function and pharmacology ( Abriel, 2010 ). The β1 ( SCN1B ) subunit alters the voltage dependence of DIII and DIV VSD activation, Nav1.5 gating kinetics, and anti-arrhythmic drug affinity ( Angsutararux et al., 2021 ; Martinez-Moreno et al., 2020 ; Moreno et al., 2019 ; Zhu et al., 2021 , 2017). Calmodulin (CaM) alters trafficking and modulates the voltage sensing kinetics of multiple channels including KCNQ1, Nav1.5, and Cav1-2 ( Ben-Johny and Yue, 2014 ; Crotti et al., 2013 ; Herzog et al., 2003 ; Houdusse et al., 1996 ; Kang et al., 2023 ; Yu et al., 2003 ). CaM itself binds to the IQ (Isoleucine-Glutamine) motif located at Nav 1908-09 on the Nav1.5 CTD ( Kim et al., 2004 ; Tan et al., 2002 ). The CaM protein itself is a ubiquitous Ca 2+ sensor that is made up of four E-F hands arranged in a dumbbell-like manner. CaM will change its conformation or binding orientation based on the addition of Ca 2+ on its C and N-lobes. Typically, CaM must be saturated with Ca 2+ at all four E-F hands to bind to various proteins including KCNQ1 and FGF12A ( Mahling et al., 2021 ; Shamgar et al., 2006 ). However, apo-CaM without Ca 2+ can interact with the Nav1.5 CTD ( Ben-Johny et al., 2016 ). The loss of CaM binding is associated with LQTS, Brugada Syndrome, and atrial fibrillation ( Kapplinger et al., 2010 ; Wilde and Amin, 2018 ). CaM binding to Nav1.5 is seen as protective against increased persistent late current and allows for increased peak channel opening ( Kang et al., 2021 ; Yan et al., 2017 ). Intracellular fibroblast growth factors (iFGFs), also known as fibroblast homologous factors (FHFs), have distinct isoforms with varying exon splicing (e.g., FGF12A and FGF12B). These iFGFs do not have the N-terminal sequence that is required for extracellular secretion and therefore do not bind to FGF receptors ( Pablo and Pitt, 2016 ). However, specific iFGFs do modulate voltage-gated ion channels including sodium and calcium channels ( Pablo and Pitt, 2016 ). iFGFs interact with the Nav1.5 channel through the iFGF β-trefoil at the EF-hand of the Nav1.5 ( Goetz et al., 2009 ; Goldfarb, 2024 ). The interaction between FGF12 and Nav1.5 is known to induce a depolarizing shift in the steady-state inactivation (SSI) of the channel, leading to a reduction in the number of channels inactivated at a resting membrane potential ( Angsutararux et al., 2023 ; Dover et al., 2010 ; Goldfarb, 2024 ). FGF12A specifically has also been shown to reduce pathogenic persistent late sodium current (I Na,L ) carried by Nav1.5 channels ( Chakouri et al., 2022 ). The Nav1.5 variant of H1849R blocks binding of iFGFs to the Nav1.5 channel which resulted in a gain-of-function increase of I Na by altering steady-state inactivation and ultimately resulting in arrythmia ( Musa et al., 2015 ). FGF12B and FGF13VY have also been shown to alter the decay phase of peak I Na after fitting the curves to the sum of two exponentials which are on the time scales of 1ms (T 1 ) and 10ms (T 10 ) time components of inactivation ( Angsutararux et al., 2023 ). All A-splice variants of iFGFs have recently been shown to have a non-canonical CaM binding site on the N-terminal portion of the protein ( Mahling et al., 2021 ). The CaM binding region on FGF12A allows for the possibility of multiple calmodulins binding to the Nav1.5 CTD. The relationship between CaM and FGF12A independently on the CTD has been analyzed ( Gade et al., 2019 ), however, the specific interactions of the CaM located on the FGF12A N-terminus with Nav1.5 gating kinetics has not been defined. We have designed experiments to test whether CaM presence on FGF12A is required for the myriad changes in Nav1.5 kinetics that have been previously reported, including shifts in activation, inactivation and reduction of I Na,L ( Chakouri et al., 2022 ). Materials and Methods Xenopus Laevis oocyte collection Xenopus laevis females with length > 9cm were purchased from Xenopus One Corp. Oocytes were collected from anesthetized frogs in accordance of previously reported protocols( Varga et al., 2015 ). All animal handling protocols were approved by Washington University’s Institutional Animal Care and Use Committee (IACUC) and were developed in compliance with the National institutes of Health (NIH) Guild for the Care and Use of Laboratory Animals. Molecular biology The molecular biology protocols used were adapted from Angsutararux et al. 2023 . Plasmids encoding human SCN5A and Fgf12A were produced on a pMAX and pBSTA backbone respectively. All Nav1.5 point mutations ( Table 1 ) and FGF12A del CaM ( Figure 1 ) were generated using High-Fidelity PCR via the Herculase II Fusion DNA Polymerase (Agilent). The scrambled FGF12A (FGF12A Scram CaM) sequence was created with GenScript ( Figure 1 ). The plasmid was constructed and packaged by VectorBuilder with the vector ID of VB231023-1405afd. cDNA plasmids were amplified from glycerol E. coli stocks with NucleoSpin Plasmid DNA purification (Macherey-Nagel). The cDNA plasmids were linearized with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). cRNA was then synthesized with the mMessage mMACHINE T7 Transcription kit (Life Technologies) and reconstituted in nuclease-free H 2 O at a concentration of approximately 1 μg/μL. View this table: View inline View popup Table 1 Download figure Open in new tab Figure 1: CaM binds to FGF12A non-canonically to confer shifts in inactvation kinetics. A) Schematic representation of the Nav1.5 C-terminus domain (CTD) with auxiliary proteins calmodulin (CaM) and intracellular fibroblast growth factor 12A (iFGF12A), not drawn to scale. B) Sequence of CaM binding region describe in Mahling et al. 2021 . Red denotes WT FGF12A, Blue: deleted CaM binding region (FGF12A del CaM), Orange: scrambled CaM binding region (FGF12A Scram CaM). C) Representative traces with corresponding auxiliary subunit schematics. D) Mean normalized ±SEM conductance-voltage (GV) and steady-state inactivation (SSI) curves for WT Nav1.5 and the addition of: FGF12A, FGF12A del CaM, and FGF12A Scram CaM. Curves are fitted single Boltzmann functions. The absence of CaM on FGF12A shows a slight shift in the inactivation of the Nav1.5 ( Table 1 ). E) Mean ±SEM T1 and T 10 inactivation time constants of I Na decay fitted to a double exponential described in Methods. WT FGF12A showed a marked increase in the slow time component at multiple holding potentials. F) Mean ±SEM recovery from inactivation from a protocol described in Methods. Cut-open oocyte voltage-clamp Collected Xenopus laevis were digested with 24mg collagenase and 3mg trypsin inhibitor in 35 mL OR2 buffer (in mM, 82.5 NaCl, 2.5 KCl, 1 MgCl 2 , 5 HEPES, pH 7.6) for 20 minutes. Oocytes were selected and kept at 5°C overnight in ND93 solution (in mM, 93 NaCl, 5 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 5 HEPES, 2.5 Na pyruvate, and 1% penicillin/streptomycin, pH 7.4). Oocytes were injected with 50ng of cRNA with a ratio of 2:1:1 (Nav1.5, β 1 , FGF12A) for co-expression or 3:1 (Nav1.5:β 1 ) and incubated for 24 hours at 18°C prior to recordings. The auxiliary subunit of β 1 ( SCN1B ) was present in each group for consistency and increased channel expression ( Martinez-Moreno et al., 2020 ). A custom cut-open Vaseline gap (COVG) rig and protocol described previously was used for oocyte voltage-clamp recordings ( Angsutararux et al., 2023 ; Rudokas et al., 2014 ; Varga et al., 2015 ). Oocytes were kept at 18 C with an HCC-100A temperature controller (Dagan Corporation) throughout recordings. A CA-1B cut-open amplifier (Dagan Corporation) with corresponding electrodes and bath-guard connected with a digitizer (1440 Digidata; Molecular Devices) to the computer software Clampex (Molecular Devices). The oocytes were recorded in an external solution (in mM, 25 N-methyl-D-glucamine (NMDG), 90 2-(N-Morpholino)ethanesulfonic acid (MES) sodium salt (Na-MES), 20 HEPES, 2 Ca-MES 2 , pH 7.4) and an internal solution (in mM, 105 NMDG, 10 Na-MES, 20 HEPES, 2 EGTA, pH 7.4). For steady-state activation, oocytes were clamped from -120 to 60mV for 100ms with a holding potential of -120mV. The peak currents were measured, normalized, and divided by the driving force to determine the conductance (G) at each voltage step. The G-V curves were fitted with the Boltzmann Equation ( Eq. 1 ): with V 1/2 as the voltage at half-activation and k is the slope. To measure the inactivation time components, a two-exponential equation ( Eq. 2 ) was fitted to the decay phase of activation at each voltage step: where A 1 and A 10 are the amplitudes of inactivaiton and T 1 and T 10 are the time components on the time scale of 1ms or 10ms respectively. Steady-state inactivation involved pre-pulsing the cells from -150 to 20mV for 200ms and then stepping to - 20mV to determine the channel availability. Recovery from inactivation was a two-pulse protocol in which the first pulse stepped up from the holding potential of -120mV to -20mV for 20ms. The second pulse was applied after a varying amount of time (2-1000ms). The ratio between the first peak (P1) and the second (P2) was used to determine channel recovery. For intermediate inactivation prolonged protocols were used during voltage-clamp experiments( Veldkamp et al., 2000 ). Intermediate inactivation followed a similar protocol to steady-state inactivation except with a 1000ms depolarizing pulse before measuring channel availability a -20mV. Intermediate recovery was similar to the recovery from inactivation protocol except the first pulse had a 1000ms duration. Intermediate onset included a two-pulse protocol where the first pulse was a step to -20mV for a duration varying from 2-1000ms. A second pulse was applied after 20ms to determine the time-course of intermediate inactivation ( Wang et al., 2000 ). Electrophysiological data analyses Electrophysiological data and statistical analysis were conducted with Clampfit (Molecular Devices), Prism 10 (Graph Pad), and JupyterLab. The statistical significance between experimental groups was determined by a Welch ANOVA unpaired t-test or Tukey’s multiple comparisons test. Supplemental material SI Figure 1 includes the conductance-voltage relationship curve (GV) and steady-state inactivation curves (SSI) for the Nav1.5 variants of Y1795C, S1904L, K1493E, and IQM compared to WT Nav1.5 and co-transfected with either WT FGF12A or FGF12A del CaM. SI Figure 2 shows the recovery from inactivation curves of the Y1795C, S1904L, and K1493E Nav1.5 variants with either WT FGF12A or FGF12A del CaM. SI Figure 3 are the inactivation time constants fitted from the decay phase of the activation pulse protocol for the aforementioned Nav1.5 variants with either WT FGF12A or FGF12A del CaM. SI Table 1 includes all of the fitted inactivation time constants at - 30mv for all constructs included in the project. SI Figure 4 shows the representative traces of Y1795C, S1904L, and K1493E co-transfected either with WT FGF12A or FGF12A del CaM. View this table: View inline View popup Supplemental Table Results A-splice variants of iFGFs have a non-canonical CaM binding region We tested whether the presence of CaM on FGF12A affects FGF12A regulation of Nav1.5. To prevent CaM interaction with FGF12A, we considered two options: deleting the entire CaM binding region ( residues 38-66 ) on FGF12A (FGF12A del CaM) or scrambling the CaM binding region section of FGF12A (FGF12A scram CaM) ( Figure 1B ). We conducted cut-open voltage clamp recordings with Nav1.5 expressed alone or with: FGF12A ( red ), FGF12A del CaM ( blue ), and FGF12A scram CaM ( orange ) ( Figure 1C ). Co-expression of WT FGF12A resulted in a small depolarizing shift in the SSI (+5.385mV ±1.546, p = 0.0462) and a hyperpolarizing shift in GV curve (−8.226mV ± 2.065, p=0.0005) ( Figure 1D and Table 1 ), consistent with previous observations ( Dover et al., 2010 ). Inactivation was well-fit with two exponentials with time constants on the order of 10ms and 2ms. The 10ms inactivation time constant (T 10 ) was significantly increased with WT FGF12A co-expression(+2.98ms ±0.667, p = 0.029, Figure 1E ). Conversely, with the CaM binding region deleted from FGF12A, no shifts in T 10 (−0.729ms ±0.462, p=0.1585) ( SI Table 1 ) or SSI V 1/2 (−0.5mV ±0.992, p=0.6179) ( Table 1 ) were observed. We continued by comparing FGF12A scram CaM with FGF12A del CaM, as the deleted section of the FGF12A protein was substantial. No significant changes in FGF12A scram CaM’s effects compared to the FGF12A del CaM protein was detected ( Figure 1C-F and Table 1 ). Therefore, we concluded that the depolarizing shift in SSI voltage-dependence and inactivation time constants were due to the lack of CaM binding itself and not changes in overall protein structure of FGF12A that were caused by the deletion of the CaM binding region. Subsequent studies used FGF12A del CaM as the sole source of CaM deletion from FGF12A. CaM’s absence from the Nav1.5 CTD allows for an increased effect due to FGF12A Results from Figure 1 show that FGF12A can only shift WT Nav1.5 inactivation when CaM is bound to the FGF12A N-terminus. Therefore, we considered whether the removal of CaM from the Nav1.5 CTD would amplify the FGF12A effect, given that the only CaM present in the Nav1.5 complex would be associated with WT FGF12A. We proceeded to test this hypothesis by using the IQ(1908-09)/AA variant that inhibits CaM binding to Nav1.5 ( Abrams et al., 2020 ) ( Figure 2A ). The IQ/AA variant of Nav1.5 independently has large hyperpolarizing and depolarizing shifts in its SSI (−10.63mV ±1.676, p<0.0001) and GV V 1/2 (+11.37mV ±2.65, p=0.003) respectively and significantly increases the persistent late current (I Na,L ) (+0.037% ±0.006, p<0.0001) compared to WT Nav1.5 ( Table 1 ). Download figure Open in new tab Figure 2: Removing CaM from the Nav1.5 CTD induces FGF12A-CaM dependent shifts in voltage-dependencies. A) Representative traces of the IQ(1908-09)AA Nav1.5 mutation which prevents binding of the CaM protein to the Nav1.5 CTD. Shown with schematic representations of the auxiliary subunits. B) Mean ±SEM GV and SSI plots with fitted Boltzmann functions. WT Nav1.5 (dashed grey), IQ/AA (black), IQ/AA+FGF12A (red), and IQ/AA+FGF12A del CaM (blue). The complete absence of CaM on the Nav1.5 CTD allows for a more dramatic CaM-dependent shift in activation and inactivation with the presence of WT FGF12A. C) Persistent late current (I Na,L ) is significantly lowered regardless of the presence of CaM with FGF12A ( Table 1 ). Percent of late I Na at -30mV holding potential analysis is described in Methods. Error bars are SEM (* p = 0.0137, ** p = 0.0062). D) Mean ±SEM recovery from inactivation. E) Mean ±SEM T 1 and T 10 inactivation time constants of I Na decay with WT FGF12A showing a significantly higher T 10 component. When co-transfected with the IQ/AA variant, a clear distinction in the SSI voltage-dependence was observed between WT FGF12A and FGF12A del CaM. Only WT FGF12A produced a significant depolarizing shift (+12.29mV ±1.787, p<0.0001) compared to the IQ/AA channel ( Figure 2B and Table 1 ). A major hyperpolarizing shift in the GV curve was also observed in WT FGF12A (+15.07mV ±2.934, p<0.0001) with FGF12A del CaM shifting the GV V 1/2 back to WT Nav1.5 values (−8.941mV ± 1.493, p<0.0001) ( Figure 2B and Table 1 ). Similarly, only WT FGF12A significantly increased T 10 (+6.074ms ±1.108, p=0.0009). However, both WT FGF12A and FGF12A del CaM slightly increased T 1 (+1.55ms ±0.123, p<0.0001) and (+1.123ms ±0.281, p=0.0052) respectively compared to the IQ/AA variant ( Figure 2E and SI Table 1 ). Accordingly, we discerned that in the absence of CaM already present on the Nav1.5 CTD, the addition of FGF12A drastically alters the inactivation gating of the Nav1.5 channel potentially through a CaM presence on the FGF12A N-terminal. Conversely, the addition WT FGF12A blocks the increased levels of I Na,L observed with the IQ/AA variant (−2.332% ±0.656, p=0.0137) as expected, however, the addition FGF12A del CaM also decreased the I Na,L (−2.753% ±0.696, p=0.0062) ( Figure 2C ). The result of the reduction of pathogenic I Na,L by either WT FGF12A or FGF12A del CaM led us to hypothesize that there are two different mechanisms by which FGF12A interacts with the Nav1.5 channel: CaM-regulated and CaM-independent. Impaired fast inactivation of Nav1.5 illustrates a CaM-independent mechanism of FGF12A The F1486Q (IQM) mutation was used to inhibit fast inactivation of the Nav1.5 channel by impairing the inactivation gating particle located on the DIII-DIV linker ( West et al., 1992 ). The marked difference in fast inactivation of the IQM variant is shown in Figure 3A . Additionally, IQM substantially increases I Na,L compared to the WT Nav1.5 channels (+12.85% ±2.165, p<0.0001) ( Table 1 ). The additions of either WT FGF12A (−6.832% ±2.16, p=0.0157) and FGF12A del CaM (−8.92% ±2.14, p=0.0396) were sufficient to reduce I Na,L without changes to the peak current expression ( Figure 3D and Table 1 ). Both WT FGF12A and FGF12A del CaM altered the time course of recovery from inactivation of the IQM variant channel in a similar manner ( Figure 3C ). GV and SSI curves in addition to fitted inactivation time constants for the IQM variant with(out) FGF12A are shown in Supplemental Information ( SI Figure 1 and 3 ) Download figure Open in new tab Figure 3: Inhibiting fast inactivation via the IQM mutation demonstrates the CaM-independent mechanism by which FGF12A inhibits I Na,L . A) Representative traces and schematic views of the F1486Q (IQM) variant, with(out) auxiliary subunits, that inhibits fast inactivation through altering the DIII-DIV linker responsible for fast inactivation after VSD DIV activation. B) Comparative traces of Nav1.5 at a step potential of -40mV. Comparison of persistent late current between Nav.15 variants includes determining the percentage of the Peak I Na that the I Na,L is. C) Recovery from inactivation shows that FGF12A with and without CaM significantly alters the relationship. D) Percentage of peak current comparing the differences in I Na,L in WT and IQM channels at multiple holding potentials (mV). Error bars are ±SEM. (* p = 0.0396, 0.0234; *** p = 0.0004; **** p < 0.0001) The reduction of I Na,L in the IQM variant by both WT FGF12A and FGF12A del CaM is consistent with the results found in our experiments with the IQ/AA variant ( Figure 2C ). However, with the IQM variant, CaM is present in the Nav1.5 CTD, therefore we conclude that the process by which FGF12A reduces pathogenic I Na,L is CaM-independent as the presence or absence of CaM does not affect I Na,L inhibition. Nav1.5 LQT3 variants show a consistent split in CaM mechanisms We continued observing FGF12A regulation of Nav1.5 voltage dependence by using LQT3 mutations located on the CTD (Y1795C and S1904L) ( Bankston et al., 2007 ; Rivolta et al., 2001 ) ( Figure 4A ). Independently, Y1795C shifts the SSI V 1/2 leftward compared to the WT Nav1.5 channel (−9.7mV ±1.628, p<0.0001) while S1904L shifts the GV V 1/2 rightward (+11.61mV ±2.575, p=0.0041) ( Table 1 ). A depolarizing (right) shift of the V 1/2 of activation can lead to late channel activation while a hyperpolarizing (left) shift of inactivation increases the number of channels activated at higher membrane potentials. Both effects can potentially lead to a prolonged cardiac action potential and subsequent arrhythmias( Han et al., 2018 ). Download figure Open in new tab Figure 4: Altering Nav1.5’s gating kinetics show both CaM independent and dependent effects. A) Schematic representation of various Nav1.5 mutation locations tested. B) Example graphs of steady state inactivation (top) and conductance vs. voltage (bottom) that illustrate how the IQ/AA variant (red) shifts the V 1/2 value in either a hyperpolarizing or depolarizing manner compared to WT Nav1.5 (black). C) FGF12A doesn’t require CaM to decrease the persistent late current from an altered Nav1.5 channel. Each Nav1.5 variant is separated into three groups: no FGF12A (grey), with WT FGF12A (red), with FGF12A del CaM (blue). Graph is box-and-whisker plot with bars from 5 th to 95 th percentile. (** p = 0.0058, *** p = 0.0002, **** p< 0.0001). D) FGF12A requires CaM to depolarize the SSI V 1/2 except when the DIII-DIV linker is unable to interact with the CTD as in the case of K1493E. Additionally, a slight hyperpolarizing shift occurs with GV V 1/2 only with WT FGF12A. Graph is a scatter dot plot with the median line shown. SSI (** p = 0.0032, 0.0019, *** p = 0.0003, **** p < 0.0001), GV (* p = 0.0289,0.0251, **** p < 0.0001) ( Table 1 ). We observed a trend in the SSI V 1/2 with WT FGF12A depolarizing the V 1/2 and FGF12A del CaM having no significant shifts in V 1/2 (( Figure 4D and Table 1 ). We also observed that WT FGF12A induces a hyperpolarizing shift in the GV V 1/2 of Y1795C, but not in S1904L ( Figure 4D and Table 1 ). Thus, FGF12A alters the two variants in an opposite manner, depolarizing the SSI in S1904L and hyperpolarizing the GV in Y1795C. Additionally, the observation of WT FGF12A affecting SSI or GV while FGF12A del CaM does not is recapitulated with the IQ/AA variant ( Figure 2B ), which indicates that the mechanism is dependent on the CaM associated with FGF12A. Conversely, this depolarizing SSI V 1/2 trend was not shown in the K1493E variant. The K1493E variant individually shifts the SSI V 1/2 to depolarized potentials (−14.3mV ±1.746, p<0.0001) compared to WT Nav1.5 ( Table 1 ). The addition of either WT FGF12A or FGF12A del CaM does alter the already shifted SSI curve ( Figure 4D ). We hypothesize that this lack of effect is due to the requirement of Nav1.5 CTD interacting with the DIII-DIV linker which is impaired in the K1493E variant ( Gade et al., 2019 ). We continued to examine the FGF12A effect on the Nav1.5 I Na,L . S1904L and Y1795C both increased I Na,L compared to WT Nav1.5 (+1.331% ±0.523, p= 0.0085) and (+2.431% ±0.562, p=0.0038) ( Table 1 ). As predicted, the additions of either WT FGF12A or FGF12A del CaM were sufficient to reduce the pathogenic I Na,L ( Figure 4C and Table 1 ) consistent with our findings with the IQ/AA variant ( Figure 2C ) and the IQM variant ( Figure 3 ). Representative traces and other measurements (GV, SSI, Recovery from Inactivation, and Inactivation time constants) are shown in the Supplemental Information Figures ( SI Figures 1 - 4 ) . Overall, there was an established difference between the FGF12A ability to alter the voltage dependence of inactivation versus the persistent late current of the Nav1.5 channel with the former requiring CaM present on the FGF12A N-terminus. Intermediate inactivation indicates how FGF12A alters inactivation of the Nav1.5 channel In addition to fast inactivation which occurs over a 1 to 10ms time domain, Na + channels display inactivation over multiple slower time domains as well ( Ruben et al., 1992 ). Intermediate inactivation is a distinct gating process that has a time period of 500ms to 1 second ( Kambouris et al., 1998 ). As we have shown that FGF12A differentially alters the decay of fast inactivation and persistent late current, we proceeded to test the CaM effect on intermediate inactivation ( Figure 5 ) utilizing protocols developed by Wang et al. 2000 . We observed a hyperpolarizing shift in the intermediate inactivation between WT Nav1.5 and FGF12A del CaM (−6.856mV ±1.882, p=0.0015) ( Figure 5B ). WT FGF12A caused a non-significant depolarizing shift in the intermediate inactivation suggesting that only FGF12A without CaM present shifts the voltage-dependency of intermediate inactivation. Download figure Open in new tab Figure 5: Intermediate inactivation of Nav1.5 shows the split mechanism between FGF12A and CaM. A) Representative trace and pulse protocols of (right to left): intermediate inactivation, intermediate onset, and intermediate recovery. B) Normalized ±SEM current vs voltage of intermediate with fitted Boltzmann function show WT (black, n=5), WT + FGF12A (red, n=4), and WT + FGF12A del CaM (blue, n=6). FGF12A requires CaM to function as a normal Nav1.5 channel. C) Normalized ±SEM values of the intermediate onset. FGF12A with and without CaM function similarly with a reduced onset compared to WT Nav1.5 without FGF12A. D) Normalized ±SEM values of intermediate recovery. However, for the intermediate inactivation onset and recovery of the Nav1.5 channel, WT FGF12A and FGF12A del CaM had similar effects ( Figure 5C-D ). The reduced onset of intermediate inactivation due to either WT FGF12A or FGF12A del CaM implies a lower channel availability over a longer time period. The intermediate inactivation shifts could be associated with the reduction in persistent late current due to FGF12A as it has been shown with slow inactivation ( Silva and Goldstein, 2013a , 2013c ). Discussion We have demonstrated the distinct differences in the CaM-independent and CaM-regulated mechanisms associated with FGF12A on the Nav1.5 channel. WT FGF12A when co-transfected with the Nav1.5 channel shifts the SSI in a depolarizing manner. This shift occurs only when CaM is present on FGF12A’s N-terminal. The effect is more pronounced when CaM is also removed from the Nav1.5 CTD. Conversely, when the fast inactivation of the channel is blocked via the IQM mutation, FGF12A, regardless of CaM’s presence, can rescue fast inactivation and reduce the persistent late current. The split mechanism trend was upheld when LQT3 variants were tested with the presence of WT FGF12A and FGF12A del CaM. The Nav1.5 CTD interacts with the DIII-DIV linker to stabilize fast inactivation Current models of Nav1.5 fast inactivation include a CTD interaction with the III-IV linker ( Biswas et al., 2024 ). Structures of Nav1.5 in the open, activated state suggest that the CTD initially interacts with the III-IV linker at the beginning of fast inactivation to stabilize the pore domain of the channel ( Biswas et al., 2024 ; Shen et al., 2017 ). Once VSD-IV has activated and the IFM motif has tightly bound itself to the IFM receptor, the CTD moves away from the linker. Gade et al. 2019 altered the CTD’s interaction with the DIII-DIV linker with point mutations at K1493 and E1784 located on the DIII-DIV linker and CTD respectively ( Gade et al., 2019 ). Changes in these residues provoked shifts in the fast inactivation of the channel and altered responses to pathogenic I Na,L and indicate a reduction in interaction between the DIII-DIV linker and the CTD for normal channel conductance ( Gade et al., 2019 ; Gardill et al., 2018 ). Increasingly, it has been shown that auxiliary subunits bound to the Nav1.5 CTD also interact with DIII-DIV linker/stabilize the overall interaction. CaM typically binds to the IQ motif on the Nav1.5 CTD ( Kim et al., 2004 ), but has also been proposed to bind to a section of the DIII-DIV linker ( Potet et al., 2009 ). Structural models have correspondingly shown both FGF12A and CaM located on the Nav1.5 CTD interacting with the DIII-DIV residues ( Gade et al., 2019 ). CaM itself has been proposed to stabilize the interaction of the CTD with the IFM motif and to allow for faster state switching ( Gabelli et al., 2014 ). FGF12A CaM-independent mechanism reduces pathogenic I Na,L Previously published literature has touched on FGF12A’s effect of I Na,L by considering its N-terminal section an inactivation particle ( Abrams et al., 2020 ; Chakouri et al., 2022 ; Dover et al., 2010 ; Gade et al., 2019 ; Goldfarb, 2024 ; Wang et al., 2012 ). In Chakouri et al. 2022 , the group proposed that the FGF12A N-terminal residues of 1-30 were sufficient to provide a protective effect against pathogenic I Na,L including late current induced by the IQ/AA variant of Nav1.5 ( Chakouri et al., 2022 ). Additionally, in Mahling et al. 2021 , iFGF A-splice variants were proposed to have a long-term inactivation particle (LTP) on their N-terminus ( Mahling et al., 2021 ). We hypothesize that FGF12A interacts with the DIII-DIV linker via its N-terminal ( residues 1-30 ) to stabilize fast and slow inactivation. FGF12A can accomplish the same effects with the CaM-binding region deleted, negating the region’s necessity in reducing I Na,L . FGF12B, which is the splice variant of FGF12 without the N-terminal section of FGF12A, does not have the same reduction of I Na,L as FGF12A ( Chakouri et al., 2022 ). Goldfarb et al. 2024 describe a similar mechanism of canonical FGF12A interaction of specifically the initial N-terminal portion of the protein interacting with the IFM motif to stabilize the inactivation state of the Nav1.5 channel ( Goldfarb, 2024 ). FGF12A CaM-regulated mechanisms alter inactivation kinetics of Nav1.5 The various iFGFs are prevalent throughout mammalian cells with FGF12 as the predominant isoform located within human cardiomyocytes andFGF13 as the most common in murine cardiomyocytes( Park et al., 2016 ; Wang et al., 2011 ). In Angsutararux et al. 2023 , they determined that both the iFGF core region and the N-terminal of the protein is necessary to confer the shifts in inactivation and activation voltage-dependencies ( Angsutararux et al., 2023 ). With regards to FGF12A, we showed that the presence of the CaM binding region altered FGF12A’s effects on both the steady-state inactivation and inactivation decay rate. FGF12B, which doesn’t have a CaM binding region on its N-terminus, can also shift both the SSI and GV in a depolarizing manner ( Angsutararux et al., 2023 ). FGF12A independent of CaM does slightly alter the gating function of Nav1.5 as seen in Figures 2 and 4 . However, FGF12A does require CaM for the full expected shifts in SSI and an increase the slow time component of inactivation. Potentially, FGF12A independently alters the VSD activations as seen in FGF12B ( Angsutararux et al., 2023 ) whereas CaM interacts directly with the III-IV linker to alter Nav1.5 inactivation. Physiological Relevance FGF12A has been shown to reduce pathogenic I Na,L and shift the voltage-dependencies of steady-state inactivation in a depolarizing manner. Both effects, through either the CaM-independent or CaM-regulated mechanism, can reduce the duration of the cardiac action potential ( Abriel, 2007 ; Han et al., 2018 ; Moreau et al., 2015 , 2013 ). Typically, these effects are most prevalent when perturbations to the Nav1.5 channel have occurred either through genetic mutations such as LQT3 variants or changes in the presences of auxiliary subunits ( Abriel, 2010 ; Han et al., 2018 ). FGF12A with respect to the persistent late current, could be viewed as a defense mechanism in restoring the normal action potential duration. With regards to the CaM present on the FGF12A N-terminal, the CaM binding site potentially could have evolved as an auxiliary protein redundancy to protect the fast inactivation of the Nav1.5 channel. The iFGF genes themselves are fairly conserved in all vertebrates with the CaM binding region being located on the N-terminal of all A-splice variants ( Goldfarb, 2005 ; Satou et al., 2002 ). The IQ/AA Nav1.5 variant without CaM present shown increased I Na,L and hyperpolarizing shifts in its SSI voltage dependencies. However, in IQ/AA transgenic mice, no effects are detected( Abrams et al., 2020 ). Abrams et al. 2020 proposed that the iFGFs endogenously present in the mice compensated for the lack of CaM on the Nav1.5 CTD ( Abrams et al., 2020 ). However, it could be that the conserved CaM binding region on the A-splice variants of iFGFs are a secondary measure to ensure the presence of CaM on the Nav1.5 CTD. Overall FGF12A mechanisms with Nav1.5 gating states As previously established by Mahling et al. 2021 , all A-splice variants of iFGFs have a CaM binding region located on the N-terminal. Our results show that there are two distinct mechanisms by which FGF12A affects Nav1.5 conduction: a CaM-regulated and a CaM-independent mechanism. Figure 6 represents these two mechanisms in a state dependent manner. At resting membrane potentials, the Nav1.5 channel is closed: all of its VSDs are in their downward positions and no Na + ions are flowing ( Chanda and Bezanilla, 2002 ; D et al., 2020). Once the membrane is depolarized, the VSD-I to III activate which translocates the coupled S5-S6 helices to open the pore, allowing for Na + ions to flow inward ( Chanda and Bezanilla, 2002 ; Jiang et al., 2021 ). Immediately after, VSD-IV activates revealing an allosteric pocket for the IFM motif allowing the III-IV linker to bind( Capes et al., 2013 ; Gardill et al., 2018 ; Varga et al., 2015 ). The binding forces the S5-S6 helices to constrict, causing fast inactivation of the channel, preventing the flow of Na + ions ( Biswas et al., 2024 ; Li et al., 2021 ). Between the activated and inactivated states of the channel, FGF12A and its associated CaM interact with the III-IV linker via the Nav1.5 CTD to alter the voltage dependencies of steady state inactivation and the fast inactivation decay rate ( Angsutararux et al., 2023 ; Gade et al., 2019 ). We propose that instead of the FGF12A protein independently, the effects on Nav1.5 gating are due to the core of FGF12A bound to the Nav1.5 CTD ( Angsutararux et al., 2023 ) and are regulated by the associated CaM protein. Once the Nav1.5 channel is inactivated, the FGF12A N-terminal will then interact with the DIII-DIV linker to stabilize the inactivated state of the channel and prevent pathogenic persistent late current consistent with previous publications ( Chakouri et al., 2022 ; Dover et al., 2010 ; Goldfarb, 2024 ). Download figure Open in new tab Figure 6: There are two mechanisms by which FGF12A interacts with the Nav1.5 channel via the CTD. A) Closed state: All VSDs are in their downward state; no Na + ions are flowing inward. B) Open state: VSDs I-III are activated; Na + ions are flowing inward. C) Inactivated State: The DIV VSD activates allowing the IFM motif to bind for fast inactivation to the allosteric pocket, thus shifted the coupled S5-S6 helices to close the pore; the Nav1.5 CTD swings to interact via CaM (on FGF12A) to modulate VSD activation. D) Stably Inactivated State: The N-terminal of FGF12A interact with the DIII-DIV linker to stabilize the inactivated state and prevent I Na,L . Supplemental Figures Download figure Open in new tab SI Figure 1: Mean normalized ±SEM conductance-voltage (GV) and steady-state inactivation (SSI) curves for WT Nav1.5 and the addition of: FGF12A, FGF12A del CaM, and FGF12A Scram CaM. Curves are fitted single Boltzmann functions.V 1/2 values are recorded in Figure 4 and Table 1 . Shifts in the SSI and GV are seen withthe addition of only WT FGF12A in the S1904L and Y1795C variants respectively, requiring CaM for the full effects. The K1493E variant inhibits intercation of the Nav1.5 CTD with the III-IV linker, therefore no shifts are observed with the addition of WT FGF12A or FGF12A del CaM. Theaddition of FGF12A to the IQM mutation rescued fast inactivation without shiftingthe voltage-dependency of inactivation. Download figure Open in new tab SI Figure 2: Mean ±SEM recovery from inactivation of the Y1795C, S1904L, and K1493E variants. No significant shifts were detected. Download figure Open in new tab SI Figure 3: Mean ±SEM 1 and 10 inactivation time constantsof peak I Na decay with the addition of WT FGF12A with the Y1795C, S1904L, K1493E, and IQM variants showinga significantly higher 10 component.Values at holding voltage of-30mV are show in SI Table 1 . Download figure Open in new tab SI Figure 4: Representative traces of variants shown in Figure 4 . Current traces were filtered via bandpass filter in Clampfit and are not normalized. Acknowledgements This work was completed in its entirety within the Silva lab at Washington University in St. Louis. It was funded by NIH-NHLBI RO1 HL136553, NIH-NHLBI RO1HL150637 and the individual predoctoral fellowship 1F31HL164080-01A1. References ↵ Abrams , J. , Roybal , D. , Chakouri , N. , Katchman , A.N. , Weinberg , R. , Yang , L. , Chen , B. , Zakharov , S.I. , Hennessey , J.A. , Avula , U.M.R. , Diaz , J. , Wang , C. , Wan , E.Y. , Pitt , G.S. , Ben-Johny , M. , Marx , S.O. , 2020 . Fibroblast growth factor homologous factors tune arrhythmogenic late NaV1.5 current in calmodulin binding–deficient channels . JCI Insight 5 . doi: 10.1172/jci.insight.141736 OpenUrl CrossRef ↵ Abriel , H. , 2010 . Cardiac sodium channel Nav1.5 and interacting proteins: Physiology and pathophysiology . J. Mol. Cell. Cardiol., Special Issue: Ion Channels 48 , 2 – 11 . doi: 10.1016/j.yjmcc.2009.08.025 OpenUrl CrossRef PubMed ↵ Abriel , H. , 2007 . Roles and regulation of the cardiac sodium channel Nav1.5: Recent insights from experimental studies . Cardiovasc. Res . 76 , 381 – 389 . doi: 10.1016/j.cardiores.2007.07.019 OpenUrl CrossRef PubMed Web of Science ↵ Ahern , C.A. , Payandeh , J. , Bosmans , F. , Chanda , B. , 2016 . The hitchhiker’s guide to the voltage-gated sodium channel galaxy . J. Gen. Physiol . 147 , 1 – 24 . doi: 10.1085/jgp.201511492 OpenUrl Abstract / FREE Full Text ↵ Angsutararux , P. , Dutta , A.K. , Marras , M. , Abella , C. , Mellor , R.L. , Shi , J. , Nerbonne , J.M. , Silva , J.R. , 2023 . Differential regulation of cardiac sodium channels by intracellular fibroblast growth factors . J. Gen. Physiol . 155 , e202213300 . doi: 10.1085/jgp.202213300 OpenUrl CrossRef PubMed ↵ Angsutararux , P. , Zhu , W. , Voelker , T.L. , Silva , J.R. , 2021 . Molecular Pathology of Sodium Channel Beta-Subunit Variants . Front. Pharmacol . 12 , 761275 . doi: 10.3389/fphar.2021.761275 OpenUrl CrossRef PubMed ↵ Bankston , J.R. , Sampson , K.J. , Kateriya , S. , Glaaser , I.W. , Malito , D.L. , Chung , W.K. , Kass , R.S. , 2007 . A Novel LQT-3 Mutation Disrupts an Inactivation Gate Complex with Distinct Rate-Dependent Phenotypic Consequences . Channels 1 , 273 – 280 . doi: 10.4161/chan.4956 OpenUrl CrossRef PubMed ↵ Ben-Johny , M. , Yue , D.N. , Yue , D.T. , 2016 . Detecting stoichiometry of macromolecular complexes in live cells using FRET . Nat. Commun . 7 , 13709 . doi: 10.1038/ncomms13709 OpenUrl CrossRef PubMed ↵ Ben-Johny , M. , Yue , D.T. , 2014 . Calmodulin regulation (calmodulation) of voltage-gated calcium channels . J. Gen. Physiol . 143 , 679 – 692 . doi: 10.1085/jgp.201311153 OpenUrl Abstract / FREE Full Text ↵ Biswas , R. , López-Serrano , A. , Huang , H.-L. , Ramirez-Navarro , A. , Grandinetti , G. , Heissler , S. , Deschênes , I. , Chinthalapudi , K. , 2024 . Structural basis of human Nav1.5 gating mechanisms . Res. Sq. rs.3.rs-3985999 . doi: 10.21203/rs.3.rs-3985999/v1 OpenUrl CrossRef ↵ Capes , D.L. , Goldschen-Ohm , M.P. , Arcisio-Miranda , M. , Bezanilla , F. , Chanda , B. , 2013 . Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels . J. Gen. Physiol . 142 , 101 – 112 . doi: 10.1085/jgp.201310998 OpenUrl Abstract / FREE Full Text ↵ Catterall , W.A. , 2023 . Voltage gated sodium and calcium channels: Discovery, structure, function, and Pharmacology . Channels 17 , 2281714 . doi: 10.1080/19336950.2023.2281714 OpenUrl CrossRef PubMed ↵ Catterall , W.A. , Goldin , A.L. , Waxman , S.G. , 2005 . International Union of Pharmacology . XLVII. Nomenclature and Structure-Function Relationships of Voltage-Gated Sodium Channels. Pharmacol. Rev . 57 , 397 – 409 . doi: 10.1124/pr.57.4.4 OpenUrl Abstract / FREE Full Text ↵ Chakouri , N. , Rivas , S. , Roybal , D. , Yang , L. , Diaz , J. , Hsu , A.L. , Mahling , R. , Chen , B.-X. , Owoyemi , J.O. , DiSilvestre , D. , Sirabella , D. , Corneo , B. , Tomaselli , G.F. , Dick , I.E. , Marx , S.O. , Ben-Johny , M. , 2022 . Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current . Nat. Cardiovasc. Res . 1 , 1 – 13 . doi: 10.1038/s44161-022-00060-6 OpenUrl CrossRef ↵ Chanda , B. , Bezanilla , F. , 2002 . Tracking Voltage-dependent Conformational Changes in Skeletal Muscle Sodium Channel during Activation . J. Gen. Physiol . 120 , 629 – 645 . doi: 10.1085/jgp.20028679 OpenUrl Abstract / FREE Full Text ↵ Crotti , L. , Johnson , C.N. , Graf , E. , De Ferrari , G.M. , Cuneo , B.F. , Ovadia , M. , Papagiannis , J. , Feldkamp , M.D. , Rathi , S.G. , Kunic , J.D. , Pedrazzini , M. , Wieland , T. , Lichtner , P. , Beckmann , B.-M. , Clark , T. , Shaffer , C. , Benson , D.W. , Kääb , S. , Meitinger , T. , Strom , T.M. , Chazin , W.J. , Schwartz , P.J. , George , A.L. , 2013 . Calmodulin mutations associated with recurrent cardiac arrest in infants . Circulation 127 , 1009 – 1017 . doi: 10.1161/CIRCULATIONAHA.112.001216 OpenUrl Abstract / FREE Full Text D, J., H, S., L, T., Tm, G.E.-D., Mj, L., Y, Z., C, Y., N, Z., Wa, C ., 2020 . Structure of the Cardiac Sodium Channel . Cell 180 . doi: 10.1016/j.cell.2019.11.041 OpenUrl CrossRef PubMed ↵ Deschênes , I. , Trottier , E. , Chahine , M. , 2001 . Implication of the C-terminal region of the alpha-subunit of voltage-gated sodium channels in fast inactivation . J. Membr. Biol . 183 , 103 – 114 . doi: 10.1007/s00232-001-0058-5 OpenUrl CrossRef PubMed Web of Science ↵ Dover , K. , Solinas , S. , D’Angelo , E. , Goldfarb , M. , 2010 . Long-term inactivation particle for voltage-gated sodium channels . J. Physiol . 588 , 3695 – 3711 . doi: 10.1113/jphysiol.2010.192559 OpenUrl CrossRef PubMed ↵ Gabelli , S.B. , Boto , A. , Kuhns , V.H. , Bianchet , M.A. , Farinelli , F. , Aripirala , S. , Yoder , J. , Jakoncic , J. , Tomaselli , G.F. , Amzel , L.M. , 2014 . Regulation of the Na V 1.5 cytoplasmic domain by calmodulin . Nat. Commun . 5 , 5126 . doi: 10.1038/ncomms6126 OpenUrl CrossRef PubMed ↵ Gade , A.R. , Marx , S.O. , Pitt , G.S. , 2019 . An interaction between the III-IV linker and CTD in NaV1.5 confers regulation of inactivation by CaM and FHF . J. Gen. Physiol . 152 , e201912434 . doi: 10.1085/jgp.201912434 OpenUrl CrossRef ↵ Gardill , B.R. , Rivera-Acevedo , R.E. , Tung , C.-C. , Okon , M. , McIntosh , L.P. , Van Petegem , F. , 2018 . The voltage-gated sodium channel EF-hands form an interaction with the III-IV linker that is disturbed by disease-causing mutations . Sci. Rep . 8 , 4483 . doi: 10.1038/s41598-018-22713-y OpenUrl CrossRef PubMed ↵ Goetz , R. , Dover , K. , Laezza , F. , Shtraizent , N. , Huang , X. , Tchetchik , D. , Eliseenkova , A.V. , Xu , C.-F. , Neubert , T.A. , Ornitz , D.M. , Goldfarb , M. , Mohammadi , M. , 2009 . Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels* . J. Biol. Chem . 284 , 17883 – 17896 . doi: 10.1074/jbc.M109.001842 OpenUrl Abstract / FREE Full Text ↵ Goldfarb , M. , 2024 . Fibroblast growth factor homologous factors: canonical and non-canonical mechanisms of action . J. Physiol . 602 , 4097 – 4110 . doi: 10.1113/JP286313 OpenUrl CrossRef PubMed ↵ Goldfarb , M. , 2005 . Fibroblast growth factor homologous factors: Evolution, structure, and function . Cytokine Growth Factor Rev . 16 , 215 – 220 . doi: 10.1016/j.cytogfr.2005.02.002 OpenUrl CrossRef PubMed Web of Science ↵ Han , D. , Tan , H. , Sun , C. , Li , G. , 2018 . Dysfunctional Nav1.5 channels due to SCN5A mutations . Exp. Biol. Med . 243 , 852 – 863 . doi: 10.1177/1535370218777972 OpenUrl CrossRef PubMed ↵ Herzog , R.I. , Liu , C. , Waxman , S.G. , Cummins , T.R. , 2003 . Calmodulin Binds to the C Terminus of Sodium Channels Nav1.4 and Nav1.6 and Differentially Modulates Their Functional Properties . J. Neurosci . 23 , 8261 – 8270 . doi: 10.1523/JNEUROSCI.23-23-08261.2003 OpenUrl Abstract / FREE Full Text ↵ Houdusse , A. , Silver , M. , Cohen , C. , 1996 . A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch . Struct. Lond. Engl. 1993 4 , 1475 – 1490 . doi: 10.1016/s0969-2126(96)00154-2 OpenUrl CrossRef ↵ Jiang , D. , Banh , R. , Gamal El-Din , T.M. , Tonggu , L. , Lenaeus , M.J. , Pomès , R. , Zheng , N. , Catterall , W.A. , 2021 . Open-state structure and pore gating mechanism of the cardiac sodium channel . Cell 184 , 5151 - 5162.e11 . doi: 10.1016/j.cell.2021.08.021 OpenUrl CrossRef PubMed ↵ Kambouris , N.G. , Hastings , L.A. , Stepanovic , S. , Marban , E. , Tomaselli , G.F. , Balser , J.R. , 1998 . Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat μ1 skeletal muscle sodium channel . J. Physiol . 512 , 693 – 705 . doi: 10.1111/j.1469-7793.1998.693bd.x OpenUrl CrossRef PubMed Web of Science ↵ Kang , P.W. , Chakouri , N. , Diaz , J. , Tomaselli , G.F. , Yue , D.T. , Ben-Johny , M. , 2021 . Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes . Proc. Natl. Acad. Sci . 118 . doi: 10.1073/pnas.2025085118 OpenUrl Abstract / FREE Full Text ↵ Kang , P. wei , Woodbury , L. , Angsutararux , P. , Sambare , N. , Shi , J. , Marras , M. , Abella , C. , Bedi , A. , Zinn , D. , Cui , J. , Silva , J.R. , 2023 . Arrhythmia-associated Calmodulin Variants Interact with KCNQ1 to Confer Aberrant Membrane Trafficking and Function . doi: 10.1101/2023.01.28.526031 OpenUrl Abstract / FREE Full Text ↵ Kapplinger , J.D. , Tester , D.J. , Alders , M. , Benito , B. , Berthet , M. , Brugada , J. , Brugada , P. , Fressart , V. , Guerchicoff , A. , Harris-Kerr , C. , Kamakura , S. , Kyndt , F. , Koopmann , T.T. , Miyamoto , Y. , Pfeiffer , R. , Pollevick , G.D. , Probst , V. , Zumhagen , S. , Vatta , M. , Towbin , J.A. , Shimizu , W. , Schulze-Bahr , E. , Antzelevitch , C. , Salisbury , B.A. , Guicheney , P. , Wilde , A.A.M. , Brugada , R. , Schott , J.-J. , Ackerman , M.J. , 2010 . An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing . Heart Rhythm 7 , 33 – 46 . doi: 10.1016/j.hrthm.2009.09.069 OpenUrl CrossRef PubMed Web of Science ↵ Kim , J. , Ghosh , S. , Liu , H. , Tateyama , M. , Kass , R.S. , Pitt , G.S. , 2004 . Calmodulin mediates Ca2+ sensitivity of sodium channels . J. Biol. Chem . 279 , 45004 – 45012 . doi: 10.1074/jbc.M407286200 OpenUrl Abstract / FREE Full Text ↵ Li , Z. , Jin , X. , Wu , T. , Zhao , X. , Wang , W. , Lei , J. , Pan , X. , Yan , N. , 2021 . Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome . Proc. Natl. Acad. Sci . 118 , e2100069118 . doi: 10.1073/pnas.2100069118 OpenUrl Abstract / FREE Full Text ↵ Mahling , R. , Rahlf , C.R. , Hansen , S.C. , Hayden , M.R. , Shea , M.A. , 2021 . Ca2+-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors . J. Biol. Chem . 296 , 100458 . doi: 10.1016/j.jbc.2021.100458 OpenUrl CrossRef ↵ Mangold , K.E. , Brumback , B.D. , Angsutararux , P. , Voelker , T.L. , Zhu , W. , Kang , P.W. , Moreno , J.D. , Silva , J.R. , 2017 . Mechanisms and models of cardiac sodium channel inactivation . Channels Austin Tex 11 , 517 – 533 . doi: 10.1080/19336950.2017.1369637 OpenUrl CrossRef PubMed ↵ Martinez-Moreno , R. , Selga , E. , Riuró , H. , Carreras , D. , Parnes , M. , Srinivasan , C. , Wangler , M.F. , Pérez , G.J. , Scornik , F.S. , Brugada , R. , 2020 . An SCN1B Variant Affects Both Cardiac-Type (NaV1.5) and Brain-Type (NaV1.1) Sodium Currents and Contributes to Complex Concomitant Brain and Cardiac Disorders . Front. Cell Dev. Biol . 8 , 528742 . doi: 10.3389/fcell.2020.528742 OpenUrl CrossRef PubMed ↵ Mizusawa , Y. , Wilde , A.A.M. , 2012 . Brugada Syndrome . Circ. Arrhythm. Electrophysiol . 5 , 606 – 616 . doi: 10.1161/CIRCEP.111.964577 OpenUrl FREE Full Text ↵ Moreau , A. , Gosselin-Badaroudine , P. , Boutjdir , M. , Chahine , M. , 2015 . Mutations in the Voltage Sensors of Domains I and II of Nav1.5 that are Associated with Arrhythmias and Dilated Cardiomyopathy Generate Gating Pore Currents . Front. Pharmacol . 6 , 301 . doi: 10.3389/fphar.2015.00301 OpenUrl CrossRef PubMed ↵ Moreau , A. , Krahn , A. , Gosselin-Badaroudine , P. , Klein , G. , Christé , G. , Vincent , Y. , Boutjdir , M. , Chahine , M. , 2013 . Sodium overload due to a persistent current that attenuates the arrhythmogenic potential of a novel LQT3 mutation . Front. Pharmacol . 4 . ↵ Moreno , J.D. , Zhu , W. , Mangold , K. , Chung , W. , Silva , J.R. , 2019 . A Molecularly Detailed NaV1.5 Model Reveals a New Class I Antiarrhythmic Target . JACC Basic Transl. Sci . 4 , 736 – 751 . doi: 10.1016/j.jacbts.2019.06.002 OpenUrl Abstract / FREE Full Text ↵ Musa , H. , Kline , C.F. , Sturm , A.C. , Murphy , N. , Adelman , S. , Wang , C. , Yan , H. , Johnson , B.L. , Csepe , T.A. , Kilic , A. , Higgins , R.S.D. , Janssen , P.M.L. , Fedorov , V.V. , Weiss , R. , Salazar , C. , Hund , T.J. , Pitt , G.S. , Mohler , P.J. , 2015 . SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia . Proc. Natl. Acad. Sci. U. S. A . 112 , 12528 – 12533 . doi: 10.1073/pnas.1516430112 OpenUrl Abstract / FREE Full Text ↵ Pablo , J.L. , Pitt , G.S. , 2016 . Fibroblast growth factor homologous factors (FHFs): new roles in neuronal health and disease . Neurosci. Rev. J. Bringing Neurobiol. Neurol. Psychiatry 22 , 19 – 25 . doi: 10.1177/1073858414562217 OpenUrl CrossRef PubMed ↵ Park , D.S. , Shekhar , A. , Marra , C. , Lin , X. , Vasquez , C. , Solinas , S. , Kelley , K. , Morley , G. , Goldfarb , M. , Fishman , G.I. , 2016 . Fhf2 gene deletion causes temperature-sensitive cardiac conduction failure . Nat. Commun . 7 , 12966 . doi: 10.1038/ncomms12966 OpenUrl CrossRef PubMed ↵ Potet , F. , Chagot , B. , Anghelescu , M. , Viswanathan , P.C. , Stepanovic , S.Z. , Kupershmidt , S. , Chazin , W.J. , Balser , J.R. , 2009 . Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin * . J. Biol. Chem . 284 , 8846 – 8854 . doi: 10.1074/jbc.M806871200 OpenUrl Abstract / FREE Full Text ↵ Rivolta , I. , Abriel , H. , Tateyama , M. , Liu , H. , Memmi , M. , Vardas , P. , Napolitano , C. , Priori , S.G. , Kass , R.S. , 2001 . Inherited Brugada and Long QT-3 Syndrome Mutations of a Single Residue of the Cardiac Sodium Channel Confer Distinct Channel and Clinical Phenotypes . J. Biol. Chem . 276 , 30623 – 30630 . doi: 10.1074/jbc.M104471200 OpenUrl Abstract / FREE Full Text ↵ Ruben , P.C. , Starkus , J.G. , Rayner , M.D. , 1992 . Steady-state availability of sodium channels . Interactions between activation and slow inactivation. Biophys. J . 61 , 941 – 955 . OpenUrl PubMed ↵ Rudokas , M.W. , Varga , Z. , Schubert , A.R. , Asaro , A.B. , Silva , J.R. , 2014 . The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry . JoVE J. Vis. Exp . e51040 . doi: 10.3791/51040 OpenUrl CrossRef ↵ Satou , Y. , Imai , K.S. , Satoh , N. , 2002 . Fgf genes in the basal chordate Ciona intestinalis . Dev. Genes Evol . 212 , 432 – 438 . doi: 10.1007/s00427-002-0266-8 OpenUrl CrossRef PubMed Web of Science ↵ Shamgar , L. , Ma , L. , Schmitt , N. , Haitin , Y. , Peretz , A. , Wiener , R. , Hirsch , J. , Pongs , O. , Attali , B. , 2006 . Calmodulin Is Essential for Cardiac IKS Channel Gating and Assembly: Impaired Function in Long-QT Mutations . Circ. Res . 98 , 1055 – 1063 . doi: 10.1161/01.RES.0000218979.40770.69 OpenUrl Abstract / FREE Full Text ↵ Shen , H. , Zhou , Q. , Pan , X. , Li , Z. , Wu , J. , Yan , N. , 2017 . Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution . Science 355 , eaal4326 . doi: 10.1126/science.aal4326 OpenUrl Abstract / FREE Full Text ↵ Silva , J.R. , Goldstein , S.A.N. , 2013a . Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: A periodic paralysis mutation in NaV1.4 (L689I) . J. Gen. Physiol . 141 , 323 – 334 . doi: 10.1085/jgp.201210910 OpenUrl Abstract / FREE Full Text Silva , J.R. , Goldstein , S.A.N. , 2013b . Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: Wild-type skeletal muscle NaV1.4 . J. Gen. Physiol . 141 , 309 – 321 . doi: 10.1085/jgp.201210909 OpenUrl Abstract / FREE Full Text ↵ Silva , J.R. , Goldstein , S.A.N. , 2013c . Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: A periodic paralysis mutation in NaV1.4 (L689I) . J. Gen. Physiol . 141 , 323 – 334 . doi: 10.1085/jgp.201210910 OpenUrl Abstract / FREE Full Text ↵ Tan , H.L. , Kupershmidt , S. , Zhang , R. , Stepanovic , S. , Roden , D.M. , Wilde , A.A.M. , Anderson , M.E. , Balser , J.R. , 2002 . A calcium sensor in the sodium channel modulates cardiac excitability . Nature 415 , 442 – 447 . doi: 10.1038/415442a OpenUrl CrossRef PubMed ↵ Varga , Z. , Zhu , W. , Schubert , A.R. , Pardieck , J.L. , Krumholz , A. , Hsu , E.J. , Zaydman , M.A. , Cui , J. , Silva , J.R. , 2015 . Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations . Circ. Arrhythm. Electrophysiol . 8 , 1228 – 1239 . doi: 10.1161/circep.115.003155 OpenUrl Abstract / FREE Full Text ↵ Veldkamp , M.W. , Viswanathan , P.C. , Bezzina , C. , Baartscheer , A. , Wilde , A.A.M. , Balser , J.R. , 2000 . Two Distinct Congenital Arrhythmias Evoked by a Multidysfunctional Na + Channel . Circ. Res . 86 . doi: 10.1161/01.RES.86.9.e91 OpenUrl Abstract / FREE Full Text ↵ Wang , C. , Chung , B.C. , Yan , H. , Lee , S.-Y. , Pitt , G.S. , 2012 . Crystal Structure of the Ternary Complex of a NaV C-Terminal Domain, a Fibroblast Growth Factor Homologous Factor, and Calmodulin . Structure 20 , 1167 – 1176 . doi: 10.1016/j.str.2012.05.001 OpenUrl CrossRef PubMed ↵ Wang , Chaojian , Wang , Chuan , Hoch , E.G. , Pitt , G.S. , 2011 . Identification of Novel Interaction Sites that Determine Specificity between Fibroblast Growth Factor Homologous Factors and Voltage-gated Sodium Channels* . J. Biol. Chem . 286 , 24253 – 24263 . doi: 10.1074/jbc.M111.245803 OpenUrl Abstract / FREE Full Text ↵ Wang , D.W. , Makita , N. , Kitabatake , A. , Balser , J.R. , George , A.L. , 2000 . Enhanced Na+ Channel Intermediate Inactivation in Brugada Syndrome . Circ. Res . 87 . doi: 10.1161/01.RES.87.8.e37 OpenUrl Abstract / FREE Full Text ↵ West , J.W. , Patton , D.E. , Scheuer , T. , Wang , Y. , Goldin , A.L. , Catterall , W.A. , 1992 . A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation . Proc. Natl. Acad. Sci. U. S. A . 89 , 10910 – 10914 . OpenUrl Abstract / FREE Full Text ↵ Wilde , A.A.M. , Amin , A.S. , 2018 . Clinical Spectrum of SCN5A Mutations . JACC Clin. Electrophysiol . 4 , 569 – 579 . doi: 10.1016/j.jacep.2018.03.006 OpenUrl Abstract / FREE Full Text ↵ Yan , H. , Wang , C. , Marx , S.O. , Pitt , G.S. , 2017 . Calmodulin limits pathogenic Na+ channel persistent current . J. Gen. Physiol . 149 , 277 – 293 . doi: 10.1085/jgp.201611721 OpenUrl Abstract / FREE Full Text ↵ Yu , F.H. , Westenbroek , R.E. , Silos-Santiago , I. , McCormick , K.A. , Lawson , D. , Ge , P. , Ferriera , H. , Lilly , J. , DiStefano , P.S. , Catterall , W.A. , Scheuer , T. , Curtis , R. , 2003 . Sodium Channel β4, a New Disulfide-Linked Auxiliary Subunit with Similarity to β2 . J. Neurosci . 23 , 7577 – 7585 . OpenUrl Abstract / FREE Full Text Zhu , W. , Voelker , T.L. , Varga , Z. , Schubert , A.R. , Nerbonne , J.M. , Silva , J.R. , 2017 . Mechanisms of noncovalent β subunit regulation of NaV channel gating . J. Gen. Physiol . 149 , 813 – 831 . doi: 10.1085/jgp.201711802 OpenUrl Abstract / FREE Full Text ↵ Zhu , W. , Wang , W. , Angsutararux , P. , Mellor , R.L. , Isom , L.L. , Nerbonne , J.M. , Silva , J.R. , 2021 . Modulation of the effects of Class-Ib antiarrhythmics on cardiac NaV1.5-encoded channels by accessory NaVβ subunits . JCI Insight . doi: 10.1172/jci.insight.143092 OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted January 03, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following FGF12A Regulates Nav1.5 via CaM-regulated and CaM-independent Mechanisms Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share FGF12A Regulates Nav1.5 via CaM-regulated and CaM-independent Mechanisms Lucy Woodbury , Paweorn Angsutararux , Martina Marras , Emily Wagner , Carlota Abella , Anna Li , Jonathan R. Silva bioRxiv 2025.01.02.631125; doi: https://doi.org/10.1101/2025.01.02.631125 Share This Article: Copy Citation Tools FGF12A Regulates Nav1.5 via CaM-regulated and CaM-independent Mechanisms Lucy Woodbury , Paweorn Angsutararux , Martina Marras , Emily Wagner , Carlota Abella , Anna Li , Jonathan R. Silva bioRxiv 2025.01.02.631125; doi: https://doi.org/10.1101/2025.01.02.631125 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biophysics Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17686) Bioengineering (13891) Bioinformatics (41932) Biophysics (21450) Cancer Biology (18587) Cell Biology (25498) Clinical Trials (138) Developmental Biology (13375) Ecology (19898) Epidemiology (2067) Evolutionary Biology (24311) Genetics (15608) Genomics (22502) Immunology (17736) Microbiology (40391) Molecular Biology (17177) Neuroscience (88589) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4823) Physiology (7641) Plant Biology (15150) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9823) Zoology (2271)