Dominant-negative Effect of MinK-A8V Impairs I Ks and Disturbs Channel Structure: Importance of Clinical Surveillance in Mild LQTS Patients and Asymptomatic Carriers

Dominant-negative Effect of MinK-A8V Impairs I Ks and Disturbs Channel Structure: Importance of Clinical Surveillance in Mild LQTS Patients and Asymptomatic Carriers | 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 Article Dominant-negative Effect of MinK-A8V Impairs I Ks and Disturbs Channel Structure: Importance of Clinical Surveillance in Mild LQTS Patients and Asymptomatic Carriers Jose G. Acuna-Ochoa, Janire Urrutia, Maribel Hernandez, Matthew B. Hillis, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9151984/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background. The slow delayed rectifier potassium current (I Ks ), formed by the assembly of the Kv7.1 ( KCNQ1 ) and MinK ( KCNE1 ) subunits, is essential for cardiac repolarization. Variants of either protein may disrupt this current and contribute to different types of Long QT Syndrome (LQTS), conditions that might lead to arrhythmias and sudden cardiac death (SCD). However, the clinical significance of certain KCNE1 mutations remains ambiguous, particularly in asymptomatic carriers. Genetic screening and proper functional characterization of such variants represent crucial steps for surveillance and clinical management. Results. Among the different LQTS-associated genes, a deleterious heterozygous mutation KCNE1 (c.23C > T)/ MinK-A8V was detected in an asymptomatic individual, which disturbs a well-conserved residue. Functional analysis in vitro demonstrated a pronounced reduction of I Ks when co-expressed in heterozygosity along with KCNQ1 , but the kinetics of activation of the I Ks did not change, suggesting defects in trafficking mechanisms. Consistently, prediction of the N-terminal portion of MinK and structure analysis of AlphaFold3-derived I Ks channels revealed less stability and structure confidence of the heterozygous form than in the WT and homozygous complexes. Conclusion. MinK-A8V evaluation demonstrated a dominant-negative effect over I Ks , which can lead to structural changes, a possible reduction in the stability of the channel complex and detrimental cardiac performance if present in an individual, depending on particular genetic, environmental or pharmacological backgrounds. Along with the results of other studies, it is suggestive that MinK-A8V variant may provoke defects in trafficking mechanisms of the I Ks channel. Altogether, the present study supports the notion that c.23C > T in KCNE1 may act as modifier allele, underlying the importance of surveillance and/or clinical management of asymptomatic individuals and mild LQTS patients when carrying a KCNE1 variant. Health sciences/Cardiology Health sciences/Diseases Biological sciences/Genetics IKs KCNE1 MinK channelopathies LQTS arrhythmias 3D Modeling AlphaFold3 AI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 BACKGROUND The delayed rectifier potassium current (I K ) is a crucial outward potassium current that plays a major role in repolarizing the membrane of cardiomyocytes, contributing to regular heart contraction in humans. This repolarization phase is integrated by several components, including the rapid delayed rectifier current (I Kr ) and the slow delayed rectifier current (I Ks ), which are distinct in their activation kinetics and pharmacological properties. To generate human cardiac I Ks, the assembly of four pore-forming Kv7.1 α-subunits (encoded by KCNQ1 ) and MinK β-subunits (encoded by KCNE1 ) is necessary. MinK can regulate this voltage-gated potassium channel by slowing its activation and regulating its conductance [ 1 ]. Mutations in the above-mentioned genes can impact the regular performance of the I Ks channel either directly through changes in stability and allosteric mechanisms, decreasing its abundance in the cellular membrane or altering its pharmacological properties, causing different types of Long QT Syndrome (LQTS) [ 2 ]. This syndrome is an inherited cardiac condition in which the QT intervals in the electrocardiogram (ECG) of an individual are prolonged, predisposing them to life-threatening arrhythmias that can lead to sudden cardiac death (SCD). In this sense, the detection of genetic variants that may impair I Ks as well as the determination of their functional/physiological implications may be beneficial for LQTS patient care and preventive follow-up of asymptomatic carriers. Determining the structure of ion channels has been crucial in understanding their properties and their role in channelopathies. The recent development of sophisticated Artificial Intelligence (AI) operated platforms for the structure prediction of proteins with biological importance represents an improved tool for the assessment of genetic variants. AlphaFold3 represents perhaps one of the most famous models of its kind, exhibiting markedly enhanced accuracy compared with several specialized docking tools [ 3 ]. Indeed, several investigations across diverse disease contexts have employed AI resources in an effort of assessing the impact of variants over 3D protein models, some of them showing a good correlation with the observed functional data [ 4 ]. The sum of these arguments prompted us to investigate whether a given KCNE1 variant could influence the normal features of the I Ks in vitro and in silico in the context of healthy carriers. RESULTS Molecular and comparative analysis revealed a single missense mutation among principal LQTS-related genes, disturbing a well-conserved residue at the N-terminus of MinK Genetic screening of the proband did not identify mutations within principal genes related to LQTS onset ( KCNQ1 , KCNH2 and SCN5A ), but a single heterozygous mutation in KCNE1 . Unfortunately, clinical or familial information of the subject was not available when the study was carried out. Chromatographic results revealed one single mutation consisting of a C to T transition at nucleotide 23 of KCNE1 (c.23 C > T, ALFA, genomAD and ExAC MAF = 0.0001) (Fig. 1 A), which leads to the substitution of alanine at position eight with valine in the N-terminal α-helix of the MinK β-subunit (Fig. 1 B, center). In turn, MinK subunits are colocalized in the outer pore space between two Kv7.1 subunits (Fig. 1 B left), close to the voltage-sensing domain (VSD) (Fig. 1 B, right). Comparative analysis of orthologous amino acid sequences revealed a high degree of preservation of that residue across species (Fig. 1 C, top), which is not the case for other human KCNE family members (Fig. 1 C, bottom). Moreover, in silico simulations of the WT and A8V structures indicate this shift provokes the formation of new hydrogen bonds (Val8—Ser4, Val8—Asn5), a possible steric clash (Val8—Pro11) and the loss of hydrogen bonding (Ala8—Phe12) (Fig. 1 D). Panel B of Fig. 1 shows different views of a representative model of WT IK s complex and the configuration of its components, originating from the present research. The MinK N-terminal α-helix is anchored to the cell membrane within the WT I Ks models After removing disorganized regions (amino acids 1–89, 404–676 from Kv7.1 and 115–129 from MinK, specified in additional file 1, Table 1), we successfully generated three-dimensional models of the three genotype groups of the I Ks channel with respect to the MinK-A8V mutation using AlphaFold3 (representative structures in additional files 2, 3 and 4). Validation of our WT models revealed mild shifts in terms of the Root Mean Square Deviation (RMSD) values (Cui et al. model, M = 1.471 ± 0.0274 Å, n = 20; Zhong et al .l model, M = 2.824 ± 0.0273 Å, n = 20) (Fig. 2 A) when comparing to the recently partially solved cryo-EM structures of the I Ks channel, indicating a moderately high structural level of similarity suitable for downstream comparative analysis. The MinK N-terminus α-helix was most frequently situated parallel to its TMD in our I Ks channel model in the closed state. After the structures were uploaded to the Swiss Model server, the cell membrane was built in the most likely coordinates with respect to the I Ks model (Fig. 2 B). The latter is supported by an increased density of hydrophobic residues falling within the membrane hydrophobic interface (Fig. 2 C). A comparison of different WT MinK protein extracted from different I Ks channel models showed the N-terminal α-helix of MinK to be anchored in the cell membrane to some extent but in different orientations, while the TMD position is highly conserved between seeds (Fig. 2 D). This embedding correlates with the visual representation of the hydrophobic/hydrophilic residues throughout independent modeling of different single-span MinK seeds, where the amphipathic nature of the N-terminal α-helix is uncovered (Fig. 2 E), allowing it to interact with the cell membrane. The A8V mutation in MinK provokes a strong dominant-negative effect on I Similar trends were observed in I Ks activation traces, the mean current density (I dev ) and the mean tail current density (I tail ) from the three experimental systems: cells expressing both WT KCNQ1 and WT KCNE1 displayed standard behavior (I Ks Fig. 3 A left, I dev Fig. 3 B, I tail Fig. 3 C. n = 16). This was contrasting with the heterozygous expression of the WT and A8V mutation, which presented a significant reduction in channel current (I Ks Fig. 3 A central-left, I dev Fig. 3 B, I tail Fig. 3 C, n = 9). Meanwhile, homozygous expression of the MinK-A8V mutant produced a modest reduction of the current (I Ks Fig. 3 A central-right, I dev Fig. 3 B, I tail Fig. 3 C, n = 7). Expression of the Kv7.1 subunit alone showed a well-known activation current behavior without the modulation of MinK (Fig. 3 A right). Statistical significance of I dev and I tail were reached only between WT and the heterozygous expression groups. Homozygous expression of the variant did not show a statistically significant shift of currents. Finally, Fig. 3 D depicts the behaviors of the normalized curves of the tail currents as the mean V h values. Normalized tail currents exhibit mild hyperpolarization in the WT/A8V heterozygous model (V h = 6.41 ± 3.01, k = 14.61 ± 1.42, n = 9), whereas the homozygous KCNE1- A8V variant is slightly more depolarized (V h =13.39 ± 3.05, k = 17.47 ± 2.38, n = 7) than the WT control values (V h = 10.96 ± 1.65, k = 14.52 ± 1.34, n = 16). Nonetheless, no significant differences between groups were found. Overall, the MinK-A8V mutant appears to significantly impact the function of the WT channel when expressed in heterozygosity. Structural stability of the AI-generated I Ks channel models correlates with the dominant-negative effect of the MinK-A8V mutation observed in electrophysiological results According to color coded per-atom confidence estimate analysis (pLDDT), the TMD portion of MinK presents changes in structural confidence of both the mutated and wild-type subunits when the variant A8V is expressed in heterozygosis (Fig. 4 A). The statistical analysis of mean (M) pLDDT values shows significant differences between WT and heterozygous groups ( p < 0.001) when considering the whole structure of the I Ks complex, but also when evaluating the Kv7.1 and MinK subunits by separate (Fig. 4 B): I Ks channel (left) (M = 68.8 ± 0.155 Vs. M = 64.6 ± 0.124, n = 20), Kv7.1 (center) (Median = 74.2 Vs. Median = 71.6, n = 20) and MinK subunits (right) (M = 54 ± 0.314 Vs. M = 46 ± 0.14, n = 20). On the other hand, structural alignments between the models derived from the same seed showed greater deviations in RMSD values between the WT/heterozygous I Ks channel groups (M = 0.593 ± 0.059 Å, n = 20) than between the WT/homozygous (M = 0.456 ± 0.050 Å, n = 20) and the heterozygous/homozygous groups (M = 0.594 ± 0.055 Å, n = 20) (Fig. 4 C), although no significant differences were found. In parallel, the Predicted Aligned Error (PAE) matrices depict differences in the expected position error, which is more noticeable between zones contemplating MinK subunits across the different zygosity states (Fig. 5 A). Accordingly, quantitative analysis of average PAE values revealed significant differences ( p < 0.003) between WT and A8V- heterozygous I Ks channel models (left) (M = 18.1 ± 0.104 Å Vs. M = 19.8 ± 0.035 Å, n = 20), but also on Kv7.1 subunits (center) (M = 14.5 ± 0.07 Å Vs. M = 15.1 ± 0.05351 Å, n = 20) and MinK subunits (right) (M = 22.3 ± 0.15 Å Vs. M = 25.2 ± 0.03 Å, n = 20) (Fig. 5 B). In the same manner, differences between the heterozygous and homozygous models were observed with respect to the full structure of the I Ks channel (left) (M = 19.8 ± 0.035 Å Vs. M = 18.3 ± 0.11 Å, n = 20) and Kv7.1 alone (center) (M = 15.1 ± 0.053 Å Vs. M = 14.7 ± 0.085 Å, n = 20). Following the same pattern, WT I Ks channels were thermodynamically more stable than the heterozygous complexes (M= -273 ± 11.245 Kcal/mol Vs. M= -238 ± 8.76 Kcal/mol, n = 20), as well as more reliable according to predicted TM-score (PTM) values (Median = 0.73 Vs. Median = 0.69, n = 20) (Fig. 5 C). Similarly, the total energy of the Kv7.1/MinK interaction increased upon the A8V heterozygous mutation (M= -315 ± 6.3 Kcal/mol Vs. M= -282 ± 7.35 Kcal/mol, n = 20), inversely aligning with lower interface Predicted Template Modelling (ipTM) values (M = 0.714 ± 0.003 Vs. M = 0.665 ± 0.001, n = 20) (Fig. 5 D), which indicated a decreased reliability of inter-subunit interactions. As expected, the average decrease in both the global free energy and interaction ΔG was greater when expressing the heterozygous MinK-A8V variant than in the homozygous state (M = 34.84 ± 12.83 Kcal/mol Vs. M = 9.73 ± 10.37 Kcal/mol, n = 20), correlating with ΔΔG of interaction between Kv7.1/MinK (M= -32.665 ± 7.65 Kcal/mol Vs. M= -2.55 ± 8.9 Kcal/mol, n = 20) (Fig. 5 E). Altogether, these results suggest a possible change in the stability of the complex and a partial loss of Kv7.1/MinK interaction. DISCUSSION Our research found a rare missense mutation in KCNE1 (c.23 C > T) that was originally linked to an LQTS case [ 5 ]. Controversially, we found a carrier of the same mutation in a control individual without cardiac malfunctions, raising questions about the penetrance of the variant and the mechanisms behind a LQTS phenotype when a mutant KCNE1 allele is present. In fact, several studies support the idea that a single mutation in KCNE1 is insufficient to drive disease presentation in most carriers, notwithstanding, such individuals may carry a hidden risk for developing an LQTS type 5 phenotype that may be unmasked by environmental or physiological triggers [ 6 , 7 , 8 , 9 , 10 ]. In this sense, this variant has multiple entries in ClinVar, some of them supporting its probable pathogenicity through different approaches. A study showed a clear co-segregation of the MinK A8V variant along with a LQTS phenotype [ 11 ]. On the other hand, Itoh et al . included in their study cohort a carrier of the same variant who developed a LQTS phenotype following accidental exposure to an arrhythmia-triggering factor (not specified), condition that remained active despite the elimination of trigger [ 12 ]. Giving these scenarios, it seems that this specific variant represents a genetic component that can be influenced by environmental or physiological factors, but little is known about the specific targeted cellular mechanisms. This reinforces the necessity of clarifying how some mutations in its component proteins can influence the behavior of the I Ks channel. KCNE1 has been found to influence I Kr channel responses to premature stimulation, pharmacological inhibition, pH sensitivity and pharmacological properties of Kv7.1 [ 13 , 14 , 15 , 16 ]. In the electrophysiological field, two main studies have assessed the influence of the MinK-A8V variant over I Ks [ 5 , 17 ]. Although different expression models were used, our results align with the other two studies in terms of I Ks not showing a significant change in the homozygous co-expression of MinK-A8V with KCNQ1 (Fig. 3 A, B). Instead, Ohno et al. 2007 found a marked suppression of I Kr when the mutant was co-expressed with KCNH2 [ 5 ]. However, none of these studies explored the actual heterozygous genotype found in the patient and therefore the influence of possibly different interactions and triggering mechanisms when WT and mutated proteins coexist in the plasma membrane. In our study, a statistically significant reduction in I Ks was evidenced by the heterozygous expression of the mutant (Fig. 3 B, C), suggesting that a disease phenotype could be caused not only by defects in I Kr but also by defects in I Ks . These findings constitute a clear example of a dominant-negative effect in channelopathies, which highlights the importance of mimicking a heterozygous genotype in vitro , representing a more reliable and close-to-reality approach in the study of channelopathies. Genetic variants related to LQTS are often loss-of-function mutations that reduce the density of the current in a dominant-negative manner either by changing the activation kinetics of the channels, changing channel conductance or trafficking defects that decrease channel abundance in the cell membrane [ 18 , 19 , 20 ]. Our data showed a reduction in both development and tail currents, with the heterozygous form being more severe than the homozygous mutant (Fig. 3 B, C). Nevertheless, the V h of activation was not significantly different between groups (Fig. 3 D), suggesting that the variant had no influence on the kinetics of the channel. This last led us to strongly hypothesize there could be less available functional channels in the cells. In this matter, we found a recent study showing a mild disruption of cell surface expression of the KCNE1 protein when the A8V variant is expressed in homozygosity [ 21 ], which supports and correlates with our hypothesis. Furthermore, a non-significant functional reduction was observed through cell fitness assay, matching our electrophysiological results from the homozygous state. Interestingly, another research found the LQTS causing T7I MinK mutation —a neighbor residue of alanine 8— and surrounding N5I/N5Q/T6P induced mutations, to disrupt normal glycosylation mechanisms that ultimately lead to decreasing the number of functional I Ks channel complexes [ 22 ]. At our end, we showed how a change from alanine to a larger valine at position eight may form new hydrogen bonds that could bend the α-helix, increase hydrophobicity, provoke steric hindrance and affect local flexibility. Given the spatial proximity of alanine eight to these last aminoacids, we speculate that a larger valine may also reduce the accessibility for enzymatic glycosylation at this site, reducing the macroscopic current in our cellular model. Unfortunately, we did not perform any trafficking assay to properly infer the precise underlaying mechanism that could corroborate our hypotheses. There are a few other reports that functionally evaluate N-terminal MinK mutations under a clinical context. A publication demonstrated that arginines in positions 32, 33 and 36 of MinK are essential for the stabilizing interaction of the N-terminal α-helix with the plasma membrane [ 19 ]. A recent robust study evaluating clinically relevant KCNE1 variants revealed that nearly half of mutations in the N-terminal portion could cause variable degrees of loss of function of I Ks [ 17 ]. While other variants did not have any functional effect in their homozygous state, that does not mean they are not pathogenic when present in heterozygosis or trigger a LQTS phenotype after an environmental exposure. Although KCNE family members have divergences, the general topology within the cell membrane among this group of proteins is highly similar. Analogously, genetic variants in the N-terminal section of KCNE2 (T8A, Q9E, T10M) have been associated with the spontaneous phenotypic development of LQTS in patients after taking certain medications, stress exposure or electrolyte imbalances [ 23 , 24 , 25 ]. These are clear examples of conferred genetic risk, which reinforces the idea that mutations in potassium channels may remain clinically unrecognized until they are unmasked by physiological or environmental stressors. Detecting these kinds of variants should be important in the clinical setting in order to avoid unintentional disease induction. Recently, high-resolution structure of the native I Ks channel has been obtained by two research groups using cryo-EM [ 26 , 27 ]. However, none of these include the N- and C-terminus of MinK since they are disorganized/dynamic areas that are hard to stabilize in laboratory procedures, precluding the modelling of mutations in those regions, as is the case of this study. Thus, we employed the newly upgraded AI-based computational method AlphaFold3 [ 3 ] as an effort to obtain a close-to-reality 3D conformation of the missing I Ks channel region of interest (residues 1 to ~ 40). Our model displays a highly similar topology to the cryo-EM structures of Kv7.1/MinK, displaying the distinctive single span transmembrane domain (TMD) of MinK flanked by intra- and extracellular α-helices joined by flexible linkers [ 28 ]. The extracellular N-terminus of MinK has been determined to play an important role in the stabilization and gating of the I Ks channel [ 28 , 29 ], in which changes in spatial orientation and local flexibility could alter the stability of the complex. The full NMR structure of MinK was determined by Kang et al. 2008 through the isolation of the molecule in micelles [ 28 ]. In these, the MinK N-terminus α-helix was observed resting on the surface of the micelle, highlighting an amphipathic nature. This is extremely consistent with our AI-generated models where we noted the same MinK portion being partially embedded in the membrane (Fig. 2 ) and may be a reason for its stabilization attributes. Indeed, NMR relaxation studies and electron paramagnetic resonance spectroscopy support the interaction of the MinK N-terminal with the plasma membrane [ 30 , 31 ], serving as an anchor-like component that is nearly as fixed as its TMD. However, these last two domains are connected through a mobile and flexible structure situated in the extracellular side of the membrane upon assembly with Kv7.1, facilitating dynamic rearrangements that could explain the different orientations of the N-terminal α-helix across our different seed models. Thus, the constructed N-terminal α-helix of MinK in our 3D models could be displaying one transition between order and disorder as part of the high dynamism of the I Ks channel. Nevertheless, the pLDDT values in that location of our models were predicted to be low (< 70) (Fig. 4 A), which may reflect either an unstable structure and/or greater chances of spurious tridimensional conformation, in which latter case this model should be interpreted conservatively. Nonetheless, the conjunction of all previous experimental data here reviewed supports the reliability of our model, which may make it a suitable approach for variant effect assessment of the I Ks channel. The confidence metrics calculated by AlphaFold3 served as a measure of the accuracy of the models [ 32 ], as well as variables subject to this study to evaluate the effect over the I Ks channel when the MinK-A8V mutation is expressed. pLDDT scores indicated greater structural confidence in the I Ks channel zones that are buried within the plasma membrane, which is not surprising because they constitute the least mobile portion of the protein. In contrast, pLDDT values were markedly lower when the MinK-A8V mutation was expressed in heterozygosity, suggesting a reduced confidence in the structural accuracy of the complete I Ks structure, Kv7.1 and MinK itself, potentially reflecting an increase in conformational flexibility, destabilization or changes in dynamic behavior. Specifically, we observed the TMD of MinK with lower pLDDT scores only at the heterozygous I Ks (Fig. 4 A). This is of special importance given that the TMD is a structurally characterized region that plays a central role in I Ks channel gating and activation [ 28 , 33 ], in which stability and position changes may affect its interaction within the I Ks channel and lead to channel performance intermittency, and may justify the mild hyperpolarization of the activation. On the other hand, the predicted pTM score reflects the confidence in the overall topology of a predicted structure. On this basis, higher pTM values may indicate better domain packing and structural stability of protein complexes, which is a fundamental principle under physiological conditions. Accordingly, we used pTM scores as an indirect measure of conformational stability across different I Ks channel models, and in conjunction with ΔG calculations, we observed less stability of the complexes and their members when the MinK-A8V mutation was expressed in heterozygosity. Likewise, the ipTM score is designed to reflect the confidence in relative subunit positioning within multimeric assemblies, and we interpreted it as a proxy for the likelihood and stability of subunit–subunit interactions. A drop in iPTM reflected a loss of confidence in the interaction between subunits, suggesting dissociation, destabilization or steric interference. Based on these findings and calculations of interaction ΔG, we observed a major general decrease in subunit interaction when the MinK-A8V mutation was expressed in heterozygosity. Finally, PAE values report the expected positional error at residue i when the model is aligned at residue j , thereby allowing the estimation of structural certainty between different regions or chains of the model. Indeed, it seems there is a correlation between two residues in different entities that present low PAE values and their probability of interaction. On this basis, we detected an increased position error in I Ks complexes and their subunits when the heterozygous MinK-A8V mutation was present, which may represent a general loss of the structural coupling and normal subunit proximity. Taking this data together and given the lack of significant differences in RMSD values between the WT, heterozygous and homozygous I Ks models (Fig. 4 C), the functional impairments observed in electrophysiological experiments in the heterozygous context suggest that the underlying mechanism may not specifically be due to shifts in the structure at atomic level. Rather, it may be related to altered symmetry or subunit arrangement within the I Ks complex, potentially disrupting subunit interaction. The heterozygous expression of a mutated MinK has also implications in the stoichiometry of the I Ks channel components, which is an important variable to consider when measuring ion currents through patch clamp because the expression of MinK regulates Kv7.1 and vice versa [ 19 ]. It seems that, depending on their concentrations, the native number of MinK subunits assembled with tetrameric Kv7.1 channels can range from one to four, with different stoichiometries having distinct gating properties [ 34 , 35 , 36 , 37 ]. In the present study we used the same expression vector backbones to achieve equal expressions and facilitate a 4:4 stoichiometry of MinK/Kv7.1, which shows a major influence over I Ks compared to other ratios. In this sense, the degree of reduction of current density we observed in our electrophysiological data could be more pronounced than in real cardiac tissue. The use of a cellular model in this work may introduce a different genetic background that influences the molecular interactions/mechanisms and may not recapitulate what happens in a native environment, thus the clinical impact may be widely variable and we should not directly extrapolate these results directly, especially because each individual may be exposed to different environmental factors. Nonetheless, we speculate that one of the reasons that an individual carrying this type of variants could suppress the development of the disease phenotype is by facilitating other stoichiometries and allele expression as compensatory mechanisms. Likewise, other genetic or epigenetic regulators may be involved. Further studies in hiPSC-derived cardiomyocytes or in vivo models are warranted to explore more accurately the clinical implications of such mutations in asymptomatic individuals and their differential phenotypes. Similarly, additional experiments providing biochemical (e.g., surface biotinylation, western blot and co-immunoprecipitation) or imaging data may concisely corroborate our observations in the future. In the present work we found that a mutation in KCNE1 may reduce I Ks by making the channel less stable, combined with presumed defects in MinK trafficking that were previously reported [ 21 , 22 ]. On this basis, our findings underline the clinical importance of detecting a KCNE1 mutation in asymptomatic individuals or mild LQTS patients, which should be then followed by appropriate additional genetic testing, careful revision of family clinical history, changes of life style and medications as needed in order to avoid known arrhythmic triggers (potential QT-prolonging drugs, electrolyte imbalances, bradycardia or stress), as well as revision of environmental expositions over time by an specialized physician. All this could lead to personalized management and therefore, an improvement in the clinical outcome. Overall, our study suggests considering the A8V mutation in MinK as modifier allele. It is worth noting considering that several limitations come in the present work that we fully recognize. The accurate modeling of the true conformation of the MinK N-terminus and other portions is still uncertain. We were not able to simulate the entire sequence of the I Ks channel due to the formation of disorganized structures and we are aware that AlphaFold3 may build structural hallucinations in disordered regions. Yet, our model fits well according to functional and structural data in this and other mentioned studies. Besides, our model only reflects static conformations, and even multiple random seed runs may not recapitulate the real dynamic behavior of biomolecular systems, as stated by AlphaFold3 developers. Nonetheless, we reinforce that this model represents a good resource to evaluate the effect of mutations within the N-terminal portion of MinK, which experimental conformation within the native I Ks channel remains unresolved. Notably, we are missing the evaluation of the studied mutation within the active/open state of the pore, as our AI-generated 3D structure represents only the closed state of the I Ks channel and we were not able to perform protein‒protein docking at this time. Unfortunately, neither were we able to control the localization of the mutant MinK chains in the generated models, so we could not decide the symmetry of the complex. Finally, we did not evaluate scenarios where only two or three MinK subunits co-assemble in the four Kv7.1 subunits, and the different combinations of mutants/WT coexisting. We hope that future advances in protein structure determination may contribute to resolve the true conformation of the remaining undetermined I Ks channel portions and thus, a better understanding of 3D structures upon mutations. In the meantime, future implementations of Molecular Dynamics (MD) of the resulting AI structures could help to refine our I Ks channel models, with the inclusion of the cellular membrane and calmodulin as active interacting components, as well as phosphatidylinositol 4,5-bisphosphate (PIP2) as an activating cofactor for channel opening, which are elements that would contribute to a more realistic system. CONCLUSIONS In this study, we demonstrate that the MinK-A8V variant exerts a dominant-negative effect on I Ks when co-expressed with Kv7.1, leading to a significant reduction in both development and tail current densities in vitro . Importantly, the activation kinetics were similar across groups, indicating that the reduction in current is unlikely due to altered gating. The conjunction of our results with mechanistic data from other studies suggest that the MinK-A8V mutation may induce trafficking defects that reduce the abundance of functional I Ks channel in the plasma membrane. A pronounced functional impairment could be aggravated only when the mutant and WT MinK proteins coexist in the I Ks channel subunits (heterozygosis), which is remarked by differential confidence and stability metrics of the I Ks channel 3D models here presented, disrupting channel structure, energetic stability, assembly, symmetry and function in an individual, when additional environmental conditions are present. The modeling of the MinK N-terminal α-helix structure and its probable interaction with the cell membrane was particularly important, as it recapitulates its amphipathic and flexibility features which could be a key property within the I Ks complex. This study may represent a clear example of how the interpretation electrophysiological data in the light of the structural composition of ion channels is crucial and imperative in order to have a clearer picture of functionality effects upon mutations. Despite the demonstrated functional deficit, the MinK-A8V variant was identified in a healthy individual, supporting the notion of representing a modifier allele rather than a fully pathogenic mutation. Nonetheless, the complex significance of KCNE1 mutations in asymptomatic individuals with or without a family history of LQTS that we describe emphasizes the importance of genetic screening and appropriate functional evaluations of these variants, which may contribute to improved clinical outcomes. Overall, these findings also underscore the importance of recapitulating the original in vivo heterozygous genotypes when translated into in vitro experiments, which could lead to a different physiologically relevant approach in the functional evaluation of ion channel variants expressed in humans. METHODS Genetic analysis. All experimental procedures were performed in accordance with the 1964 Declaration of Helsinki and after approval of the Main Line Hospitals Institutional Review Board (MLH IRB, approval number E-22-5283). Informed consent was obtained to collect a blood sample from the proband. Genomic DNA was extracted from peripheral blood leucocytes with a commercial kit (Puregene, Gentra Systems, Inc., Minneapolis, MN). The DNA was amplified by PCR on the GeneAmp ® PCR System 9700 (Applied Biosystems, Foster City, CA). All exons and intron borders of the following LQTS susceptibility genes were amplified and analyzed by direct sequencing: KCNQ1, KCNH2, KCNE1, KCNE2 and SCN5A . PCR products were purified with a commercial reagent (ExoSAP-IT, USB, Cleveland, OH) and directly sequenced from both directions using the ABI PRISM 3100 Automatic DNA sequencer (Applied Biosystems, Foster City, CA). Electropherograms were visually examined for heterozygous peaks and compared with reference sequences for homozygous variations employing CodonCode Aligner Ver. 2.0.4 (CodonCode Corporation, Dedham, MA). Site-directed mutagenesis and cell transfection. Site-directed mutagenesis was performed with QuikChange (Stratagene, La Jolla, CA) on full-length human KCNE1- WT to introduce the c.23C > T mutation (A8V amino acid change). The KCNQ1- WT, KCNE1- WT and KCNE1- A8V cDNA sequences were subsequently cloned by separate in a pcDNA3.1 vector. The KCNE1 -A8V plasmid was sequenced to ensure the presence of the mutation without spurious substitutions. I Ks channels were expressed in the modified human embryonic kidney (HEK) cell line TSA201 (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol for transient transfection using Fugene6 (Roche Diagnostics, Indianapolis, IN). Briefly, constructs of KCNQ1- WT and KCNE1- WT or/and KCNE1- A8V were employed at a ratio of 1µg:1µg for homozygous expression and 1µg: 0.5µg :0.5µg to mimic the heterozygous form of the mutation. GFP cDNA was co-transfected as well as reporter gene to visually identify transfected cells. After 24h of incubation, cells were split into polylysine-coated 35 mm culture dishes (Cell+, Sarstedt, Newton, NC) at approximately 10% confluence. Forty-eight to 72 hours after transfection, fluorescent cells were used for patch-clamp studies. The conditions of cell maintenance included the use of GIBCO Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and penicillin [100 U/mL]/streptomycin [100 µg/mL], and incubation in a 5% CO 2 chamber at 37°C. This procedure was performed four different times for each experimental group. Electrophysiological studies. Membrane currents were measured using whole-cell patch-clamp technique to at least five cells per transfection, per group. All recordings were obtained at body temperature (36–37°C) using an Axopatch 200B amplifier equipped with a CV-201A head stage (Axon Instruments. Union 5 City, CA). Macroscopic whole-cell K + current was recorded from cells bathed in solution containing the following concentration (mmol/L): 132 NaCl, 4.8 KCl, 2 CaCl 2 , 1.2 MgCl 2 , 10 HEPES and 10 glucose (pH 7.4). Patch pipettes were pulled from borosilicate glass (Model PP-89, Narashige, Tokyo, Japan), obtaining a resistance of 2–3 MΩ when filled with a solution containing (mmol/L): 110 K-Aspartate, 1 MgCl 2 , 11 EGTA, 5 MgATP and 10 HEPES (pH 7.35). Currents were elicited by depolarizing pulses from a holding potential of -80 mV to test potentials between − 40mV and + 80 mV with a 10 mV step increment, followed by repolarization to -40 mV to measure the tail current amplitude. Current densities (pA/pF) were calculated by normalizing current amplitude to cell capacitance. Tail amplitudes were normalized to maximum amplitude in order to calculate the activation curve and fitted to Boltzmann’s function to calculate the voltage of half activation (V h ). Currents were digitized at 5 kHz. Generation and assessment of three-dimensional I Ks model groups To distinguish disorganized from well-structured reliable portions of the I Ks channel, we first performed a screening run with the full sequence of four Kv7.1 and four MinK subunits (NCBI Ref. Seq. NP_000209.2 and NP_000210.2, respectively) on the official AlphaFold3 server (DeepMind/Isomorphic Labs), adjusted to find PDB templates to the most recent possible date (02/02/2025). Additionally, this method was performed separately for a single-span MinK subunit. Visually unstructured regions (lose ribbon-like structures with very low pLDDT values that are usually removed in computationally generated models to avoid undesirable noise and inaccurate data) flanking the N- and C- terminus were removed as possible, preserving the central functional topology of the channel according to the available experimental structure data. To ensure statistical representation, twenty different seeds were used for each I Ks genotype group (see additional file 1), prompting a 4:4 stoichiometry and ensuring that the heterozygous form expressed two WT and two A8V MinK subunits. Each run generated five predictions, and the top ranked model from each seed was selected and added to the dataset for analysis. Generated structural predictions are subject to the AlphaFold Server Output Terms of Use. Quality control metrics such as pLDDT, pTM, ipTM and PAE were analyzed to assess the confidence of each model and treated as indicator variables of protein stability and subunit proximity. 3D models were uploaded to the SwissModel server via the user template approach to obtain MolProbity parameters as an additional layer of quality control. Simultaneously, .cif files were opened in YASARA View version 25.1.13 (YASARA Biosciences GmbH, Radboud University, Nijmegen, The Netherlands) [ 38 ], and the recently updated FoldX 5.1 plugin (Centre for Genomic Regulation, Barcelona, Spain) was used to optimize protein structure, calculate thermodynamic stability (ΔG) and interaction energy between Kv7.1/MinK subunits [ 39 , 40 ]. Then we compared our WT models against the two available experimental structures of the I Ks channel (PDB ID 9u7f and 9vec) as an approach of validation [ 26 , 27 ]. This comparison was performed by aligning the structures in PyMOL version 3.1.6.1 (The PyMOL Molecular Graphics System, Schrödinger LLC., New York) [ 41 ] and obtaining Root Mean Square Deviation values (RMSD) considering only the sequence in common (cycles = 5, WT/WT cutoff=2Å and WT/mutant cutoff = 1.5Å). The same strategy was adopted for comparison with the mutant groups, using our WT model as the reference. Finally, local structural changes were examined using Dynamut2 web server (Institute, University of Melbourne, Melbourne, Victoria, Australia) [ 42 ]. The model with the closest ΔG to the median value within the WT group and its mutant versions was used to obtain representative images/diagrams. Please refer to additional file 1, Table 2 for the complete dataset of evaluated quantitative parameters. Data and statistical analysis Electrophysiologic data acquisition and analysis were performed using the suite of pCLAMP programs 9.2 (Axon Instruments, Union City, CA), Excel (Microsoft Corp., Redmond, WA) and OriginPro, version 2025 (OriginLab Corporation, Northampton, MA). Two-way ANOVA, Student's t -test or Mann-Whitney Rank Sum test was performed employing SigmaStat 3.5 statistical software (Systat Software, Inc., Chicago, IL). Comparisons of structural and stability metrics were performed applying paired t -test or Wilcoxon Signed-Rank test. The summary of statistical analysis is available in additional file 1, tabs 3 and 4. Declarations Ethics approval and consent to participate The procedures performed in this study involving human participants were conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. All the patients were allowed personal data processing and informed consent was obtained from the participants before proceeding to any methodology. All the experimental protocols in this study were reviewed and approved by the Main Line Hospitals Institutional Review Board (MLH IRB), with approval number E-22-5283. Consent for publication Not applicable. Funding This work was supported by the W.W. Smith Charitable Trust (HBM, JGAO, MBH, MH: H2205), Women’s Board from Lankenau Medical Center (HBM, JGAO, MBH, MH: 25401, 4101) and the Sharpe-Strumia Research Foundation of the Bryn Mawr Hospital (HBM, JGAO, MH, AWA: SSRF2025-07, SSRF2024-06). Author Contribution HBM led the study. HBM, JU, DJ, and JGAO conceived and designed the research. HBM, JU, AWA and JGAO analyzed and interpreted the data. JU, EB, GC and RP performed the experiments. HBM, JU, AWA and JGAO drafted the manuscript and substantively reviewed it. MH and MBH contributed to patient selection. All authors reviewed and approved the definitive version of the manuscript for submission to BMC Biology. Every author validated their own contribution. Acknowledgement We thank Terri Olshefski for her valuable job in the proof-reading of the manuscript. Data Availability The report of the presented variant is available in ClinVar (accession number SCV007542593). Representative models of the generated 3D structures were deposited in ModelArchive (https://modelarchive.org/) with the following accession IDs: WT: ma-2l8hk; homozygous: ma-eru9d; heterozygous: ma-a3ecv. Raw data and other information are available upon request.All AlphaFold 3 structural predictions reported here were obtained via the official AlphaFold Server (DeepMind/Isomorphic Labs) and are provided under, and subject to, the AlphaFold Server Output Terms of Use. Minor modifications to the output, including energy minimization and visualization coloring, were applied prior to publication. References Sanguinetti, M. C. et al. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKs potassium channel. Nature 384 (6604), 80–83. 10.1038/384080a0 (1996). Brewer, K. R. et al. Structures Illuminate Cardiac Ion Channel Functions in Health and in Long QT Syndrome. Front. Pharmacol. 11 , 550. 10.3389/fphar.2020.00550 (2020). Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630 , 493–500. 10.1038/s41586-024-07487-w (2024). McBride, J. M. et al. AlphaFold2 can predict single-mutation effects. Phys. Rev. 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Supplementary Files Additionalfile1.xlsx Additionalfile2.pdb Additionalfile3.pdb Additionalfile4.pdb Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 04 May, 2026 Editor assigned by journal 04 May, 2026 Editor invited by journal 04 May, 2026 Submission checks completed at journal 30 Apr, 2026 First submitted to journal 30 Apr, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9151984","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":635694876,"identity":"f66ba188-aa61-4c02-b37b-f8a778a370e8","order_by":0,"name":"Jose G. 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This last is a single span transmembrane domain (TMD) flanked by intra- and extracellular\u0026nbsp;α-helices joined by flexible linkers (center). Rotation of the channel shows its extracellular aerial view, exposing another view of the localization of its elements (VSD of Kv7.1: A-B, MinK: a-b) (right). \u003cstrong\u003eC \u003c/strong\u003eAlignment of MinK amino acid sequences showing that the wild-type alanine at position eight is well conserved among different species, while it is not conserved across the \u003cem\u003eKCNE\u003c/em\u003e family. \u003cstrong\u003eD\u003c/strong\u003e Local atomic view of the A8V transition in MinK suggests mild internal subdomain rearrangements.\u003csub\u003e \u003c/sub\u003e\u003cem\u003eCreated in BioRender. Acuna, J. (2026) https://BioRender.com/jjvo9q2\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/dd005aecf2c7a3895aac770e.jpg"},{"id":109119342,"identity":"7540fd73-790c-47e7-a412-234a53030046","added_by":"auto","created_at":"2026-05-12 16:57:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5757819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTridimensional modeling predicts the MinK N-terminal α-helix is partially embedded in the cell membrane. A \u003c/strong\u003eJuxtaposition of our WT I\u003csub\u003eKs\u003c/sub\u003e models against the cryo-EM structures of IK\u003csub\u003es \u003c/sub\u003echannel exhibits a good grade of similarity but with mild structural deviations. \u003cstrong\u003eB\u003c/strong\u003e Visualization of the WT I\u003csub\u003eKs \u003c/sub\u003ecomplex inserted in the cell membrane displaying a clearer visualization of its components. \u003cstrong\u003eC\u003c/strong\u003e Color-coded representation of the channel indicating the hydrophobic nature of each amino acid, denoting the correlation of its position with respect to the cell membrane. \u003cstrong\u003eD \u003c/strong\u003ePositioning of a selection of different WT MinK subunits originated from different I\u003csub\u003eKs\u003c/sub\u003e seeds in AlphaFold3. \u003cstrong\u003eE \u003c/strong\u003eRelative positioning of a selection of different WT MinK subunits originated from different MinK\u003csub\u003e \u003c/sub\u003eseeds in AlphaFold3. Black bar within data points represents the mean. Created in BioRender. Acuna, J. (2026) https://BioRender.com/jjvo9q2\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/871632f39d4958d639a64f34.jpg"},{"id":109204888,"identity":"5a2283cb-a9c0-4d8f-8c4c-d14cc8f88235","added_by":"auto","created_at":"2026-05-13 15:02:44","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1180773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologous expression of the heterozygous MinK-A8V-I\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eKs\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e channel results in significant differences in current\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003edensities.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Representative comparison between \u003cem\u003ein vitro\u003c/em\u003e current traces from the study groups reveals a significant and mild reduction of I\u003csub\u003eKs \u003c/sub\u003ewhen expressing the heterozygous \u003cem\u003eKCNE1\u003c/em\u003e-A8V mutation and in its homozygous state, respectively. \u003cstrong\u003eB \u003c/strong\u003eCurrent-voltage relationship data of developing I\u003csub\u003eKs \u003c/sub\u003ein experimental groups reveals to be significantly lower in the heterozygous \u003cem\u003eKCNE1\u003c/em\u003e-A8V expression system. \u003cstrong\u003eC\u003c/strong\u003e The mean tail current densities are plotted as a function of test pulse from -40 to +80 mV, showing an evident reduction in the heterozygous \u003cem\u003eKCNE1\u003c/em\u003e-A8V variant. \u003cstrong\u003eD \u003c/strong\u003eTrends of the V\u003csub\u003eh\u003c/sub\u003e of the activation average outline some degree of hyperpolarization of the heterozygous model, but no significant differences were found. Sample size per group: WT n=16, homozygous n=7, heterozygous n=9. Data points are presented as mean ± SEM, statistical differences are denoted with *, with \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 compared with the WT. Created in BioRender. Acuna, J. (2026) https://BioRender.com/jjvo9q2\u003c/p\u003e","description":"","filename":"Figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/73dcbe6bb07e9e6b42c03af8.jpeg"},{"id":109204963,"identity":"dd59a456-20c9-4004-af21-46a22898fbcc","added_by":"auto","created_at":"2026-05-13 15:03:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4255513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTridimensional analysis of I\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eKs\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e models identifies structural deviations and confidence shifts upon expression of MinK-A8V. A \u003c/strong\u003eRepresentative illustration of the I\u003csub\u003eKs \u003c/sub\u003emodels obtained from the AlphaFold3 server from the same seed shows less structural confidence of MinK subunits in heterozygous A8V variant. \u003cstrong\u003eB \u003c/strong\u003epLDDT of the global I\u003csub\u003eKs\u003c/sub\u003e channel (left), the Kv7.1 (center) and MinK subunits (right) are significantly reduced when the heterozygous MinK-A8V mutant is present. \u003cstrong\u003eC \u003c/strong\u003eRSMD analysis shows greater deviations between the WT/A8V-heterozygous I\u003csub\u003eKs \u003c/sub\u003epair\u003csub\u003e \u003c/sub\u003ethan the WT/A8V-homozygous I\u003csub\u003eKs\u003c/sub\u003e pair. Black bar within each data set represents the mean or median and significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001) are denoted with *. Created in BioRender. Acuna, J. (2026) https://BioRender.com/jjvo9q2\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/0f7115e127eb86b6c41dd4a2.jpg"},{"id":109119348,"identity":"889167ee-a4ba-47c2-b53f-1ad10e1ce469","added_by":"auto","created_at":"2026-05-12 16:57:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5223905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of heterozygous MinK-A8V may disturb Kv7.1/MinK interaction and generate less stable I\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eKs\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e complexes. A \u003c/strong\u003eRepresentative PAE plots illustrate the heterozygous MinK-A8V-I\u003csub\u003eKs\u003c/sub\u003e channel with greater positioning error, particularly in zones contemplating MinK in relation to adjacent MinK/Kv7.1 subunits. Lower values (darker green) indicate higher confidence in the relative spatial positioning. \u003cstrong\u003eB \u003c/strong\u003eHeterozygous expression of MinK-A8V affects the position confidence of the whole I\u003csub\u003eKs\u003c/sub\u003e channel (left) and its subunits (center and right). \u003cstrong\u003eC \u003c/strong\u003eThe thermodynamic stability and overall pTM indicated that both the WT and A8V-homozygous states were more stable than the A8V-heterozygous. \u003cstrong\u003eD\u003c/strong\u003e Consistently, global interactions between Kv7.1 and MinK subunits were more stable within the WT structures than in the heterozygous models and iPTM values indicated decreased reliability of inter-subunit interactions. \u003cstrong\u003eE\u003c/strong\u003e Overall, a tendency towards greater ΔΔG was observed in I\u003csub\u003eKs\u003c/sub\u003e complexes expressing the MinK-A8V mutation in heterozygosity than in the homozygous state, and ΔΔG of interaction between Kv7.1/MinK was significantly greater in the heterozygous models. Significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.003) are denoted with * and †. Created in BioRender. Acuna, J. (2026) https://BioRender.com/jjvo9q2\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/4bcec8eebe7e55ec8d5a647c.jpg"},{"id":109206678,"identity":"4872059f-1214-4001-9e2e-9bd232732dfc","added_by":"auto","created_at":"2026-05-13 15:15:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23099894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/a14a24fa-100a-4666-bd9f-696a7713486e.pdf"},{"id":109119346,"identity":"f1b9f616-d378-459b-82b8-6a989b971a40","added_by":"auto","created_at":"2026-05-12 16:57:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":68669,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/c963079a0cce249f8f5b6600.xlsx"},{"id":109204932,"identity":"747718b7-6f61-4cfc-8d85-df51f580c27a","added_by":"auto","created_at":"2026-05-13 15:02:54","extension":"pdb","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1108721,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.pdb","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/596823a77b31770c46b77870.pdb"},{"id":109119349,"identity":"db4271c5-e9a7-4345-9527-98291471cd2a","added_by":"auto","created_at":"2026-05-12 16:57:16","extension":"pdb","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1109041,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.pdb","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/4e8738cfeb5d8adda0e833a1.pdb"},{"id":109119345,"identity":"f9e8f057-8f1f-4f54-8ad2-2cfad1e5a69d","added_by":"auto","created_at":"2026-05-12 16:57:16","extension":"pdb","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1109361,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4.pdb","url":"https://assets-eu.researchsquare.com/files/rs-9151984/v1/7f72f16e18879e0f2b9cf751.pdb"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dominant-negative Effect of MinK-A8V Impairs I Ks and Disturbs Channel Structure: Importance of Clinical Surveillance in Mild LQTS Patients and Asymptomatic Carriers","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eThe delayed rectifier potassium current (I\u003csub\u003eK\u003c/sub\u003e) is a crucial outward potassium current that plays a major role in repolarizing the membrane of cardiomyocytes, contributing to regular heart contraction in humans. This repolarization phase is integrated by several components, including the rapid delayed rectifier current (I\u003csub\u003eKr\u003c/sub\u003e) and the slow delayed rectifier current (I\u003csub\u003eKs\u003c/sub\u003e), which are distinct in their activation kinetics and pharmacological properties. To generate human cardiac I\u003csub\u003eKs,\u003c/sub\u003e the assembly of four pore-forming Kv7.1 α-subunits (encoded by \u003cem\u003eKCNQ1\u003c/em\u003e) and MinK β-subunits (encoded by \u003cem\u003eKCNE1\u003c/em\u003e) is necessary. MinK can regulate this voltage-gated potassium channel by slowing its activation and regulating its conductance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMutations in the above-mentioned genes can impact the regular performance of the I\u003csub\u003eKs\u003c/sub\u003e channel either directly through changes in stability and allosteric mechanisms, decreasing its abundance in the cellular membrane or altering its pharmacological properties, causing different types of Long QT Syndrome (LQTS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This syndrome is an inherited cardiac condition in which the QT intervals in the electrocardiogram (ECG) of an individual are prolonged, predisposing them to life-threatening arrhythmias that can lead to sudden cardiac death (SCD). In this sense, the detection of genetic variants that may impair I\u003csub\u003eKs\u003c/sub\u003e as well as the determination of their functional/physiological implications may be beneficial for LQTS patient care and preventive follow-up of asymptomatic carriers.\u003c/p\u003e \u003cp\u003eDetermining the structure of ion channels has been crucial in understanding their properties and their role in channelopathies. The recent development of sophisticated Artificial Intelligence (AI) operated platforms for the structure prediction of proteins with biological importance represents an improved tool for the assessment of genetic variants. AlphaFold3 represents perhaps one of the most famous models of its kind, exhibiting markedly enhanced accuracy compared with several specialized docking tools [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Indeed, several investigations across diverse disease contexts have employed AI resources in an effort of assessing the impact of variants over 3D protein models, some of them showing a good correlation with the observed functional data [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The sum of these arguments prompted us to investigate whether a given \u003cem\u003eKCNE1\u003c/em\u003e variant could influence the normal features of the I\u003csub\u003eKs\u003c/sub\u003e \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein silico\u003c/em\u003e in the context of healthy carriers.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMolecular and comparative analysis revealed a single missense mutation among principal LQTS-related genes, disturbing a well-conserved residue at the N-terminus of\u003c/b\u003e \u003cb\u003eMinK\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenetic screening of the proband did not identify mutations within principal genes related to LQTS onset (\u003cem\u003eKCNQ1\u003c/em\u003e, \u003cem\u003eKCNH2\u003c/em\u003e and \u003cem\u003eSCN5A\u003c/em\u003e), but a single heterozygous mutation in \u003cem\u003eKCNE1\u003c/em\u003e. Unfortunately, clinical or familial information of the subject was not available when the study was carried out. Chromatographic results revealed one single mutation consisting of a C to T transition at nucleotide 23 of \u003cem\u003eKCNE1\u003c/em\u003e (c.23 C\u0026thinsp;\u0026gt;\u0026thinsp;T, ALFA, genomAD and ExAC MAF\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which leads to the substitution of alanine at position eight with valine in the N-terminal α-helix of the MinK β-subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, center). In turn, MinK subunits are colocalized in the outer pore space between two Kv7.1 subunits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB left), close to the voltage-sensing domain (VSD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, right). Comparative analysis of orthologous amino acid sequences revealed a high degree of preservation of that residue across species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, top), which is not the case for other human \u003cem\u003eKCNE\u003c/em\u003e family members (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, bottom). Moreover, \u003cem\u003ein silico\u003c/em\u003e simulations of the WT and A8V structures indicate this shift provokes the formation of new hydrogen bonds (Val8\u0026mdash;Ser4, Val8\u0026mdash;Asn5), a possible steric clash (Val8\u0026mdash;Pro11) and the loss of hydrogen bonding (Ala8\u0026mdash;Phe12) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Panel B of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows different views of a representative model of WT IK\u003csub\u003es\u003c/sub\u003e complex and the configuration of its components, originating from the present research.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe MinK N-terminal α-helix is anchored to the cell membrane within the WT I\u003csub\u003eKs\u003c/sub\u003e models\u003c/h2\u003e \u003cp\u003eAfter removing disorganized regions (amino acids 1\u0026ndash;89, 404\u0026ndash;676 from Kv7.1 and 115\u0026ndash;129 from MinK, specified in additional file 1, Table\u0026nbsp;1), we successfully generated three-dimensional models of the three genotype groups of the I\u003csub\u003eKs\u003c/sub\u003e channel with respect to the MinK-A8V mutation using AlphaFold3 (representative structures in additional files 2, 3 and 4). Validation of our WT models revealed mild shifts\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ein terms of the Root Mean Square Deviation (RMSD) values (Cui \u003cem\u003eet\u003c/em\u003e al. model, M\u0026thinsp;=\u0026thinsp;1.471\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0274 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20; Zhong \u003cem\u003eet al\u003c/em\u003e.l model, M\u0026thinsp;=\u0026thinsp;2.824\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0273 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) when comparing to the recently partially solved cryo-EM structures of the I\u003csub\u003eKs\u003c/sub\u003e channel, indicating a moderately high structural level of similarity suitable for downstream comparative analysis.\u003c/p\u003e \u003cp\u003eThe MinK N-terminus α-helix was most frequently situated parallel to its TMD in our I\u003csub\u003eKs\u003c/sub\u003e channel model in the closed state. After the structures were uploaded to the Swiss Model server, the cell membrane was\u003c/p\u003e \u003cp\u003ebuilt in the most likely coordinates with respect to the I\u003csub\u003eKs\u003c/sub\u003e model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The latter is supported by an increased density of hydrophobic residues falling within the membrane hydrophobic interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). A comparison of different WT MinK protein extracted from different I\u003csub\u003eKs\u003c/sub\u003e channel models showed the N-terminal α-helix of MinK to be anchored in the cell membrane to some extent but in different orientations, while the TMD position is highly conserved between seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This embedding correlates with the visual representation of the hydrophobic/hydrophilic residues throughout independent modeling of different single-span MinK seeds, where the amphipathic nature of the N-terminal α-helix is uncovered (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), allowing it to interact with the cell membrane.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe A8V mutation in MinK provokes a strong dominant-negative effect on I\u003c/h3\u003e\n\u003cp\u003eSimilar trends were observed in I\u003csub\u003eKs\u003c/sub\u003e activation traces, the mean current density (I\u003csub\u003edev\u003c/sub\u003e) and the mean tail current density (I\u003csub\u003etail\u003c/sub\u003e) from the three experimental systems: cells expressing both WT \u003cem\u003eKCNQ1\u003c/em\u003e and WT \u003cem\u003eKCNE1\u003c/em\u003e displayed standard behavior (I\u003csub\u003eKs\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA left, I\u003csub\u003edev\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, I\u003csub\u003etail\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. n\u0026thinsp;=\u0026thinsp;16). This was contrasting with the heterozygous expression of the WT and A8V mutation, which presented a significant reduction in channel current (I\u003csub\u003eKs\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA central-left, I\u003csub\u003edev\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, I\u003csub\u003etail\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, n\u0026thinsp;=\u0026thinsp;9). Meanwhile, homozygous expression of the MinK-A8V mutant produced a modest reduction of the current (I\u003csub\u003eKs\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA central-right, I\u003csub\u003edev\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, I\u003csub\u003etail\u003c/sub\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, n\u0026thinsp;=\u0026thinsp;7). Expression of the Kv7.1 subunit alone showed a well-known activation current behavior without the modulation of MinK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA right).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStatistical significance of I\u003csub\u003edev\u003c/sub\u003e and I\u003csub\u003etail\u003c/sub\u003e were reached only between WT and the heterozygous expression groups. Homozygous expression of the variant did not show a statistically significant shift of currents.\u003c/p\u003e \u003cp\u003eFinally, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD depicts the behaviors of the normalized curves of the tail currents as the mean V\u003csub\u003eh\u003c/sub\u003e values. Normalized tail currents exhibit mild hyperpolarization in the WT/A8V heterozygous model (V\u003csub\u003eh\u003c/sub\u003e= 6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01, \u003cem\u003ek\u0026thinsp;=\u003c/em\u003e\u0026thinsp;14.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42, n\u0026thinsp;=\u0026thinsp;9), whereas the homozygous \u003cem\u003eKCNE1-\u003c/em\u003eA8V variant is slightly more depolarized (V\u003csub\u003eh\u003c/sub\u003e=13.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05, \u003cem\u003ek\u0026thinsp;=\u003c/em\u003e\u0026thinsp;17.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38, n\u0026thinsp;=\u0026thinsp;7) than the WT control values (V\u003csub\u003eh\u003c/sub\u003e= 10.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65, \u003cem\u003ek\u0026thinsp;=\u003c/em\u003e\u0026thinsp;14.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34, n\u0026thinsp;=\u0026thinsp;16). Nonetheless, no significant differences between groups were found. Overall, the MinK-A8V mutant appears to significantly impact the function of the WT channel when expressed in heterozygosity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStructural stability of the AI-generated I\u003c/b\u003e \u003csub\u003e \u003cb\u003eKs\u003c/b\u003e \u003c/sub\u003e \u003cb\u003echannel models correlates with the dominant-negative effect of the MinK-A8V mutation observed in electrophysiological results\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to color coded per-atom confidence estimate analysis (pLDDT), the TMD portion of MinK presents changes in structural confidence of both the mutated and wild-type subunits when the variant A8V is expressed in heterozygosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The statistical analysis of mean (M) pLDDT values shows significant differences between WT and heterozygous groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when considering the whole structure of the I\u003csub\u003eKs\u003c/sub\u003e complex, but also when evaluating the Kv7.1 and MinK subunits by separate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB): I\u003csub\u003eKs\u003c/sub\u003e channel (left) (M\u0026thinsp;=\u0026thinsp;68.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.155 Vs. M\u0026thinsp;=\u0026thinsp;64.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.124, n\u0026thinsp;=\u0026thinsp;20), Kv7.1 (center) (Median\u0026thinsp;=\u0026thinsp;74.2 Vs. Median\u0026thinsp;=\u0026thinsp;71.6, n\u0026thinsp;=\u0026thinsp;20) and MinK subunits (right) (M\u0026thinsp;=\u0026thinsp;54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.314 Vs. M\u0026thinsp;=\u0026thinsp;46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, n\u0026thinsp;=\u0026thinsp;20). On the other hand, structural alignments between the models derived from the same seed showed greater deviations in RMSD values between the WT/heterozygous I\u003csub\u003eKs\u003c/sub\u003e channel groups (M\u0026thinsp;=\u0026thinsp;0.593\u0026thinsp;\u0026plusmn;\u0026thinsp;0.059 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) than between the WT/homozygous (M\u0026thinsp;=\u0026thinsp;0.456\u0026thinsp;\u0026plusmn;\u0026thinsp;0.050 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) and the heterozygous/homozygous groups (M\u0026thinsp;=\u0026thinsp;0.594\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), although no significant differences were found. In parallel, the Predicted Aligned Error (PAE) matrices depict differences in the expected position error, which is more noticeable between zones contemplating MinK subunits across the different zygosity states (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Accordingly, quantitative analysis of average PAE values revealed significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.003) between WT and A8V-\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eheterozygous I\u003csub\u003eKs\u003c/sub\u003e channel models (left) (M\u0026thinsp;=\u0026thinsp;18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.104 \u0026Aring; Vs. M\u0026thinsp;=\u0026thinsp;19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20), but also on Kv7.1 subunits (center) (M\u0026thinsp;=\u0026thinsp;14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026Aring; Vs. M\u0026thinsp;=\u0026thinsp;15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05351 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) and MinK subunits (right) (M\u0026thinsp;=\u0026thinsp;22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026Aring; Vs. M\u0026thinsp;=\u0026thinsp;25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the same manner, differences between the heterozygous and homozygous models were observed with respect to the full structure of the I\u003csub\u003eKs\u003c/sub\u003e channel (left) (M\u0026thinsp;=\u0026thinsp;19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035 \u0026Aring; Vs. M\u0026thinsp;=\u0026thinsp;18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20) and Kv7.1 alone (center) (M\u0026thinsp;=\u0026thinsp;15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053 \u0026Aring; Vs. M\u0026thinsp;=\u0026thinsp;14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.085 \u0026Aring;, n\u0026thinsp;=\u0026thinsp;20).\u003c/p\u003e \u003cp\u003eFollowing the same pattern, WT I\u003csub\u003eKs\u003c/sub\u003e channels were thermodynamically more stable than the heterozygous complexes (M= -273\u0026thinsp;\u0026plusmn;\u0026thinsp;11.245 Kcal/mol Vs. M= -238\u0026thinsp;\u0026plusmn;\u0026thinsp;8.76 Kcal/mol, n\u0026thinsp;=\u0026thinsp;20), as well as more reliable according to predicted TM-score (PTM) values (Median\u0026thinsp;=\u0026thinsp;0.73 Vs. Median\u0026thinsp;=\u0026thinsp;0.69, n\u0026thinsp;=\u0026thinsp;20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similarly, the total energy of the Kv7.1/MinK interaction increased upon the A8V heterozygous mutation (M= -315\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3 Kcal/mol Vs. M= -282\u0026thinsp;\u0026plusmn;\u0026thinsp;7.35 Kcal/mol, n\u0026thinsp;=\u0026thinsp;20), inversely aligning with lower interface Predicted Template Modelling (ipTM) values (M\u0026thinsp;=\u0026thinsp;0.714\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 Vs. M\u0026thinsp;=\u0026thinsp;0.665\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), which indicated a decreased reliability of inter-subunit interactions. As expected, the average decrease in both the global free energy and interaction ΔG was greater when expressing the heterozygous MinK-A8V variant than in the homozygous state (M\u0026thinsp;=\u0026thinsp;34.84\u0026thinsp;\u0026plusmn;\u0026thinsp;12.83 Kcal/mol Vs. M\u0026thinsp;=\u0026thinsp;9.73\u0026thinsp;\u0026plusmn;\u0026thinsp;10.37 Kcal/mol, n\u0026thinsp;=\u0026thinsp;20), correlating with ΔΔG of interaction between Kv7.1/MinK (M= -32.665\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65 Kcal/mol Vs. M= -2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9 Kcal/mol, n\u0026thinsp;=\u0026thinsp;20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Altogether, these results suggest a possible change in the stability of the complex and a partial loss of Kv7.1/MinK interaction.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur research found a rare missense mutation in \u003cem\u003eKCNE1\u003c/em\u003e (c.23 C\u0026thinsp;\u0026gt;\u0026thinsp;T) that was originally linked to an LQTS case [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Controversially, we found a carrier of the same mutation in a control individual without cardiac malfunctions, raising questions about the penetrance of the variant and the mechanisms behind a LQTS phenotype when a mutant \u003cem\u003eKCNE1\u003c/em\u003e allele is present. In fact, several studies support the idea that a single mutation in \u003cem\u003eKCNE1\u003c/em\u003e is insufficient to drive disease presentation in most carriers, notwithstanding, such individuals may carry a hidden risk for developing an LQTS type 5 phenotype that may be unmasked by environmental or physiological triggers [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this sense, this variant has multiple entries in ClinVar, some of them supporting its probable pathogenicity through different approaches. A study showed a clear co-segregation of the MinK A8V variant along with a LQTS phenotype [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. On the other hand, Itoh \u003cem\u003eet al\u003c/em\u003e. included in their study cohort a carrier of the same variant who developed a LQTS phenotype following accidental exposure to an arrhythmia-triggering factor (not specified), condition that remained active despite the elimination of trigger [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Giving these scenarios, it seems that this specific variant represents a genetic component that can be influenced by environmental or physiological factors, but little is known about the specific targeted cellular mechanisms. This reinforces the necessity of clarifying how some mutations in its component proteins can influence the behavior of the I\u003csub\u003eKs\u003c/sub\u003e channel.\u003c/p\u003e \u003cp\u003e \u003cem\u003eKCNE1\u003c/em\u003e has been found to influence I\u003csub\u003eKr\u003c/sub\u003e channel responses to premature stimulation, pharmacological inhibition, pH sensitivity and pharmacological properties of Kv7.1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the electrophysiological field, two main studies have assessed the influence of the MinK-A8V variant over I\u003csub\u003eKs\u003c/sub\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although different expression models were used, our results align with the other two studies in terms of I\u003csub\u003eKs\u003c/sub\u003e not showing a significant change in the homozygous co-expression of MinK-A8V with \u003cem\u003eKCNQ1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Instead, Ohno \u003cem\u003eet al.\u003c/em\u003e 2007 found a marked suppression of I\u003csub\u003eKr\u003c/sub\u003e when the mutant was co-expressed with \u003cem\u003eKCNH2\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, none of these studies explored the actual heterozygous genotype found in the patient and therefore the influence of possibly different interactions and triggering mechanisms when WT and mutated proteins coexist in the plasma membrane. In our study, a statistically significant reduction in I\u003csub\u003eKs\u003c/sub\u003e was evidenced by the heterozygous expression of the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C), suggesting that a disease phenotype could be caused not only by defects in I\u003csub\u003eKr\u003c/sub\u003e but also by defects in I\u003csub\u003eKs\u003c/sub\u003e. These findings constitute a clear example of a dominant-negative effect in channelopathies, which highlights the importance of mimicking a heterozygous genotype \u003cem\u003ein vitro\u003c/em\u003e, representing a more reliable and close-to-reality approach in the study of channelopathies.\u003c/p\u003e \u003cp\u003eGenetic variants related to LQTS are often loss-of-function mutations that reduce the density of the current in a dominant-negative manner either by changing the activation kinetics of the channels, changing channel conductance or trafficking defects that decrease channel abundance in the cell membrane [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our data showed a reduction in both development and tail currents, with the heterozygous form being more severe than the homozygous mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Nevertheless, the V\u003csub\u003eh\u003c/sub\u003e of activation was not significantly different between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting that the variant had no influence on the kinetics of the channel. This last led us to strongly hypothesize there could be less available functional channels in the cells. In this matter, we found a recent study showing a mild disruption of cell surface expression of the \u003cem\u003eKCNE1\u003c/em\u003e protein when the A8V variant is expressed in homozygosity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which supports and correlates with our hypothesis. Furthermore, a non-significant functional reduction was observed through cell fitness assay, matching our electrophysiological results from the homozygous state. Interestingly, another research found the LQTS causing T7I MinK mutation \u0026mdash;a neighbor residue of alanine 8\u0026mdash; and surrounding N5I/N5Q/T6P induced mutations, to disrupt normal glycosylation mechanisms that ultimately lead to decreasing the number of functional I\u003csub\u003eKs\u003c/sub\u003e channel complexes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. At our end, we showed how a change from alanine to a larger valine at position eight may form new hydrogen bonds that could bend the α-helix, increase hydrophobicity, provoke steric hindrance and affect local flexibility. Given the spatial proximity of alanine eight to these last aminoacids, we speculate that a larger valine may also reduce the accessibility for enzymatic glycosylation at this site, reducing the macroscopic current in our cellular model. Unfortunately, we did not perform any trafficking assay to properly infer the precise underlaying mechanism that could corroborate our hypotheses.\u003c/p\u003e \u003cp\u003eThere are a few other reports that functionally evaluate N-terminal MinK mutations under a clinical context. A publication demonstrated that arginines in positions 32, 33 and 36 of MinK are essential for the stabilizing interaction of the N-terminal α-helix with the plasma membrane [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A recent robust study evaluating clinically relevant \u003cem\u003eKCNE1\u003c/em\u003e variants revealed that nearly half of mutations in the N-terminal portion could cause variable degrees of loss of function of I\u003csub\u003eKs\u003c/sub\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While other variants did not have any functional effect in their homozygous state, that does not mean they are not pathogenic when present in heterozygosis or trigger a LQTS phenotype after an environmental exposure.\u003c/p\u003e \u003cp\u003eAlthough KCNE family members have divergences, the general topology within the cell membrane among this group of proteins is highly similar. Analogously, genetic variants in the N-terminal section of \u003cem\u003eKCNE2\u003c/em\u003e (T8A, Q9E, T10M) have been associated with the spontaneous phenotypic development of LQTS in patients after taking certain medications, stress exposure or electrolyte imbalances [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These are clear examples of conferred genetic risk, which reinforces the idea that mutations in potassium channels may remain clinically unrecognized until they are unmasked by physiological or environmental stressors. Detecting these kinds of variants should be important in the clinical setting in order to avoid unintentional disease induction.\u003c/p\u003e \u003cp\u003eRecently, high-resolution structure of the native I\u003csub\u003eKs\u003c/sub\u003e channel has been obtained by two research groups using cryo-EM [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, none of these include the N- and C-terminus of MinK since they are disorganized/dynamic areas that are hard to stabilize in laboratory procedures, precluding the modelling of mutations in those regions, as is the case of this study. Thus, we employed the newly upgraded AI-based computational method AlphaFold3 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] as an effort to obtain a close-to-reality 3D conformation of the missing I\u003csub\u003eKs\u003c/sub\u003e channel region of interest (residues 1 to ~\u0026thinsp;40). Our model displays a highly similar topology to the cryo-EM structures of Kv7.1/MinK, displaying the distinctive single span transmembrane domain (TMD) of MinK flanked by intra- and extracellular α-helices joined by flexible linkers [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The extracellular N-terminus of MinK has been determined to play an important role in the stabilization and gating of the I\u003csub\u003eKs\u003c/sub\u003e channel [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], in which changes in spatial orientation and local flexibility could alter the stability of the complex.\u003c/p\u003e \u003cp\u003eThe full NMR structure of MinK was determined by Kang \u003cem\u003eet al.\u003c/em\u003e 2008 through the isolation of the molecule in micelles [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In these, the MinK N-terminus α-helix was observed resting on the surface of the micelle, highlighting an amphipathic nature. This is extremely consistent with our AI-generated models where we noted the same MinK portion being partially embedded in the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and may be a reason for its stabilization attributes. Indeed, NMR relaxation studies and electron paramagnetic resonance spectroscopy support the interaction of the MinK N-terminal with the plasma membrane [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], serving as an anchor-like component that is nearly as fixed as its TMD. However, these last two domains are connected through a mobile and flexible structure situated in the extracellular side of the membrane upon assembly with Kv7.1, facilitating dynamic rearrangements that could explain the different orientations of the N-terminal α-helix across our different seed models. Thus, the constructed N-terminal α-helix of MinK in our 3D models could be displaying one transition between order and disorder as part of the high dynamism of the I\u003csub\u003eKs\u003c/sub\u003e channel. Nevertheless, the pLDDT values in that location of our models were predicted to be low (\u0026lt;\u0026thinsp;70) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which may reflect either an unstable structure and/or greater chances of spurious tridimensional conformation, in which latter case this model should be interpreted conservatively. Nonetheless, the conjunction of all previous experimental data here reviewed supports the reliability of our model, which may make it a suitable approach for variant effect assessment of the I\u003csub\u003eKs\u003c/sub\u003e channel.\u003c/p\u003e \u003cp\u003eThe confidence metrics calculated by AlphaFold3 served as a measure of the accuracy of the models [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], as well as variables subject to this study to evaluate the effect over the I\u003csub\u003eKs\u003c/sub\u003e channel when the MinK-A8V mutation is expressed. pLDDT scores indicated greater structural confidence in the I\u003csub\u003eKs\u003c/sub\u003e channel zones that are buried within the plasma membrane, which is not surprising because they constitute the least mobile portion of the protein. In contrast, pLDDT values were markedly lower when the MinK-A8V mutation was expressed in heterozygosity, suggesting a reduced confidence in the structural accuracy of the complete I\u003csub\u003eKs\u003c/sub\u003e structure, Kv7.1 and MinK itself, potentially reflecting an increase in conformational flexibility, destabilization or changes in dynamic behavior. Specifically, we observed the TMD of MinK with lower pLDDT scores only at the heterozygous I\u003csub\u003eKs\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This is of special importance given that the TMD is a structurally characterized region that plays a central role in I\u003csub\u003eKs\u003c/sub\u003e channel gating and activation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], in which stability and position changes may affect its interaction within the I\u003csub\u003eKs\u003c/sub\u003e channel and lead to channel performance intermittency, and may justify the mild hyperpolarization of the activation. On the other hand, the predicted pTM score reflects the confidence in the overall topology of a predicted structure. On this basis, higher pTM values may indicate better domain packing and structural stability of protein complexes, which is a fundamental principle under physiological conditions. Accordingly, we used pTM scores as an indirect measure of conformational stability across different I\u003csub\u003eKs\u003c/sub\u003e channel models, and in conjunction with ΔG calculations, we observed less stability of the complexes and their members when the MinK-A8V mutation was expressed in heterozygosity. Likewise, the ipTM score is designed to reflect the confidence in relative subunit positioning within multimeric assemblies, and we interpreted it as a proxy for the likelihood and stability of subunit\u0026ndash;subunit interactions. A drop in iPTM reflected a loss of confidence in the interaction between subunits, suggesting dissociation, destabilization or steric interference. Based on these findings and calculations of interaction ΔG, we observed a major general decrease in subunit interaction when the MinK-A8V mutation was expressed in heterozygosity. Finally, PAE values report the expected positional error at residue \u003cem\u003ei\u003c/em\u003e when the model is aligned at residue \u003cem\u003ej\u003c/em\u003e, thereby allowing the estimation of structural certainty between different regions or chains of the model. Indeed, it seems there is a correlation between two residues in different entities that present low PAE values and their probability of interaction. On this basis, we detected an increased position error in I\u003csub\u003eKs\u003c/sub\u003e complexes and their subunits when the heterozygous MinK-A8V mutation was present, which may represent a general loss of the structural coupling and normal subunit proximity. Taking this data together and given the lack of significant differences in RMSD values between the WT, heterozygous and homozygous I\u003csub\u003eKs\u003c/sub\u003e models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), the functional impairments observed in electrophysiological experiments in the heterozygous context suggest that the underlying mechanism may not specifically be due to shifts in the structure at atomic level. Rather, it may be related to altered symmetry or subunit arrangement within the I\u003csub\u003eKs\u003c/sub\u003e complex, potentially disrupting subunit interaction.\u003c/p\u003e \u003cp\u003eThe heterozygous expression of a mutated MinK has also implications in the stoichiometry of the I\u003csub\u003eKs\u003c/sub\u003e channel components, which is an important variable to consider when measuring ion currents through patch clamp because the expression of MinK regulates Kv7.1 and \u003cem\u003evice versa\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It seems that, depending on their concentrations, the native number of MinK subunits assembled with tetrameric Kv7.1 channels can range from one to four, with different stoichiometries having distinct gating properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study we used the same expression vector backbones to achieve equal expressions and facilitate a 4:4 stoichiometry of MinK/Kv7.1, which shows a major influence over I\u003csub\u003eKs\u003c/sub\u003e compared to other ratios. In this sense, the degree of reduction of current density we observed in our electrophysiological data could be more pronounced than in real cardiac tissue. The use of a cellular model in this work may introduce a different genetic background that influences the molecular interactions/mechanisms and may not recapitulate what happens in a native environment, thus the clinical impact may be widely variable and we should not directly extrapolate these results directly, especially because each individual may be exposed to different environmental factors. Nonetheless, we speculate that one of the reasons that an individual carrying this type of variants could suppress the development of the disease phenotype is by facilitating other stoichiometries and allele expression as compensatory mechanisms. Likewise, other genetic or epigenetic regulators may be involved. Further studies in hiPSC-derived cardiomyocytes or \u003cem\u003ein vivo\u003c/em\u003e models are warranted to explore more accurately the clinical implications of such mutations in asymptomatic individuals and their differential phenotypes. Similarly, additional experiments providing biochemical (e.g., surface biotinylation, western blot and co-immunoprecipitation) or imaging data may concisely corroborate our observations in the future.\u003c/p\u003e \u003cp\u003eIn the present work we found that a mutation in \u003cem\u003eKCNE1\u003c/em\u003e may reduce I\u003csub\u003eKs\u003c/sub\u003e by making the channel less stable, combined with presumed defects in MinK trafficking that were previously reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. On this basis, our findings underline the clinical importance of detecting a \u003cem\u003eKCNE1\u003c/em\u003e mutation in asymptomatic individuals or mild LQTS patients, which should be then followed by appropriate additional genetic testing, careful revision of family clinical history, changes of life style and medications as needed in order to avoid known arrhythmic triggers (potential QT-prolonging drugs, electrolyte imbalances, bradycardia or stress), as well as revision of environmental expositions over time by an specialized physician. All this could lead to personalized management and therefore, an improvement in the clinical outcome. Overall, our study suggests considering the A8V mutation in MinK as modifier allele.\u003c/p\u003e \u003cp\u003eIt is worth noting considering that several limitations come in the present work that we fully recognize. The accurate modeling of the true conformation of the MinK N-terminus and other portions is still uncertain. We were not able to simulate the entire sequence of the I\u003csub\u003eKs\u003c/sub\u003e channel due to the formation of disorganized structures and we are aware that AlphaFold3 may build structural hallucinations in disordered regions. Yet, our model fits well according to functional and structural data in this and other mentioned studies. Besides, our model only reflects static conformations, and even multiple random seed runs may not recapitulate the real dynamic behavior of biomolecular systems, as stated by AlphaFold3 developers. Nonetheless, we reinforce that this model represents a good resource to evaluate the effect of mutations within the N-terminal portion of MinK, which experimental conformation within the native I\u003csub\u003eKs\u003c/sub\u003e channel remains unresolved.\u003c/p\u003e \u003cp\u003eNotably, we are missing the evaluation of the studied mutation within the active/open state of the pore, as our AI-generated 3D structure represents only the closed state of the I\u003csub\u003eKs\u003c/sub\u003e channel and we were not able to perform protein‒protein docking at this time. Unfortunately, neither were we able to control the localization of the mutant MinK chains in the generated models, so we could not decide the symmetry of the complex. Finally, we did not evaluate scenarios where only two or three MinK subunits co-assemble in the four Kv7.1 subunits, and the different combinations of mutants/WT coexisting.\u003c/p\u003e \u003cp\u003eWe hope that future advances in protein structure determination may contribute to resolve the true conformation of the remaining undetermined I\u003csub\u003eKs\u003c/sub\u003e channel portions and thus, a better understanding of 3D structures upon mutations. In the meantime, future implementations of Molecular Dynamics (MD) of the resulting AI structures could help to refine our I\u003csub\u003eKs\u003c/sub\u003e channel models, with the inclusion of the cellular membrane and calmodulin as active interacting components, as well as phosphatidylinositol 4,5-bisphosphate (PIP2) as an activating cofactor for channel opening, which are elements that would contribute to a more realistic system.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn this study, we demonstrate that the MinK-A8V variant exerts a dominant-negative effect on I\u003csub\u003eKs\u003c/sub\u003e when co-expressed with Kv7.1, leading to a significant reduction in both development and tail current densities \u003cem\u003ein vitro\u003c/em\u003e. Importantly, the activation kinetics were similar across groups, indicating that the reduction in current is unlikely due to altered gating. The conjunction of our results with mechanistic data from other studies suggest that the MinK-A8V mutation may induce trafficking defects that reduce the abundance of functional I\u003csub\u003eKs\u003c/sub\u003e channel in the plasma membrane. A pronounced functional impairment could be aggravated only when the mutant and WT MinK proteins coexist in the I\u003csub\u003eKs\u003c/sub\u003e channel subunits (heterozygosis), which is remarked by differential confidence and stability metrics of the I\u003csub\u003eKs\u003c/sub\u003e channel 3D models here presented, disrupting channel structure, energetic stability, assembly, symmetry and function in an individual, when additional environmental conditions are present. The modeling of the MinK N-terminal α-helix structure and its probable interaction with the cell membrane was particularly important, as it recapitulates its amphipathic and flexibility features which could be a key property within the I\u003csub\u003eKs\u003c/sub\u003e complex. This study may represent a clear example of how the interpretation electrophysiological data in the light of the structural composition of ion channels is crucial and imperative in order to have a clearer picture of functionality effects upon mutations.\u003c/p\u003e \u003cp\u003eDespite the demonstrated functional deficit, the MinK-A8V variant was identified in a healthy individual, supporting the notion of representing a modifier allele rather than a fully pathogenic mutation. Nonetheless, the complex significance of \u003cem\u003eKCNE1\u003c/em\u003e mutations in asymptomatic individuals with or without a family history of LQTS that we describe emphasizes the importance of genetic screening and appropriate functional evaluations of these variants, which may contribute to improved clinical outcomes.\u003c/p\u003e \u003cp\u003eOverall, these findings also underscore the importance of recapitulating the original \u003cem\u003ein vivo\u003c/em\u003e heterozygous genotypes when translated into \u003cem\u003ein vitro\u003c/em\u003e experiments, which could lead to a different physiologically relevant approach in the functional evaluation of ion channel variants expressed in humans.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cb\u003eGenetic analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll experimental procedures were performed in accordance with the 1964 Declaration of Helsinki and after approval of the Main Line Hospitals Institutional Review Board (MLH IRB, approval number E-22-5283). Informed consent was obtained to collect a blood sample from the proband. Genomic DNA was extracted from peripheral blood leucocytes with a commercial kit (Puregene, Gentra Systems, Inc., Minneapolis, MN). The DNA was amplified by PCR on the GeneAmp\u003cb\u003e\u0026reg;\u003c/b\u003e PCR System 9700 (Applied Biosystems, Foster City, CA). All exons and intron borders of the following LQTS susceptibility genes were amplified and analyzed by direct sequencing: \u003cem\u003eKCNQ1, KCNH2, KCNE1, KCNE2\u003c/em\u003e and \u003cem\u003eSCN5A\u003c/em\u003e. PCR products were purified with a commercial reagent (ExoSAP-IT, USB, Cleveland, OH) and directly sequenced from both directions using the ABI PRISM 3100 Automatic DNA sequencer (Applied Biosystems, Foster City, CA). Electropherograms were visually examined for heterozygous peaks and compared with reference sequences for homozygous variations employing CodonCode Aligner Ver. 2.0.4 (CodonCode Corporation, Dedham, MA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSite-directed mutagenesis and cell transfection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSite-directed mutagenesis was performed with QuikChange (Stratagene, La Jolla, CA) on full-length human \u003cem\u003eKCNE1-\u003c/em\u003eWT to introduce the c.23C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation (A8V amino acid change). The \u003cem\u003eKCNQ1-\u003c/em\u003eWT, \u003cem\u003eKCNE1-\u003c/em\u003eWT and \u003cem\u003eKCNE1-\u003c/em\u003eA8V cDNA sequences were subsequently cloned by separate in a pcDNA3.1 vector. The \u003cem\u003eKCNE1\u003c/em\u003e-A8V plasmid was sequenced to ensure the presence of the mutation without spurious substitutions. I\u003csub\u003eKs\u003c/sub\u003e channels were expressed in the modified human embryonic kidney (HEK) cell line TSA201 (Sigma-Aldrich, St. Louis, MO) following the manufacturer\u0026rsquo;s protocol for transient transfection using Fugene6 (Roche Diagnostics, Indianapolis, IN). Briefly, constructs of \u003cem\u003eKCNQ1-\u003c/em\u003eWT and \u003cem\u003eKCNE1-\u003c/em\u003eWT or/and \u003cem\u003eKCNE1-\u003c/em\u003eA8V were employed at a ratio of 1\u0026micro;g:1\u0026micro;g for homozygous expression and 1\u0026micro;g: 0.5\u0026micro;g :0.5\u0026micro;g to mimic the heterozygous form of the mutation. GFP cDNA was co-transfected as well as reporter gene to visually identify transfected cells. After 24h of incubation, cells were split into polylysine-coated 35 mm culture dishes (Cell+, Sarstedt, Newton, NC) at approximately 10% confluence. Forty-eight to 72 hours after transfection, fluorescent cells were used for patch-clamp studies. The conditions of cell maintenance included the use of GIBCO Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and penicillin [100 U/mL]/streptomycin [100 \u0026micro;g/mL], and incubation in a 5% CO\u003csub\u003e2\u003c/sub\u003e chamber at 37\u0026deg;C. This procedure was performed four different times for each experimental group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElectrophysiological studies.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMembrane currents were measured using whole-cell patch-clamp technique to at least five cells per transfection, per group. All recordings were obtained at body temperature (36\u0026ndash;37\u0026deg;C) using an Axopatch 200B amplifier equipped with a CV-201A head stage (Axon Instruments. Union 5 City, CA). Macroscopic whole-cell K\u003csup\u003e+\u003c/sup\u003e current was recorded from cells bathed in solution containing the following concentration (mmol/L): 132 NaCl, 4.8 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 MgCl\u003csub\u003e2\u003c/sub\u003e, 10 HEPES and 10 glucose (pH 7.4). Patch pipettes were pulled from borosilicate glass (Model PP-89, Narashige, Tokyo, Japan), obtaining a resistance of 2\u0026ndash;3 MΩ when filled with a solution containing (mmol/L): 110 K-Aspartate, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 11 EGTA, 5 MgATP and 10 HEPES (pH 7.35). Currents were elicited by depolarizing pulses from a holding potential of -80 mV to test potentials between \u0026minus;\u0026thinsp;40mV and +\u0026thinsp;80 mV with a 10 mV step increment, followed by repolarization to -40 mV to measure the tail current amplitude. Current densities (pA/pF) were calculated by normalizing current amplitude to cell capacitance. Tail amplitudes were normalized to maximum amplitude in order to calculate the activation curve and fitted to Boltzmann\u0026rsquo;s function to calculate the voltage of half activation (V\u003csub\u003eh\u003c/sub\u003e). Currents were digitized at 5 kHz.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and assessment of three-dimensional I\u003csub\u003eKs\u003c/sub\u003e model groups\u003c/h2\u003e \u003cp\u003eTo distinguish disorganized from well-structured reliable portions of the I\u003csub\u003eKs\u003c/sub\u003e channel, we first performed a screening run with the full sequence of four Kv7.1 and four MinK subunits (NCBI Ref. Seq.\u0026nbsp;NP_000209.2 and NP_000210.2, respectively) on the official AlphaFold3 server (DeepMind/Isomorphic Labs), adjusted to find PDB templates to the most recent possible date (02/02/2025). Additionally, this method was performed separately for a single-span MinK subunit. Visually unstructured regions (lose ribbon-like structures with very low pLDDT values that are usually removed in computationally generated models to avoid undesirable noise and inaccurate data) flanking the N- and C- terminus were removed as possible, preserving the central functional topology of the channel according to the available experimental structure data. To ensure statistical representation, twenty different seeds were used for each I\u003csub\u003eKs\u003c/sub\u003e genotype group (see additional file 1), prompting a 4:4 stoichiometry and ensuring that the heterozygous form expressed two WT and two A8V MinK subunits. Each run generated five predictions, and the top ranked model from each seed was selected and added to the dataset for analysis. Generated structural predictions are subject to the AlphaFold Server Output Terms of Use. Quality control metrics such as pLDDT, pTM, ipTM and PAE were analyzed to assess the confidence of each model and treated as indicator variables of protein stability and subunit proximity. 3D models were uploaded to the SwissModel server via the user template approach to obtain MolProbity parameters as an additional layer of quality control. Simultaneously, .cif files were opened in YASARA View version 25.1.13 (YASARA Biosciences GmbH, Radboud University, Nijmegen, The Netherlands) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and the recently updated FoldX 5.1 plugin (Centre for Genomic Regulation, Barcelona, Spain) was used to optimize protein structure, calculate thermodynamic stability (ΔG) and interaction energy between Kv7.1/MinK subunits [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Then we compared our WT models against the two available experimental structures of the I\u003csub\u003eKs\u003c/sub\u003e channel (PDB ID 9u7f and 9vec) as an approach of validation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This comparison was performed by aligning the structures in PyMOL version 3.1.6.1 (The PyMOL Molecular Graphics System, Schr\u0026ouml;dinger LLC., New York) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and obtaining Root Mean Square Deviation values (RMSD) considering only the sequence in common (cycles\u0026thinsp;=\u0026thinsp;5, WT/WT cutoff=2\u0026Aring; and WT/mutant cutoff\u0026thinsp;=\u0026thinsp;1.5\u0026Aring;). The same strategy was adopted for comparison with the mutant groups, using our WT model as the reference. Finally, local structural changes were examined using Dynamut2 web server (Institute, University of Melbourne, Melbourne, Victoria, Australia) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The model with the closest ΔG to the median value within the WT group and its mutant versions was used to obtain representative images/diagrams. Please refer to additional file 1, Table\u0026nbsp;2 for the complete dataset of evaluated quantitative parameters.\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eData and statistical analysis\u003c/h2\u003e\n\u003cp\u003eElectrophysiologic data acquisition and analysis were performed using the suite of pCLAMP programs 9.2 (Axon Instruments, Union City, CA), Excel (Microsoft Corp., Redmond, WA) and OriginPro, version 2025 (OriginLab Corporation, Northampton, MA). Two-way ANOVA, Student\u0026apos;s \u003cem\u003et\u003c/em\u003e-test or Mann-Whitney Rank Sum test was performed employing SigmaStat 3.5 statistical software (Systat Software, Inc., Chicago, IL). Comparisons of structural and stability metrics were performed applying paired \u003cem\u003et\u003c/em\u003e-test or Wilcoxon Signed-Rank test. The summary of statistical analysis is available in additional file 1, tabs 3 and 4.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe procedures performed in this study involving human participants were conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. All the patients were allowed personal data processing and informed consent was obtained from the participants before proceeding to any methodology. All the experimental protocols in this study were reviewed and approved by the Main Line Hospitals Institutional Review Board (MLH IRB), with approval number E-22-5283.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the W.W. Smith Charitable Trust (HBM, JGAO, MBH, MH: H2205), Women\u0026rsquo;s Board from Lankenau Medical Center (HBM, JGAO, MBH, MH: 25401, 4101) and the Sharpe-Strumia Research Foundation of the Bryn Mawr Hospital (HBM, JGAO, MH, AWA: SSRF2025-07, SSRF2024-06).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eHBM led the study. HBM, JU, DJ, and JGAO conceived and designed the research. HBM, JU, AWA and JGAO analyzed and interpreted the data. JU, EB, GC and RP performed the experiments. HBM, JU, AWA and JGAO drafted the manuscript and substantively\u0026nbsp;reviewed it. MH and MBH contributed to patient selection. All authors reviewed and approved the definitive version of the manuscript for submission to BMC Biology. Every author validated their own contribution.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank Terri Olshefski for her valuable job in the proof-reading of the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe report of the presented variant is available in ClinVar (accession number SCV007542593). Representative models of the generated 3D structures were deposited in ModelArchive (https://modelarchive.org/) with the following accession IDs: WT: ma-2l8hk; homozygous: ma-eru9d; heterozygous: ma-a3ecv. Raw data and other information are available upon request.All AlphaFold 3 structural predictions reported here were obtained via the official AlphaFold Server (DeepMind/Isomorphic Labs) and are provided under, and subject to, the AlphaFold Server Output Terms of Use. Minor modifications to the output, including energy minimization and visualization coloring, were applied prior to publication.\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;\u003c/h3\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSanguinetti, M. C. et al. 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DynaMut2: Assessing changes in stability and flexibility upon single and multiple point missense mutations. \u003cem\u003eProtein Sci.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (1), 60\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/pro.3942\u003c/span\u003e\u003cspan address=\"10.1002/pro.3942\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"IKs, KCNE1, MinK, channelopathies, LQTS, arrhythmias, 3D Modeling, AlphaFold3, AI","lastPublishedDoi":"10.21203/rs.3.rs-9151984/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9151984/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eThe slow delayed rectifier potassium current (I\u003csub\u003eKs\u003c/sub\u003e), formed by the assembly of the Kv7.1 (\u003cem\u003eKCNQ1\u003c/em\u003e) and MinK (\u003cem\u003eKCNE1\u003c/em\u003e) subunits, is essential for cardiac repolarization. Variants of either protein may disrupt this current and contribute to different types of Long QT Syndrome (LQTS), conditions that might lead to arrhythmias and sudden cardiac death (SCD). However, the clinical significance of certain \u003cem\u003eKCNE1\u003c/em\u003e mutations remains ambiguous, particularly in asymptomatic carriers. Genetic screening and proper functional characterization of such variants represent crucial steps for surveillance and clinical management.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e \u003cp\u003eAmong the different LQTS-associated genes, a deleterious heterozygous mutation \u003cem\u003eKCNE1\u003c/em\u003e (c.23C\u0026thinsp;\u0026gt;\u0026thinsp;T)/ MinK-A8V was detected in an asymptomatic individual, which disturbs a well-conserved residue. Functional analysis \u003cem\u003ein vitro\u003c/em\u003e demonstrated a pronounced reduction of I\u003csub\u003eKs\u003c/sub\u003e when co-expressed in heterozygosity along with \u003cem\u003eKCNQ1\u003c/em\u003e, but the kinetics of activation of the I\u003csub\u003eKs\u003c/sub\u003e did not change, suggesting defects in trafficking mechanisms. Consistently, prediction of the N-terminal portion of MinK and structure analysis of AlphaFold3-derived I\u003csub\u003eKs\u003c/sub\u003e channels revealed less stability and structure confidence of the heterozygous form than in the WT and homozygous complexes.\u003c/p\u003e\u003ch2\u003eConclusion.\u003c/h2\u003e \u003cp\u003eMinK-A8V evaluation demonstrated a dominant-negative effect over I\u003csub\u003eKs\u003c/sub\u003e, which can lead to structural changes, a possible reduction in the stability of the channel complex and detrimental cardiac performance if present in an individual, depending on particular genetic, environmental or pharmacological backgrounds. Along with the results of other studies, it is suggestive that MinK-A8V variant may provoke defects in trafficking mechanisms of the I\u003csub\u003eKs\u003c/sub\u003e channel. Altogether, the present study supports the notion that c.23C\u0026thinsp;\u0026gt;\u0026thinsp;T in \u003cem\u003eKCNE1\u003c/em\u003e may act as modifier allele, underlying the importance of surveillance and/or clinical management of asymptomatic individuals and mild LQTS patients when carrying a \u003cem\u003eKCNE1\u003c/em\u003e variant.\u003c/p\u003e","manuscriptTitle":"Dominant-negative Effect of MinK-A8V Impairs I Ks and Disturbs Channel Structure: Importance of Clinical Surveillance in Mild LQTS Patients and Asymptomatic Carriers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 16:56:40","doi":"10.21203/rs.3.rs-9151984/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-04T14:36:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T14:22:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-05-04T13:34:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T18:41:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-30T17:14:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"575e780d-d576-46ae-b257-e721d6e7a9e5","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"7","date":"2026-05-04T14:36:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T14:22:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-05-04T13:34:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T18:41:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-30T17:14:56+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67658715,"name":"Health sciences/Cardiology"},{"id":67658716,"name":"Health sciences/Diseases"},{"id":67658717,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2026-05-12T16:56:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 16:56:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9151984","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9151984","identity":"rs-9151984","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}