Identification of structures for ion channel kinetic models.

Markov models of ion channel dynamics have evolved as experimental advances have improved our understanding of channel function. Past studies have examined limited sets of various topologies for Markov models of channel dynamics. We present a systematic method for identification of all possible Markov model topologies using experimental data for two types of native voltage-gated ion channel currents: mouse atrial sodium currents and human left ventricular fast transient outward potassium currents. Successful models identified with this approach have certain characteristics in common, suggesting that aspects of the model topology are determined by the experimental data. Incorporating these channel models into cell and tissue simulations to assess model performance within protocols that were not used for training provided validation and further narrowing of the number of acceptable models. The success of this approach suggests a channel model creation pipeline may be feasible where the structure of the model is not specified a priori.

Predicting Patient Response to the Antiarrhythmic Mexiletine Based on Genetic Variation.

RATIONALE: Mutations in the SCN5A gene, encoding the α subunit of the Nav1.5 channel, cause a life-threatening form of cardiac arrhythmia, long QT syndrome type 3 (LQT3). Mexiletine, which is structurally related to the Na+ channel-blocking anesthetic lidocaine, is used to treat LQT3 patients. However, the patient response is variable, depending on the genetic mutation in SCN5A. OBJECTIVE: The goal of this study is to understand the molecular basis of patients' variable responses and build a predictive statistical model that can be used to personalize mexiletine treatment based on patient's genetic variant. METHODS AND RESULTS: We monitored the cardiac Na+ channel voltage-sensing domain (VSD) conformational dynamics simultaneously with other gating properties for the LQT3 variants. To systematically identify the relationship between mexiletine block and channel biophysical properties, we used a system-based statistical modeling approach to connect the multivariate properties to patient phenotype. We found that mexiletine altered the conformation of the Domain III VSD, which is the same VSD that many tested LQT3 mutations affect. Analysis of 15 LQT3 variants showed a strong correlation between the activation of the Domain III-VSD and the strength of the inhibition of the channel by mexiletine. Based on this improved molecular-level understanding, we generated a systems-based model based on a dataset of 32 LQT3 patients, which then successfully predicted the response of 7 out of 8 patients to mexiletine in a blinded, retrospective trial. CONCLUSIONS: Our results imply that the modulated receptor theory of local anesthetic action, which confines local anesthetic binding effects to the channel pore, should be revised to include drug interaction with the Domain III-VSD. Using an algorithm that incorporates this mode of action, we can predict patient-specific responses to mexiletine, improving therapeutic decision making.

Gating control of the cardiac sodium channel Nav1.5 by its ß3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

The auxiliary ß3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some ß3 mutations predispose individuals to cardiac arrhythmias. The ß3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the ß3 transmembrane region and its potential for functionally synergizing with the ß3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT ß3-subunit and in a ß3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed ß3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT ß3-subunit. Removal of the ß3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the ß3-Glu-176 residue and the ß3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation.

How to Connect Cardiac Excitation to the Atomic Interactions of Ion Channels.

Many have worked to create cardiac action potential models that explicitly represent atomic-level details of ion channel structure. Such models have the potential to define new therapeutic directions and to show how nanoscale perturbations to channel function predispose patients to deadly cardiac arrhythmia. However, there have been significant experimental and theoretical barriers that have limited model usefulness. Recently, many of these barriers have come down, suggesting that considerable progress toward creating these long-sought models may be possible in the near term.

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel.

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

BACKGROUND: Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. METHODS AND RESULTS: We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. CONCLUSIONS: The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism.

Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening.

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein-protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP2. This result is due to loss of coupling because PIP2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP2-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K(+) channels, suggesting that lipids play an important role in coupling in many ion channels.

Modeling K,ATP--dependent excitability in pancreatic islets.

In pancreatic ?-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic ?-cell excitability reproduces the ?-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca(2+) channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of ?-cell excitability and suggests future experiments that will lead to improved characterization of ?-cell excitability and the control of insulin secretion.

Creating Computational Models of Ion Channel Dynamics.

Markov models are widely used to represent ion channel protein configurations as different states in the model's topology. Such models allow for dynamic simulation of ion channel kinetics through the simulated application of voltage potentials across a cell membrane. In this chapter, we present a general method for creating Markov models of ion channel kinetics using computational optimization alongside a fully featured example model of a cardiac potassium channel. Our methods cover designing training protocols, iteratively testing potential model topologies for structure identification, creation of algorithms for model simulation, as well as methods for assessing the quality of fit for a finalized model.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

OBJECTIVE: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. APPROACH AND RESULTS: Whole-cell voltage-clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance, and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae, and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF. CONCLUSIONS: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs.

Emerging methods to model cardiac ion channel and myocyte electrophysiology.

In the field of cardiac electrophysiology, modeling has played a central role for many decades. However, even though the effort is well-established, it has recently seen a rapid and sustained evolution in the complexity and predictive power of the models being created. In particular, new approaches to modeling have allowed the tracking of parallel and interconnected processes that span from the nanometers and femtoseconds that determine ion channel gating to the centimeters and minutes needed to describe an arrhythmia. The connection between scales has brought unprecedented insight into cardiac arrhythmia mechanisms and drug therapies. This review focuses on the generation of these models from first principles, generation of detailed models to describe ion channel kinetics, algorithms to create and numerically solve kinetic models, and new approaches toward data gathering that parameterize these models. While we focus on application of these models for cardiac arrhythmia, these concepts are widely applicable to model the physiology and pathophysiology of any excitable cell.

Arrhythmia-associated Calmodulin Variants Interact with KCNQ1 to Confer Aberrant Membrane Trafficking and Function.

Rationale: Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates. As CaM regulates several key cardiac ion channels, a mechanistic understanding of CaM variant-associated arrhythmias requires elucidating individual CaM variant effect on distinct channels. One key CaM regulatory target is the KCNQ1 (K V 7.1) voltage-gated potassium channel that underlie the I Ks current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function. Objective: To observe how arrhythmia-associated CaM variants affect binding to KCNQ1, channel membrane trafficking, and KCNQ1 function. Methods and Results: We combine a live-cell FRET binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants effect on KCNQ1. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared to CaM wild-type over physiological Ca 2+ ranges. We further identify several CaM variants that affect KCNQ1 and I Ks membrane trafficking and/or baseline current activation kinetics, thereby contextualizing KCNQ1 dysfunction in calmodulinopathy. Lastly, we delineate CaM variants with no effect on KCNQ1 function. Conclusions: This study provides comprehensive functional data that reveal how CaM variants contribute to creating a pro-arrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function. This study provides a new approach to collecting details of CaM binding that are key for understanding how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels.

FHF2 phosphorylation and regulation of native myocardial Na V 1.5 channels.

Phosphorylation of the cardiac Na V 1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of Na V 1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of Na V channel complexes purified from mouse left ventricles, we identified nine phosphorylation sites on Fibroblast growth factor Homologous Factor 2 (FHF2). To determine the roles of phosphosites in regulating Na V 1.5, we developed two models from neonatal and adult mouse ventricular cardiomyocytes in which FHF2 expression is knockdown and rescued by WT, phosphosilent or phosphomimetic FHF2-VY. While the increased rates of closed-state and open-state inactivation of Na V channels induced by the FHF2 knockdown are completely restored by the FHF2-VY isoform in adult cardiomyocytes, sole a partial rescue is obtained in neonatal cardiomyocytes. The FHF2 knockdown also shifts the voltage-dependence of activation towards hyperpolarized potentials in neonatal cardiomyocytes, which is not rescued by FHF2-VY. Parallel investigations showed that the FHF2-VY isoform is predominant in adult cardiomyocytes, while expression of FHF2-VY and FHF2-A is comparable in neonatal cardiomyocytes. Similar to WT FHF2-VY, however, each FHF2-VY phosphomutant restores the Na V channel inactivation properties in both models, preventing identification of FHF2 phosphosite roles. FHF2 knockdown also increases the late Na + current in adult cardiomyocytes, which is restored similarly by WT and phosphosilent FHF2-VY. Together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate differential roles for FHF2 in regulating neonatal and adult mouse ventricular Na V 1.5, and suggest that the regulation of Na V 1.5 by FHF2 phosphorylation is highly complex. eTOC Summary: Lesage et al . identify the phosphorylation sites of FHF2 from mouse left ventricular Na V 1.5 channel complexes. While no roles for FHF2 phosphosites could be recognized yet, the findings demonstrate differential FHF2-dependent regulation of neonatal and adult mouse ventricular Na V 1.5 channels.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

Objective: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (K ATP ) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with distinct hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Approach and Results: Whole-cell voltage-clamp of isolated aortic and mesenteric VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no difference in voltage-gated K + (K v ) or Ca 2+ currents. K v and Ca 2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. Pinacidil-sensitive K ATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, and were considerably larger in CS hiPSC-VSMCs. Consistent with lack of any compensatory modulation of other currents, this resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Increased compliance and dilation in isolated CS mouse aortae, was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular K ATP GoF. Conclusions: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. The results further indicate that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by K ATP overactivity within VSMCs.

Use of a mixed reality system for navigational mapping during cardiac electrophysiological testing does not prolong case duration: A subanalysis from the Cardiac Augmented REality study.

Background: CommandEP™ is a mixed reality (MXR) system for cardiac electrophysiological (EP) procedures that provides a real-time 3-dimensional digital image of cardiac geometry and catheter locations. In a previous study, physicians using the system demonstrated improved navigational accuracy. This study investigated the impact of the CommandEP system on EP procedural times compared to the standard-of-care electroanatomic mapping system (EAMS) display. Objective: The purpose of this retrospective case-controlled analysis was to evaluate the impact of a novel MXR interface on EP procedural times compared to a case-matched cohort. Methods: Cases from the Cardiac Augmented REality (CARE) study were matched for diagnosis and weight using a contemporary cohort. Procedural time was compared from the roll-in and full implementation cohort. During routine EP procedures, operators performed tasks during the postablation waiting phase, including creation of cardiac geometry and 5-point navigation under 2 conditions: (1) EAMS first; and (2) CommandEP. Results: From a total of 16 CARE study patients, the 10 full implementation patients were matched to a cohort of 20 control patients (2 controls:1 CARE, matched according to pathology and age/weight). No statistical difference in total case times between CARE study patients vs control group (118 ± 29 minutes vs 97 ± 20 minutes; P = .07) or fluoroscopy times (6 ± 4 minutes vs 7 ± 6 minutes; P = .9). No significant difference in case duration for CARE study patients comparing roll-in vs full-implementation cohort (121 ± 26 minutes vs 118 ± 29 minutes; P = .96). CommandEP wear time during cases was significantly longer in full implementation cases (53 ± 24 minutes vs 24 ± 5 minutes; P = .0009). During creation of a single cardiac geometry, no significant time difference was noted between CommandEP vs EAMS (284 ± 45 seconds vs 268 ± 43 seconds; P = .1) or fluoroscopy use (9 ± 19 seconds vs 6 ± 18 seconds; P = .25). During point navigation tasks, there was no difference in total time (CommandEP 31 ± 14 seconds vs EAMS 28 ± 15 seconds; P = .16) or fluoroscopy time (CommandEP 0 second vs EAMS 0 second). Conclusion: MXR did not prolong overall procedural time compared to a matched cohort. There was no prolongation in study task completion time. Future studies with experienced CommandEP users directly assessing procedural time and task completion time in a randomized study population would be of interest.

Differential regulation of cardiac sodium channels by intracellular fibroblast growth factors.

Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I-IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11-iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.

Arrhythmia-associated calmodulin variants interact with KCNQ1 to confer aberrant membrane trafficking and function.

Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates ("calmodulinopathy"). As CaM regulates many key cardiac ion channels, an understanding of disease mechanism associated with CaM variant arrhythmias requires elucidating individual CaM variant effects on distinct channels. One key CaM regulatory target is the KCNQ1 (KV7.1) voltage-gated potassium channel that carries the IKs current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function. Here, we take a multipronged approach employing a live-cell fluorescence resonance energy transfer binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants for effect on KCNQ1 CaM binding, membrane trafficking, and channel function. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared with CaM wild-type over physiological Ca2+ ranges. We further identify several CaM variants that affect KCNQ1 and IKs membrane trafficking and/or baseline current activation kinetics, thereby delineating KCNQ1 dysfunction in calmodulinopathy. Lastly, we identify CaM variants with no effect on KCNQ1 function. This study provides extensive functional data that reveal how CaM variants contribute to creating a proarrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function, suggesting how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels. Classification: Biological, Health, and Medical Sciences, Physiology.

Determinants of iFGF13-mediated regulation of myocardial voltage-gated sodium (NaV) channels in mouse.

Posttranslational regulation of cardiac NaV1.5 channels is critical in modulating channel expression and function, yet their regulation by phosphorylation of accessory proteins has gone largely unexplored. Using phosphoproteomic analysis of NaV channel complexes from adult mouse left ventricles, we identified nine phosphorylation sites on intracellular fibroblast growth factor 13 (iFGF13). To explore the potential roles of these phosphosites in regulating cardiac NaV currents, we abolished expression of iFGF13 in neonatal and adult mouse ventricular myocytes and rescued it with wild-type (WT), phosphosilent, or phosphomimetic iFGF13-VY. While the increased rate of closed-state inactivation of NaV channels induced by Fgf13 knockout in adult cardiomyocytes was completely restored by adenoviral-mediated expression of WT iFGF13-VY, only partial rescue was observed in neonatal cardiomyocytes after knockdown. The knockdown of iFGF13 in neonatal ventricular myocytes also shifted the voltage dependence of channel activation toward hyperpolarized potentials, a shift that was not reversed by WT iFGF13-VY expression. Additionally, we found that iFGF13-VY is the predominant isoform in adult ventricular myocytes, whereas both iFGF13-VY and iFGF13-S are expressed comparably in neonatal ventricular myocytes. Similar to WT iFGF13-VY, each of the iFGF13-VY phosphomutants studied restored NaV channel inactivation properties in both models. Lastly, Fgf13 knockout also increased the late Na+ current in adult cardiomyocytes, and this effect was restored with expression of WT and phosphosilent iFGF13-VY. Together, our results demonstrate that iFGF13 is highly phosphorylated and displays differential isoform expression in neonatal and adult ventricular myocytes. While we found no roles for iFGF13 phosphorylation, our results demonstrate differential effects of iFGF13 on neonatal and adult mouse ventricular NaV channels.

A multiscale model linking ion-channel molecular dynamics and electrostatics to the cardiac action potential.

Ion-channel function is determined by its gating movement. Yet, molecular dynamics and electrophysiological simulations were never combined to link molecular structure to function. We performed multiscale molecular dynamics and continuum electrostatics calculations to simulate a cardiac K(+) channel (I(Ks)) gating and its alteration by mutations that cause arrhythmias and sudden death. An all-atom model of the I(Ks) alpha-subunit KCNQ1, based on the recent Kv1.2 structure, is used to calculate electrostatic energies during gating. Simulations are compared with experiments where varying degrees of positive charge-added via point mutation-progressively reduce current. Whole-cell simulations show that mutations cause action potential and ECG QT interval prolongation, consistent with clinical phenotypes. This framework allows integration of multiscale observations to study the molecular basis of excitation and its alteration by disease.

Updates on the inherited cardiac ion channelopathies: from cell to clinical.

OPINION STATEMENT: The inherited channelopathies are a rare, heterogeneous group of diseases with widely variable clinical presentations and courses. Systematic clinical and experimental work has led to identification of disease-causing genetic mutations and their biophysical manifestation. The process by which the knowledge base is developed, from genetic mutation, to cardiac myocyte, to whole heart, and finally to clinical presentation, has dramatically expanded our understanding of these diseases. Most importantly, we can now begin to comprehend how small changes at the genetic level can dramatically influence a patient's clinical course.