In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

The Ca 2+ -binding protein calmodulin has emerged as a pivotal player in tuning Na + channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the Na V 1.5 interactome in regulating late Na + current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human Na V 1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na + current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human Na V 1.5 demonstrated increased late Na + current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na + current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the Na V 1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing Na V 1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na + current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na + current.

Rationale: Changing activity of cardiac Ca V 1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently up-regulated via activation of protein kinase A, essential molecular details remained stubbornly enigmatic. Objective: The primary goal of this study was to determine how various factors converging at the Ca V 1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. Methods and Results: We generated transgenic mice with expression of Ca V 1.2 α 1C subunits with: 1) mutations ablating interaction between α 1C and β subunits; 2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α 1C ); or 3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 1C up-regulated in the hypertrophied heart. Introducing three glycine residues that disrupt a rigid IS6-AID helix markedly reduced basal open probability despite intact binding of Ca V β to α 1C I-II loop, and eliminated β-adrenergic agonist stimulation of Ca V 1.2 current. In contrast, introduction of the exon 9* splice variant in α 1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β 2B and Rad or transgenically expressed in cardiomyocytes. Conclusions: Ca 2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α 1C I-II loop. Ca V β binding to α 1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca 2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β binding to α 1C .

Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of Na V 1.5 inactivation results in a small persistent Na influx known as late Na current ( I Na,L ), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. Here, using low-noise multi-channel recordings in heterologous systems, LQTS3 patient-derived iPSCs cardiomyocytes, and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1–4) tune pathogenic I Na,L in an isoform-specific manner. This scheme suggests a complex orchestration of I Na,L in cardiomyocytes that may contribute to variable disease expressivity of Na V 1.5 channelopathies. We further leverage these observations to engineer a peptide-inhibitor of I Na,L with a higher efficacy as compared to a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of I Na,L in pathophysiology and outline potential therapeutic avenues.

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback. Yet, biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments suggesting that functional Ca2+-regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaM to the CaV nanodomain by tethering either wild-type or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+-regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxy-tail switched the apparent functional stoichiometry permitting two CaMs to mediate functional regulation. In all, Ca2+-feedback of CaV channels depends exquisitely on a single CaM pre-associated to the α-subunit carboxy-tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-depended feedback of channel gating but may support alternate regulatory functions.

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channel b-subunit genes revealed a mutation in the SCN2B gene, which we have functionally tested. Current-clamp analysis revealed that this mutant b2 subunit rendered small-diameter dorsal root ganglion (DRG) neurons hyperexcitable compared to DRG neurons expressing the wild-type b2 subunit. Additionally, b-subunits are known to increase sodium current density and alter biophysical properties of sodium channels. Voltageclamp analysis of small DRG neurons expressing the variant b2 subunit showed increased total sodium current density. As b-subunits can interact with all Na v channels, we subsequently investigated the effect of this mutation on TTXsensitive vs. TTX-resistant sodium channels. Results of this analysis and on the effect of the expression of the mutant b-subunit on gating properties of sodium channels will be presented.