Elementary mechanisms of calmodulin regulation of Na _V 1.5 producing divergent arrhythmogenic phenotypes

Abstract

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.

Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current

“…A prime example of such incongruity stems from our prior analysis of calmodulin (CaM) regulation of Na V 1.5. In heterologous cells, mutations that weaken CaM binding yield a consistent increase in I Na,L 27 , 28 ; yet, ablating CaM binding to Na V 1.5 paradoxically showed no such change in murine ventricular cardiomyocytes 25 . Elucidating cellular mechanisms that orchestrate I Na,L is a high priority for two reasons.…”

Calmodulin Interactions with Voltage-Gated Sodium Channels

“…Mutation at the position of 1904 (S1904L) perturbed the Na v 1.5 channel interaction with CaM. In the absence of Ca 2+ , a flow cytometric FRET two-hybrid analysis between the S1904L and CaM demonstrated a weaker affinity compared to the wild-type Na v 1.5 channel [15]. Additionally, S1904L induced an enhanced late sodium current, indicating a delayed inactivation of the channel caused by CaM dysregulation of the channel in this mutation [76].…”

“…CaM binds to the voltage-gated sodium channel and modulates the sodium channel gating process [15], alters sodium current density [18], and regulates sodium channel protein trafficking and expression [19].…”

“…The residues in the IQ domain form a seven-turn α-helix binding to CaM in a Ca 2+ -independent manner. In addition to the IQ domain, a globular domain in the C-terminus of voltage-gated sodium channel consists of EF hand-like (EFL) motifs that interact with CaM in the absence of Ca 2+ [15,16], whereas an intracellular loop connecting domains III and IV (III-IV linker) in some isoforms of the sodium channel is shown to interact with CaM in the presence of Ca 2+ [17].…”

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias

“…Furthermore, calmodulin (CaM), a ubiquitous Ca2 + -sensing protein, has been reported to interact with Na v 1.5 N-and C-terminal regions [294][295][296][297] and the DIII-IV linker [174,295]. This interaction has been demonstrated to enhance slow inactivation and modulate Na v 1.5 gating [296], while disruption of CaM binding to Na v 1.5 decreases channel activity and enhances the propensity for persistent Na + current, all resulting from a switch in the Na V inactivation mechanism [297]. Na v 1.5-CaM interaction has been further studied in a mutational context related to cardiac sodium channelopathies (See Section 4).…”