ELUCIDATING THE DIFFERENTIAL IMPACTS OF EQUIVALENT GATING-CHARGE MUTATIONS IN VOLTAGE-GATED SODIUM CHANNELS

Abstract

Voltage-gated sodium (Na_v) channels are pivotal for cellular signaling and mutations in Na_v channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSD_I and VSD_II of the cardiac sodium channel Na_v1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 μs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure-function relationships in Na_v channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

Keywords: Disease-associated mutation; Nav channel; gating property; gating-pore current; molecular dynamics simulation.

Figure 1.. Stepwise deactivation of VSDs of Na_v channels.

Cartoon depiction of the sequential deactivation of the Na_v1.5 VSD in response to membrane potential changes. The S4 helix (blue) moves relative to static S1-S3 (gray), with negatively (−) charged countercharged residues (red) and the hydrophobic constriction site (HCS) residue (green). The S4 positively (+) charged gating charge residues adopt an up conformation under depolarized potential and transition to a down conformation as the potential recovers, illustrating the VSD’s dynamic nature.

Figure 2.. Multiple sequence alignment of VSDs from four repeats of nine isoforms in the human Na_v channel family

, highlighting countercharge residues (red), HCS (green), and gating charge residues (blue).

Figure 3.. Mapping the mutation hotspots in VSDs of Na_v channels.

The annotated disease-associated mutations from UniProt are mapped along the multiple sequence alignment (MSA) of VSDs in 9 human Na_v channels. The number of mutations at the equivalent position in 4 VSDs (A), the number of phenotypes in VSD_I (B), and that in VSD_II (C) are used to demonstrate the mutation hotspots. (B, C) pathogenic missense mutations are colored according to GoF (green bars), LoF (red), mixed (yellow), and uncertain (black) effects. Hotspots are labeled in this figure with either the order of gating charges (A) or residue IDs from Na_v1.5 (B, C).

Figure 4.. State transitions of VSD in a μs-scale MD simulation.

The dynamic behavior of VSD_II under an external electric field of 500mV in opposite directions. Traces of z positions for Cα atoms of the gating charge residues (R1-K5) in different colors in VSD_II relative to the HCS are shown to track the structural changes. Molecular images of representative VSD_II snapshots (0, 3, 6, 9, and 13 μs) are presented in a white ribbon representation, with licorice representations of gating charges (blue), countercharges (red), and the HCS (green). The direction and magnitude of the external electric field applied are depicted by arrows (blue and green).

Figure 5.. Differential impacts of R-to-Q mutations in VSD_I and VSD_II during up-to-down transitions.

The differential dynamic behaviors of R-to-Q mutations (R1, R2, and R3) in VSD_I (A) and VSD_II (B) in comparison to the wild type (WT) under an external electric field of −500mV. Traces of z positions for Cα atoms of the gating-charge residues in S4 relative to that of the HCS are shown in each panel to track the structural changes. Molecular images of VSDs are presented in a white cartoon representation, illustrating the initial and final frames with licorice representations of gating charges (blue), countercharges (red), and the HCS (green). Only one representative trajectory among three independent runs for each mutant is illustrated here.

Figure 6.. Comparative analysis of MD up-to-down transition rates for WT and R-to-Q mutations in VSD_I and VSD_II.

(A) Dynamic behaviors of VSD_I transitions, showing WT (black), R219Q (blue), R222Q (green), and R225Q (red). (B) Dynamic behaviors of VSD_II transitions, showing WT (black), R808Q (blue), R811Q (green), and R814Q (red). Data points represent results from three independent simulation runs, depicted by filled circles, empty squares, and filled squares. All simulations begin in the up state (light blue) under an applied external electric field of −500 mV, with transitions observed towards the intermediate state (orange) and down state (gray).

Figure 7.. Pore opening of mutants in VSD_I and VSD_II during MD simulations.

(A and B) Each panel depicts the time series of the minimum radius of the aqueous pathway of gating pore through VSDs during simulations. A 1.5 Å dashed line represents the potential pore opening threshold for water and ion permeation through VSDs. WT traces are presented in black, R1 mutants (R219Q and R808Q) in blue, R2 mutants (R222Q and R811Q) in green, and R3 mutants (R225Q and R814Q) in red. (C-D) Each panel shows molecular images of VSD_I (C) and VSD_II (D) WT and Mutants (S3 removed for clarity). In each snapshot, gating charges (blue), countercharges (red), HCS (green) residues are illustrated in licorice representation. Sodium (yellow) and chloride (gray) ions are depicted in spheres. Snapshots are selected at the time points indicated by black arrows in A and B. The water molecules are illustrated as a transparent red surface. The black dashed circles indicate the non-accessible solvent region, separating the extracellular and intracellular water milieu.

Figure 8.. State-dependent salt-bridge network analysis for R-to-Q mutants in VSD_I and VSD_II.

Comprehensive analysis for WT and mutants (R1, R2, and R3) in VSD_I (A) and VSD_II (B). Each panel depicts the map of electrostatic interactions at various states (up, intermediate, and down) within each VSD. Nodes represent residues of gating charges (blue), mutated residues (orange), countercharges (red), HCS (green), and lipids (black). Edges represent the salt bridges, with numbers indicating the occupancy percentage of the interaction during each state. Countercharges are labeled with residue IDs from Na_v1.5.