Investigation and Computational Prediction of Gating Pore Currents in NaV1.2 Mutations Across Clinical Phenotypes

Figure 1. Inward gating pore currents (Igp) induced by NaV1.2 mutations. A) Representative traces of Igp conducted by NaV1.2 (R220G, R223I, and R223Q) compared to nonspecific leak current by WT. B) Current–voltage (I–V) curves of NaV1.2 mutants (R220G, R223I, and R223Q) and leak current of WT NaV1.2. Cells were held at −80 mV, and 20 ms voltage steps from −200 mV to −10 mV in 10 mV increments were applied. Inward Igp carried by Na+ started at −60 mV for R220G, while it started at −100 mV for both R223I and R223Q. n = 7, 8, 9, and 8 for R220G, R223I, R223Q, and WT, respectively. C) I–V curves of NaV1.2(R220G, R223I, and R223Q) and nonspecific leak current for NaV1.2(WT) when cells were held at −40 mV. Inward Igp carried by Na+ were induced by R220G, R223I, and R223Q starting at −80 mV and increased at negative potentials. n = 5, 7, 8, and 6 for R220G, R223I, R223Q, and WT, respectively. Error bars indicate SEM. The extracellular patch-clamp solution contained: 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES. D) R220G, R223I, and R223Q did not produce outward Igp when cells were held at −40 mV. E) In the absence of CaCl2 and MgCl2 in the extracellular solution, the amplitudes of Igp…

Figure 2. Voltage-dependent activation, inactivation and recovery from inactivation of NaV1.2 mutants. A) Representative families of current traces elicited by voltage steps from −100 to +40 mV from a holding potential of −120 mV induced by human NaV1.2WT, R220G, R223I, and R223Q. B, C) Voltage-dependent steady-state activation curves show a significant hyperpolarizing shift in NaV1.2(R220G) and NaV1.2(R223I) mutants and a depolarizing shift in NaV1.2(R223Q). D, E) The voltage dependence of inactivation was determined using 100-ms conditioning pulses to various potentials ranging from −120 to +70 mV in 10 mV increments. The remaining sodium current was subsequently elicited via a 20 ms long test pulse at −20 mV. Voltage-dependent steady-state inactivation curves indicate a significant hyperpolarizing shift in the inactivation of NaV1.2(R220G and R223I), while R223Q showed a similar inactivation pattern to NaV1.2WT. F) To determine the time constants of fast inactivation (th), a second-order exponential function was fit to the time course of fast inactivation from the peak current at each pulse to 20 ms after the onset of the depolarization, yielding two time constants, with only the fast…

Figure 3. Pore opening of VSDI mutants during MD simulations. Time series of the minimum radius of the aqueous pathway of gating pore through the VSD during trajectory simulations. Each panel represents the minimum pore radius over 1 μs for different systems: WT (A, black), R220G (B, blue), R223I (C, green), and R223Q (D, red). A 1.5 Å threshold (dashed line) is indicated as a potential criterion for water and ion permeation. The rightmost panels show the histogram of minimum radius of gating pore, representing collective frequency of the gating pore-opening events among three independent runs.

Figure 4. Differential dynamics of VSDI gating charges across conformational states in WT and mutant NaV1.2 channels. A) Z-position traces of the Cα atoms of gating-charge residues relative to the HCS (Y166) over 1 μs of simulation under an applied electric field of −200 mV. Each panel corresponds to a representative trajectory for WT (Traj 2), R220G (Traj 4), R223I (Traj 7), and R223Q (Traj 10). Gray shaded regions denote time intervals assigned to each labeled conformational state (down, pre, or intermediate) based on the z-position of the gating charges relative to Y166 (see Materials and methods). B) Molecular representations of VSDI in the Down, Pre, and Intermediate conformational ensembles for WT, R220G, R223I, and R223Q. VSDI is shown in white cartoon representation. Gating-charge residues (R217, R220, R223, and K226) are depicted as blue licorice; the HCS residue Y166 is shown in green; acidic counter-charge residues E159 and E169 are shown in red; and mutant side chains (G220, I223, and Q223) are highlighted in orange. Conformational ensembles were computationally inferred from simulation trajectories and do not represent experimentally resolved structures.

Figure 5. State-dependent interaction network analysis for gating charges mutants in VSDI. State-dependent comprehensive interaction map with occupancy of WT and mutants. This figure illustrates the key interactions observed in different structural states (down, pre, and intermediate) of the WT and mutant systems. The percentage values in black represent the proportion of time the system spends in each state during simulations, while the percentage values in red indicate the occurrence of gating pore opening within that state. Black lines: salt-bridge interactions. Light blue lines: hydrogen bond interactions. Purple dashed lines: cation–π interactions. * Orange dashed lines: collective hydrophobic interactions between I223 and residues W191, L194, and V198. Interactions <5% are not illustrated. ** State labels (down, pre, and intermediate) refer to MD-derived VSDI conformational ensembles defined by the z-position of R220 relative to the HCS residue Y166 (see Materials and methods) and do not represent distinct experiment.