Charge Reversal of the Uppermost Arginine in Sliding Helix S4-I Affects Gating of Cardiac Sodium Channel

Figures

• 

  Figure 1. (a) Membrane view of the cryoEM structure with activated VSDs (PDB ID: 6lqa). Repeats I, II, III, and IV are shown in pink, yellow, green, and gray, respectively. R219 forms a polar contact with glutamine Q859 in helix S5I of the pore domain, while glutamate E161 forms a salt bridge with arginine R222. (b) Deactivated VSDI visualized using coordinates from [18]. R219 forms a salt bridge with glutamate E161. Mutation R219E would cause electrostatic repulsion between the two glutamates.

• 

  Figure 2. Activated and deactivated VSDs in cryoEM structures of toxin-bound rNav1.5 and mutated hNav1.7. Shown are membrane (a,b) and extracellular (c,d) views of 3D-aligned cryo-EM structures with activated (green) and deactivated (magenta) VSDs. Upon VSD deactivation, state-dependent contacts of the uppermost arginines (R1) contribute to VSD stabilization. (a,c) rNav1.5 in the apo state (PDB ID: 6uz3) and in complex with gating-modifying toxin LQHIII (PDB ID: 7xsu). The toxin (cyan) downshifts S4IV by two helical turns (a). It also twists S4IV anticlockwise (c) and somewhat shifts helices in the membrane plane (c). In the toxin-bound deactivated state, R1 interacts with the uppermost D1_S2, which sequentially aligns with Nav1.5 glutamate E1_S2I. (c,d) WT channel hNav1.7 (PDB ID: 7xvf) and mutant channel with deactivated VSDI (PDB ID: 7xve). In the deactivated VSDI, R1 forms salt bridges with an asparagine in helix S3 and a glutamate in helix S2.

• 

  Figure 3. (a) VSDI in cryo-EM structure of hNav1.5 (PDB ID: 6lqa, green) and AF3 model of lone VSDI from hNav1.5 (gray). Arginine and glutamate residues are shown as sticks. (b) 3D aligned VSDI models generated by AF3: WT (gray), R1E (cyan), R1H (yellow), R1C (green), and R1P (magenta). R1 in the WT model and Glu in the R1E model do not make intersegment contacts. Cys in the R1C model formed a disulfide bond with a cysteine in helix S1, causing a downshift of S4I by two helical turns.

• 

  Figure 4. In silico deactivation of VSDI in the WT channel hNav1.5 (a,c) and mutant channel R219E (b,d). The backbones are shown by ribbons, with some parts removed for clarity. (a,b) membrane views. (c,d) Extracellular views. Note green color of sidechain carbon atoms in R2.

• 

  Figure 5. Generating mutant construct R219E-Nav1.5. (a) E161 and R219 in the topological model of the Nav1.5 channel. (b) Results of site-specific mutagenesis verified using the Sanger sequencing method.

• 

  Figure 6. Changes in Na+ current due to mutation of R219E. (a) Representative recordings of Na+ current for the WT and mutant channel R219E. (b) Distribution of Na+ current density at peak values. There was no significant difference for the WT and R219E channels. Each circle corresponds to an experiment with one cell (WT: n = 32; R219E: n = 21). (c) Volt-ampere characteristics of Na+ currents. Mutation R219E caused significant changes in the current density at some voltages. Asterisks (*) indicate significantly different values. (d) The voltage dependence of time to peak Na+ current for the WT and R219E channels. Mutation R219E caused a marked reduction in time to peak in the range from −50 to −35 mV. (e) Time of 50% decay for WT (black squares) and R219E (red circles) channels. Mutation R219E did not significantly influence the time of 50% decay.

• 

  Figure 7. Biophysical characteristics of wild-type WT (black squares) and R219E (red circles) channels. (a) Steady-state activation. Mutation R219E caused a significant shift (−9.6 mV) of the steady-state activation (WT: n = 32; R219E: n = 21). (b) Steady-state inactivation for the WT (black squares) and R219E (red circles) channels. Mutation caused a negative shift of steady-state inactivation (WT: n = 24; R219E: n = 15). (c) Steady-state fast inactivation for the WT (black squares) and R219E (red circles) channels. Mutation R219E cased a significant shift of (15 mV) of the fast inactivation curve towards hyperpolarized potentials (WT: n = 14; R219E: n = 14). (d) Time course of recovery from inactivation. Mutation R219E markedly decelerated recovery from inactivation.