Substitutions of the S4DIV R2 residue (R1451) in NaV1.4 lead to complex forms of paramyotonia congenita and periodic paralyses

Figure 1. Whole-cell properties of the WT, R1451C, and R1451L channels. (A) Representative whole-cell traces recorded from tsA201 cells expressing WT, R1451C, or R1451L channel. Currents were elicited from a holding potential of −140 mV and were depolarized to potentials ranging from −140 to +60 mV in 10-mV increments in 50-ms steps (see protocol in the inset under the R1451L traces). The dashed line represents zero current. (B) Normalized current/voltage relationships of the WT (n = 10), R1451C (n = 18), and R1451L (n = 18) channels. Current densities were measured by normalizing current amplitudes to membrane capacitance and were plotted against voltage. The experimental protocol was the same as in (A). (C) Histogram showing the average peak current densities recorded at −10 mV of the WT, R1451C, and R1451L channels. The R1451C and R1451L current densities decreased by 63% (***P < 0.001) and 46% (***P < 0.001), respectively, compared to the WT channel. R1451C current density decreased by 31% (*P < 0.05) compared to the R1451L channel.

Figure 2. Gating properties of the channels. (A) The voltage dependence of the inactivation time constants of the WT (n = 9), R1451C (n = 8), and R1451L (n = 15) channels. The time course of the current decay elicited at depolarized voltages was best fitted to a single exponential function, and the resulting time constants (τh) were plotted against voltage. The kinetics of fast inactivation were slower for the R1451C and R1451L channels, with R1451L being the slowest. The inset shows representative normalized Na+ currents elicited at −10 mV. The statistical significances of the differences between WT and R1451C/L (**P < 0.001, ***P < 0.001) and between R1451C and R1451L (#P < 0.05, ##P < 0.001, ###P < 0.001) are shown. (B) Steady-state activation and inactivation of the WT and the mutants. No significant shifts in the V1/2 of the R1451C/L channels were observed. Steady-state inactivation was determined using 10 ms test pulses to −10 mV after a 500-ms pre-pulse to potentials ranging from −140 mV to −30 mV (see protocol in the inset under the inactivation curves). The inactivation curves of R1451C and R1451L channels were left-shifted by −6 mV and −20 mV, respectively. The V1/2, the kv and…

Figure 3. Slow inactivation properties of WT, R1451C and R1451L channels. (A) Onsets of slow inactivation of WT (n = 10), R1451C (n = 7), and R1451L (n = 8) channels. The entry into slow inactivation was measured using a double-pulse protocol. The cells were first depolarized to −10 mV for a variable duration (1 ms to 30 s) from a holding potential of −120 mV. Following a 20-ms interpulse at −120 mV to allow complete recovery from fast inactivation, a brief 5-ms test pulse to −10 mV was applied to measure the available Na+ current (fraction not slow inactivated). The resulting curves were fitted to a double exponential equation, which yielded two time constants: τfast and τslow. There was no significant difference in the time constants of WT, R1451C, and R1451L channels. (B,C) Recovery from slow inactivation of WT (n = 7), R1451C (n = 6), and R1451L (n = 7) channels. The cells were depolarized to −10 mV for 4 s (B) or 30 s (C) from a holding potential of −120 mV to inactivate all Na+ channels. Test pulses were then applied for 20 ms to −10 mV to measure current amplitudes, with an interval ranging from 0.5 ms to 10 s. The resulting curves were fitted to a double exponential equation, which…

Figure 4. Effect of temperature on WT, R1451C and R1451L channels. (A) Representative whole-cell traces recorded at −10 mV of WT, R1451L and R1451C channels at 10 °C (blue), 15 °C (purple), 22 °C (black/orange/green) and 28 °C (red). The dashed line represents zero current. (B) Voltage dependence of inactivation time constants of WT (n = 9), R1451C (n = 8) and R1451L (n = 7) channels recorded at 10 °C. The time course of the current decay elicited at depolarized voltages was best fitted to a single exponential function, and the resulting time constants (τh) were plotted against voltage. The kinetics of fast inactivation were slower for all channels at all voltages by cooling to 10 °C. The statistical significances of the differences between the WT and R1451C/L channels (*P < 0.05, **P < 0.001, ***P < 0.001) are shown. (C) Arrhenius plots showing the temperature sensitivities of WT and R1451C/L channels. Unlike R1451C, the slope of the R1451L was significantly different from that of the WT as revealed by ANCOVA statistical test (**P < 0.005). The R1451 Each data point is a mean of 6–15 cells. (D) Steady-state inactivation (left curves) after a 500-ms prepulse was left-shifted for WT (n =…

Figure 5. Structural model of the resting state VSD of DIV of the WT, R1451C, and R1451L channels. (A) The backbones of the S1, S2, S3, and S4 transmembrane segments are represented as gray ribbons. The positively charged Arginine (R) residues of S4 are represented as blue balls, and the negative counter charges are in red. The conserved residues of S2 that define the hydrophobic seal are in yellow. The R1451C and R1451L substitutions are in green and orange, respectively. The hourglass-like solvent accessible volume is shown as a transparent turquoise surface. The hydrogen bonds between the charged residues are shown as pink dotted lines. For clarity, the dotted lines are between αC of the residues instead of the side chains. (B) Chart representing the water profile in the VSD. The number of water molecules is shown as a function of distance, where 0 Å is the center of the membrane. The dotted line indicates the limit of the hydrophobic septum. Less than one water molecule is found below this line. (C) Table indicating the hydrogen bonds shown in (A) and their distances in Å. Red circles highlight differences between mutant and WT channels.