Figure 2

Voltage-clamp properties ofNa _V 1.7/I228Mchannels in HEK293 cells. Electrophysiological analysis of I228M variant: (A) Representative current traces recorded from HEK293 cells expressing wild type Nav1.7 (WT) (top) or I228M (bottom) channels, evoked by voltage steps (100 mec) from -80 to +40 mV in 5 mV increments, from a holding potential of -120 mV. (B) Normalized I-V curves for WT and I228M expressing cells. (C) Activation and steady-state fast-inactivation for WT (black squares) and I228M (red circles). Fast-inactivation was examined using a series of 500 msec prepulses from -140 to 0 mV followed by test pulses to -10 mV. Left inset: midpoint values for fast inactivation (V_{1/2, fast-inact}) of WT (black) and I228M (red). Right inset: midpoint values for activation (V_{1/2, act}) of WT (black) and I228M (red). (D) Steady-state slow-inactivation of WT (black squares) and I228M (red circles). Slow-inactivation was assessed using a 20 msec pulse to -10 mV after a 30 second prepulse to potentials from -130 to 10 mV followed by a 100 msec pulse to -120 mV to remove fast-inactivation. Inset: midpoint values of slow-inactivation (V_{1/2, slow-inact}) (WT: black; I228M: red); *p < 0.05. V_1/2 represents voltage midpoint, I/I_maxrepresents normalized current, and G/G_max represents normalized conductance for fast-activation, slow-inactivation, and activation. (E) The kinetics of inactivation were analyzed by fitting data with a single exponential function for WT and I228M currents. (F) The kinetics of deactivation for WT and I228M expressing cells were obtained by holding the cells at -120 mV and tail currents were generated by a brief 0.5 ms depolarization to -20 mV followed by a series of repolarizations ranging from -120 to -40 mV. The closing rate of the channels was obtained by fitting the tail currents with a single exponential function.