Gating pore currents in R666G at depolarized and hyperpolarized holding potentials. Currents were recorded in the presence of 1 µM TTX in response to a series of 50-ms voltage steps ranging from −160 mV to 50 mV in 5-mV increments from a holding potential of −100 mV (left panels and black symbols) or 0 mV (right panels and red symbols) without any leak subtraction. The external solution contained 120 mM NaOH, 1.8 mM Ca(OH)2, and 10 mM HEPES, pH 7.4, with methanesulfonic acid. Currents through the wild-type NaV1.4 (A) and R666G (B) channels are shown on a scale where both gating charge displacement and gating pore currents can be observed and compared. Every fourth trace is shown for clarity. (C) Records from B are shown on an expanded scale, focusing on gating pore currents. Every second trace is shown for clarity. (D) Voltage dependence of representative currents in A and B before leak subtraction (open symbols): diamonds, NaV1.4 wild type; circles, R666G. Dotted lines are linear fits to the data in the voltage range between 0 and 40 mV used for offline subtraction of linear leak through cell membrane. (E) Voltage dependence of leak-subtracted gating pore currents (closed symbols): diamonds, NaV1.4 wild type; circles, R666G.
