Fig. 4.

Functional characterization of WT and T592I Nav1.4 channel variants expressed in HEK283T cells. (A) Representative sodium current traces for T592I and WT. Sodium currents were elicited according the protocol shown inset. Interval between each pulse was 10 s. (B) Time constants for sodium channel entry in fast inactivation calculated from current decay monoexponential fit. (C) Normalized current-voltage (I-V) relationships were drawn from the sodium current traces recorded as shown in A. (D) Voltage dependence of activation and fast inactivation. For activation, the conductance was calculated from I-V relationships using the equation gNa = INa/(V –ENa), where gNa is the sodium conductance, INa is the peak sodium current, V is the membrane voltage, and ENa is the electrochemical gradient at equilibrium for sodium ions calculated from Nernst equation (ENa = +68.4 mV). The relationships (in green and black) were fitted to the Boltzmann equation gNa/gNa,max = 1/{1 + exp.((V-aV50)/Ka)}, where aV50 is the half-maximum activation voltage and Ka is the slope factor. Voltage dependence of fast inactivation was studied using a two-pulse voltage clamp protocol shown inset. The relationships (in blue and red), showing the normalized peak current amplitude recorded during the second pulse versus the first pulse membrane potential, were fitted to the Boltzmann equation INa/INa,max = 1 / {1 + exp.((V-fV50)/Kf)}, where fV50 is the half-maximum inactivation voltage, and Kf is the slope factor. (E) Window current resulting from the overlap of activation and fast inactivation relationships. (F) The voltage dependence of slow inactivation was studied using a three-pulse voltage clamp protocol. The normalized peak current amplitude during the third pulse was plotted versus the membrane potential of the first pulse. The relationships were fitted to the Boltzmann equation INa/INa,max = IR + (1 - IR)/{1 + exp.((V-sV50)/Ks)}, where sV50 is the half-maximum inactivation voltage, Ks is the slope factor, and IR is the steady fraction of non-inactivating channels. Data points in the relationships are mean±SEM from n cells. The fit parameter values are given in Table 2.