FIGURE 3.
Biophysical properties of Kv3. 3 channels. A, representative current traces and current-voltage relationship for Kv3.3 currents recorded from CHO cells in the whole-cell configuration. Currents were evoked by applying 800-ms voltage steps from a holding potential of -70 to + 70 mV in 10-mV increments. Conductance values were obtained by dividing current with electrochemical driving force (IK/(Vm - EK)). Normalized conductance-voltage plots were obtained by dividing conductance (G) with maximal conductance (Gmax) and fitting with Boltzmann equation. The plots of τ of activation (B, upper panel) and τ of inactivation (B, lower panel) were determined, respectively, by fitting the rising and inactivating phases of currents obtained in A with a single term exponential equation. C, steady-state inactivation (h730 ms) of Kv3.3 was determined by holding the membrane potential from a pre-pulse potential ranging from -100 to +50 mV in 10-mV increments for 730 ms to a test voltage step of +20 mV for 250 ms. Current amplitude was normalized to the maximum current, and the resulting plot was fit with the Boltzmann equation. D, τ of recovery from inactivation was determined by stepping the cells from -100 mV to 20 mV for 2000 ms to a -100-mV step of variable durations in 100- or 7.5-ms increments immediately followed by a test step to 40 mV for 350 ms. Normalized amplitudes (I) to maximum (Imax) values at 40-mV steps were plotted against the inter-pulse interval and fitted with a single exponential decay equation. E, for determining the τ of deactivation, the cells were held at -70 mV, stepped to +50 mV for 5 ms, and then stepped from -110 to 40 mV in 10-mV increments for 10 ms. The deactivating phase of the currents was fit with a single function exponential equation. F and G show single channel properties of Kv3.3 in CHO cells. F, representative cell-attached single channel current traces and their corresponding amplitude histograms at different voltages. Only three of the 10 traces obtained for each voltage are shown; however, all 10 were used for analysis. The patch was held at -80 mV and stepped to different voltages for 1500 ms. G, mean current-voltage (I-V) relationships of channels obtained by determining current amplitude at first level at different voltages (n = 7). The slope of the line fitted on this I-V plot is 15.17 ± 1.9 pS. H, normalized N(Po) values determined for different voltages (n = 6).