Figure 5.

The gating of S460G:A463C:T469V and A463C in symmetrical potassium solutions. All experiments were performed in symmetrical 140 mM KCl. Representative activation current families for S460G:A463C:T469V (A) and A463C (B). The currents were elicited by 50-ms steps from −100 to +60 mV, followed by a 25-ms step to −65 (A) and −75 (B) mV. (C) Conductance–voltage relationships for ShBΔ6-46 (♦), S460G:A463C:T469V (•), and A463C (▴) were calculated from tail currents such as those in A and B. Error bars represent SEM. The smooth curves through the data represent fourth power Boltzmann fits. The ShBΔ6-46 data points (n = 10) were best fit with V_1/2 = −75.7 mV and z = 2.4 e. The triple mutant data (n = 5) were best fit with V_1/2 = −60.2 mV and z = 1.8 e. The A463C data (n = 7) were best fit with V_1/2 = −61.7 mV and z = 1.1 e. The steady state GV relationships for S460G and T469V were best fitted with V_1/2 = −71.5 mV (n = 8) and −76.5 mV (n = 3), respectively (data not shown). Deactivation was studied by tail current families generated by stepping to +50 mV for 25 ms, followed by 25-ms steps from +50 to −200 mV in 10-mV decrements for S460G:A463C:T469V (D) and 15-ms repolarizing steps for A463C (E). Deactivation time constants were obtained from single exponential fits to tail currents such as those shown in D and E. The wild-type (ShBΔ6-46) (♦; n = 7) time constant of deactivation changes e-fold per 30.0 mV, the triple mutant (•, n = 7) changes e-fold per 27.9 mV, and A463C (▴; n = 9) time constant of deactivation changes e-fold per 32 mV. The time constants of deactivation changes for S460G and T469V were e-fold per 31.7 (n = 9) and 28.9 (n = 5) mV (data not shown).