Inward NMDG⁺ currents recorded from HEK cells expressing Kv3.2b channels. (A) The pulse protocol used in the experiments shown in B–D. The cells were held at −100 mV and depolarized for 12 ms to potentials between −40 and +20 mV in 20-mV steps. The depolarization was applied at 0.5 Hz. (B–D) Current recordings from an untransfected cell (B) or a cell transiently expressing Kv3.2b channels (C and D) with symmetrical 140 mM NMDG⁺ containing solutions (B and C) or with 140 mM NMDG⁺ containing internal solution and 140 mM TEA⁺ containing external solution (D). The upward current deflections during the depolarizations in C and D are on-gating currents. The short dashed lines denote the zero current level in this and subsequent figures. (E) Averaged ratio of Q_off/Q_on as a function of the pulse potential. The amount of charge displaced during depolarization and repolarization (Q_on and Q_off) was obtained from the time integral of the currents during depolarizations and repolarizations shown in C and D (n = 5–8). The horizontal dotted line indicates Q_off/Q_on = 1.0. The solid line overlaying NMDG⁺ data are a fitting of normalized Kv3.2b channel tail currents (right ordinate) obtained with 135 mM K⁺_i/5 mM K⁺_o to a single Boltzmann function. The half-activation voltage (V_1/2) was 2.3 ± 3.0 mV, and the slope factor (k) was 9.1 ± 1.0 mV (n = 5).