Figure 2.

Fast- and slow-gating properties of K165 (filled symbols) and K165C* channels (open symbols) derived from macroscopic current recordings. (A) Comparison of the fast-gate P _o-V curves at 100.6 (circles) and 4.6 mM (squares) of [Cl⁻]_o. Solid curves were drawn according to a Boltzmann equation: P _o = P _min + (1 − P _min)/{1 + exp[−zF(V − V_1/2)/RT]}, with z = 0.8–1.2, P _min = 0.05–0.08. V_1/2s in 100.6 and 4.6 mM [Cl⁻]_o were: (K165) −76 and −14 mV; (K165C*) −31 and 35 mV. (B) V_1/2s of the fast-gate P _o-V curves as a function of [Cl⁻]_o (n = 3–8). (C) Opening rates, α, of the fast gate for K165 and K165C* channels in 100.6 and 4.6 mM [Cl⁻]_o. Symbols are the same as in A (n = 3–8). (D) Opening rates of the channels as a function of [Cl⁻]_o. Data points taken at −40 (•) and −80 mV (▪) for the K165 channel and at 0 (○) and −40 mV (□) for the K165C* channel. Solid and dotted curves were the best fit to the hyperbolic equation, α = α_max[Cl⁻]_o/(K_1/2 + [Cl⁻]_o). The fitted α_maxs are (ms⁻¹): K165, 0.29 (−40 mV) and 0.12 (−80 mV); K165C*, 0.12 (0 mV) and 0.04 (−40 mV). The fitted K_1/2s are (mM): K165, 107 (−40 mV) and 121 (−80 mV); K165C*, 103 (0 mV) and 108 (−40 mV). (E) Slow-gating transition examined by voltage activation. 30–40 μM MTSEA in the bath solution. Numbers 1 and 2 are the pulsing protocols (see materials and methods) used in the indicated periods (n = 3). Current amplitudes normalized to that of the point right before pulsing protocol 1. Dotted line represents zero-current level. (F) Temperature jump experiment revealed that the probability of closing the slow gate was only minimal upon raising the bath temperature. Pulsing protocol 1. 30–40 μM MTSEA was present. Current amplitudes normalized to that of the first point. T₁ = 21.4°C, T₂ = 27.5°C (n = 3).