FIGURE 8.

(A) Kinetic modeling of the modulation of I_US by Cd²⁺. Hypothetical scheme of the proposed kinetic states and transitions. Upper row depicts Cd²⁺-free states: NI = noninactivated, I_S = slow inactivated, I_US = ultraslow inactivated. Lower row: Cd²⁺-bound states. (B) Time course of recovery from slow inactivation. From a holding potential of −120 mV, channels were depolarized to −20 mV for 25 s. Thereafter, the membrane potential was returned to −120 mV for the indicated time periods after which channel availability was tested by a 20-ms pulse to −20 mV. Peak inward currents were normalized to the value after full recovery. The time course of recovery was determined during a control period and during superfusion with 50 μM Cd²⁺. Data points were fitted with double exponential functions (Eq. 5). The parameters were drug free: A₁ = 0.62 ± 0.04, τ₁ = 573.2 ± 32.3 ms, A₂ = 0.30 ± 0.02, τ₂ = 6493.4 ± 56.3 ms; Cd²⁺: A₁ = 0.61 ± 0.07, τ₁ = 663 ± 64.3 ms, A₂ = 0.29 ± 0.03 (P = n.s.), τ₂ = 6828.5 ± 90.4 ms, n = 3; P = n.s. (C) Time course of relief from block by 50 μM Cd²⁺ in K1237C channels. K1237C channels were heterologously expressed in tsA201 cells and examined by the whole-cell patch-clamp technique. Cells were held at −120 mV, and inward currents were elicited by 10 ms depolarizations to −20 mV at 20-s intervals. Data points were fitted with a monoexponential function (Eq. 6). See text for fitting parameters. (D) Model A: Modulation of I_US by low-affinity binding of Cd²⁺ to the I_US state. Data points are time courses of recovery during control and during exposure to 20 μM Cd²⁺ (i.e., a concentration close to the K_D), taken from Fig. 2 C. Lines are model calculations assuming rates of unbinding of Cd²⁺ from the I_US state to be reduced with respect to the unbinding rates from the I_S and NI state by factors 0, 2, 5, 10, 100, and 1000 (lines in order from right to left). The lines representing model calculations assuming unbinding rates 100-fold and 1000-fold lower for I_US than for I_S and NI are superimposed. (E) Model B: Modulation of I_US by high-affinity binding of Cd²⁺ to the I_S state. The affinity of Cd²⁺ to the I_S state was assumed to be 10 times higher than the affinities for the NI and the I_US states. Data points are the same as in Fig. 2 D. Lines from right to left represent calculations assuming the following concentrations of Cd²⁺: 0 μM, 10 μM, 20 μM, and 50 μM.