CTX binding to closed channels. (A and B), Two-pulse protocol to measure CTX-blockade kinetics to resting channels in Hi-Na⁺ (A) and Hi-K⁺ (B) solutions (for A and B, T° ∼25°C). A 500-ms prepulse to +50 mV was followed by a second 200-ms test pulse to +50 mV after a variable interpulse interval at −90 mV (top inset). The kinetics of reinhibition at resting produced by 10 nM CTX in the bath was estimated by measuring the average currents in a 5-ms interval between 5 and 10 ms into the test pulse current. These inhibition values are plotted as filled blue circles along each set of traces and fitted to a single exponential to estimate the time constant of toxin rebinding. (C) Time constants in Hi-K⁺ are more T° sensitive in the 15–33°C interval. (D) Steady-state resting inhibition, measured at the point of convergence of the traces as shown in Figs. 1 and 2 (green arrows). (E) Summary of temperature dependence of the association (open symbols) and dissociation rate constants (filled symbols) in Hi-Na⁺ and Hi-K⁺ obtained from equation system #1 for several temperatures. The solid lines were traced from Eq. 5 with ΔH^≠ = 17.3 ± 6.7 kJ/mol and ΔH^≠ = 11.3 ± 6.2 kJ/mol for k_on and k_off, respectively, in Hi-Na⁺ solutions, and ΔH^≠ = 45.7 ± 8.7 kJ/mol and ΔH^≠ = 27.1 ± 6.7 kJ/mol for k_on and k_off, respectively, in Hi-K⁺ solutions (see main text). See also Datas S5 and S6 for underlying data.