Temperature dependence of CTX-induced relaxations in the presence of Hi-Na ⁺ and Hi-K ⁺ external solutions. (A and A′) TEVC traces of measurements taken at temperatures separated by ∼20°C in Hi-Na⁺ (A) and Hi-K⁺ (A′). Voltage pulse protocols and color coding are as in Fig. 1. Control: Traces in the absence of CTX; 10 nM CTX: Traces under continuous superfusion of recording solution plus 10 nM CTX; fractional current: Point-by-point quotients at different applied voltages at the temperatures shown on top of each panel. Upward relaxations indicate higher toxin affinity for closed channels in Hi-Na⁺ solutions, while downward relaxations in Hi-K⁺ indicate a higher affinity for the open channels. Eq. 2 fits are in blue. (B and B′) Comparison of the time constants, τ, in Hi-Na⁺ and Hi-K⁺, respectively. (C and C′) Comparison of the open channel asymptotic inhibition at different voltages (ss; open symbols) and closed state inhibition (ssc; green arrow; filled symbols). (D and D′)K_D calculated from Eq. 3 for open (open symbols) and closed channels (filled symbols). (E and E′) Rate constants as a function of voltage for the higher and lower temperature experiments. The continuous lines were drawn according to Eq. 4. The fitting values are the following: Hi-Na⁺; 11.8 ± 1.6°C: k_offo = 0.33 ± 0.07 s⁻¹, zδ = 0.55 ± 0.07; k_ono = 0.14 ± 0.04 nM⁻¹s⁻¹, zδ = −0.11 ± −0.073; 30.7 ± 0.4°C: k_offo = 0.37 ± 0.05 s⁻¹, zδ = 0.59 ± 0.02; k_ono = 0.17 ± 0.05 nM⁻¹s⁻¹, zδ = −0.067 ± 0.032; Hi-K⁺; 13.1 ± 0.9°C: k_offo = 0.47 ± 0.21 s⁻¹, zδ = 0.49 ± 0.08; k_ono = 0.075 ± 0.014 nM⁻¹s⁻¹, zδ = −0.01 ± 0.05; 32.4 ± 2.3°C: k_offo = 0.66 ± 0.1 s⁻¹, zδ = 0.64 ± 0.16; k_ono = 0.21 ± 0.02 nM⁻¹s⁻¹, zδ = 0.13 ± 0.06. All data points represent between three and six measurements. The parameter errors are the SD of the individual fits obtained with the Levenberg–Marquardt algorithm. See also Data S3 for underlying data.