Voltage-dependent block of E166G by CPA and octanoate (OA). (A) Steady-state I-V curves of E166G and E166A in the absence (control) and presence of various concentrations of intracellular CPA (top) and octanoate (bottom). Recordings were made in symmetrical 140 mM Cl⁻, and the current was normalized to that obtained in control at +80 mV. Red curves are the best fit to Eqs. 1 (for E166A) and 2 (for E166G). For the E166A mutant (right), curve fittings gave z = 0.69 ± 0.03 and K_1/2(0) = 0.75 ± 0.08 mM, and the OA block gave z = 0.68 ± 0.03 and K_1/2(0) = 0.12 ± 0.02 mM. For the E166G mutant (left), the parameter θ_Cl(0), which is a Michaelis constant of the Cl⁻ binding to the pore, was set at 60 mM (Chen and Miller, 1996). The fitted parameters were as follows: CPA: z = 0.68 ± 0.08, K_1/2(0) = 0.35 ± 0.04 mM, δ = −0.41 ± 0.03, and θ_CPA(0) = 17.5 ± 2.5 μM; OA: z = 0.35 ± 0.08, K_1/2(0) = 0.16 ± 0.01 mM, δ = −0.61 ± 0.07, and θ_OA(0) = 3.0 ± 1.5 μM. (B) Relationship between the membrane voltage and the blocker affinity. Notice that the highest apparent affinity of the E166G mutant (under the recording condition with symmetrical 140 mM [Cl⁻]) occurs at −80 and −100 mV for CPA and octanoate, respectively.