Trans-ion effects on the CPA block of E166A. (A) Reduction of [Cl⁻]_o increases the steady-state CPA-blocking affinity in E166A. (B) Recovery of the current from the CPA block. The excised patch was voltage clamped at a prepulse voltage at −160 mV to allow a CPA block to reach a steady state, followed by a voltage step to a positive voltage (from +80 to +20 mV). Intracellular [CPA] was 1 mM in all three experiments. (C) Comparison of the averaged current recovery time constants in the three ionic conditions shown in B. The color of the symbols are the same as those in B: black, symmetrical 140 mM Cl⁻; green, [Cl⁻]_o = 140 mM, [Cl⁻]_i = 15 mM; red, [Cl⁻]_o = 15 mM, [Cl⁻]_i = 140 mM. Notice that altering [Cl⁻]_o significantly changed the recovery time constants, whereas altering the [Cl⁻]_i had little effect. In the condition of [Cl⁻]_o = 15 mM and [Cl⁻]_i = 140 mM, the current relaxation was not prominent at +40 mV because the voltage was closed to the reversal potential. The time constant of the current recovery was therefore not analyzed.