Extracellular CPA effects on the E166G and E166A mutants. (A) Recordings of the E166G mutant in two ionic conditions as indicated, with 10 mM CPA in the pipette. Voltage protocol is the same as that shown in the inset of Fig. 2. The percentage of the hyperpolarization-induced current out of the whole steady-state inward current ranged from 5.1–7.5% in the symmetrical 140-mM Cl⁻ condition and from 20.5–23.2% in the 140-/14-mM Cl⁻ condition. (B) Recordings of E166A with 10 mM CPA in the pipette. No hyperpolarization-activated current is noted at very negative voltages, even in low [Cl⁻]_o (10 mM). (C) Time constants of the current-increasing process shown in A (color symbols) upon stepping the potential from +80 mV to the various test voltages. Only the last several hyperpolarization voltages were able to generate a prominent current activation for a single-exponential fit. For comparison, the time constants presented in Fig. 5 B are reproduced (open symbols).