Effects of mutations at the intracellular pore entrance on the k_on and k_off of the CPA block. All mutants were with the E166C background mutation. (A) Recording traces of the E166C (black trace) and the E166C/K519E (red trace) mutants. On the left are the original current traces of these two mutants obtained by stepping the membrane voltage from +80 to −160 mV. On the right, the initial current amplitude (after the capacitance spike) of the E166C/K519E mutant at −160 mV is scaled to the same initial value as that of the E166C mutant. The inverse of the time constant of the current inhibition course is plotted against [CPA] in the bottom panel to evaluate k_on and k_off according to Eq. 3. (B) Analysis of the apparent k_on and k_off of the CPA block at −160 mV among three channels in which K, M, and E are at position 519 of the E166C mutant, respectively. The fitted k_on and k_off values were: E166C/K519: 6.3 × 10⁵ M⁻¹s⁻¹ and 17.2 s⁻¹; E166C/K519M: 1.7 × 10⁵ M⁻¹s⁻¹ and 10.5 s⁻¹; and E166C/K519E: 1.1 × 10⁵ M⁻¹s⁻¹ and 9.5 s⁻¹. Notice that both k_on and k_off are reduced. Thus, the effects of these mutations on the apparent blocker affinity (shown in Fig. 6) are small. (C) Comparison of the apparent k_on and k_off of the CPA block at −160 mV among four E127Q/K519M combinatorial mutations in the E166C background. The k_on and k_off values were: E166C/E127Q: 7.6 × 10⁵ M⁻¹s⁻¹ and 17.9 s⁻¹; and E166C/E127Q/K519M: 4.6 × 10⁵ M⁻¹s⁻¹ and 11.4 s⁻¹.