Alterations in the activation and inactivation curves by the interaction between the countercharges introduced to Y1618 and the pore loop residues in domain 4. The averaged activation curves (A) and inactivation curves (B; the inactivating pulse duration = 100 ms) were done by the protocols described in Materials and methods (n = 3–5). The lines in the left panel are fits with a Boltzmann function with V_1/2 values (in mV) of −24.5, −23.4, −21.4, −27.7, −20.5, and −29.7, and k values of 4.6, 4.2, 4.0, 5.2, 5.2, and 4.6 for Y1618K, Y1618K plus G1715E, Y1618K plus G1718E, Y1618K plus L1719E, Y1618K plus L1720E, and Y1618D plus L1719K mutant channels, respectively. The data for the wild-type channel are from Fig. 1 A. The lines in the right panel are fits with a Boltzmann function with V_1/2 values (in mV) of −47.2, −46.1, −48.6, −47.8, −75.0, −48.5, and −89.1, and k values of 6.0, 7.3, 6.9, 6.8, 6.7, 7.0, and 5.3 for the wild-type, 1618K, Y1618K plus G1715E, Y1618K plus G1718E, Y1618K plus L1719E, Y1618K plus L1720E, and Y1618D plus L1719K mutant channels, respectively. We also did the inactivation curves with 9-s inactivating pulses and found that the inactivation curve is still leftward shifted by ∼30 mV in the Y1618K/L1719E double-mutant channel compared with the wild-type channel. The data for the wild-type channel are shown as gray circles with gray lines.