Effect of introducing charged residues at the internal entrance of P475D channels. (Left) Leak- and capacitance-subtracted single-channel traces of (A) P475D/V476D and (B) P475D/S479D in the 30-1,000-mM range of symmetrical [K⁺]. (Right) I-V relations from the unitary amplitudes of both variants. The slope third-order polynomial fits were used to obtain the conductance values at zero voltage. (C) Zero-voltage slope unitary conductance obtained from the I-V relations displayed in A and B plotted versus [K⁺]. Data of P475D/S479D were fitted to a Langmuir isotherm (Kd = 22 ± 7 mM and g_max = 179 ± 10 pS), whereas the data of P475D/V476D could not be fitted by a Langmuir isotherm; instead, we choose to average the unitary conductance of the channel at all K⁺ concentrations (g_max = 87 ± 1 pS). For better comparison, we included the fit of P475D from Moscoso et al. (2012) (Kd = 48 ± 6 mM and g_max = 186 ± 2 pS; discontinued line).