A naturally occurring, charge-neutralizing mutation (R174W) impairs VSD-I activation and voltage-dependent channel opening, leaving VSDs II, III, and IV unaffected. (A) Voltage dependence of pore opening, as measured by normalized Ba²⁺ current tails for WT or R174W channels. Lines are fits to single Boltzmann distributions. Note that the R174W mutation caused a rightward-shift of the current voltage dependence G(V). (B) Mean voltage dependence of VSD activation constructed from experiments as in Fig. S4. Lines are fits to single Boltzmann distributions. Note that the R174W mutation impaired VSD-I activation, as revealed by a more depolarized voltage dependence, leaving VSDs II, III, and IV unaffected. Tables 1 and 3 report fitting parameters of Boltzmann functions describing G(V) and F(V) curves for R174W Ca_V1.1 channels, respectively.