Topology of Ca_V1.1 channels, S4 segments sequence alignment, and voltage dependence of Ca_V1.1 channel opening for WT and Cys mutants. (A) The topology of the pore-forming α_1S subunit of skeletal Ca_V1.1 channels is shown: four concatenated homologous but nonidentical repeats (I–IV) form the α_1S subunit. Each repeat includes six transmembrane segments (S1–S6): S1–S4 make the VSD (colored cylinders), while S5 and S6 contribute to one quarter of the pore (gray cylinders). (B) A sequence alignment of the four S4 segments is based on the most recent structure of Ca_V1.1 channels (bold font corresponds to residues included in S4 helices; Protein Data Bank accession no. 6JP5; Zhao et al., 2019). Part of S3 and S4 loops are also reported to illustrate the location of the amino acids substituted into cysteines for fluorophore conjugation (pink, with a rhodamine molecule above them). Putative voltage-sensing, charged amino acids are in blue. (C) Mean voltage dependence of channel opening (G(V)) for WT and Cys mutants is shown (n = 3–7). G(V)s for WT channels have been measured from oocytes with (pink circle) or without (black circles) fluorophore labeling. G(V)s from Cys mutants are all from fluorophore-labeled channels. Note that fluorophore conjugation to the 13 endogenous extracellular cysteines (WT channels) did not modify channel activation. (D) Mean voltage dependence of gating charge (Q(V)) for WT and Cys mutants was calculated by integrating 10-ms gating current recordings. Lines in C and D are fits to single Boltzmann distributions (fitting parameters are reported in Tables 1 and 2, respectively). Note that Cys substitutions in the four VSDs did not perturb Ca_V1.1 voltage dependence of G(V) and Q(V).