A scan of the Shaker and Kv1.2 S3–S4 linkers reveals differences in fluorescence phenotypes. (A and B) Representative fluorescence traces collected in a cysteine scan of five consecutive homologous residues in the S3–S4 linker and N-terminal end of S4 in Shaker (A) and Kv1.2 (B). Cells expressing these constructs were held at −80 mV and depolarized to 60 mV for 100 ms. (C–F) Normalized G-V (closed symbols) and F-V (open symbols) relationships for four of the five Shaker (squares) and Kv1.2 (circles) constructs expressing changes in fluorescence upon depolarization, as labeled in the top left corner of each panel. For Kv1.2 S289C (D), the gray circles and accompanying fit denote the Boltzmann fit to the voltage-dependent fluorescence observed between −140 and −20 mV. Mean half-activation and slope data obtained from the fits to all four mutant constructs for each channel can be found in Table I. Data are shown as mean ± SEM and are the mean of three to eight cells collected from each mutant.