Functional effects of Cys substitution in the intracellular flanks of the β1 TM helices. Eight Cys-substituted mutants were expressed with pWT α in HEK 293 cells. Recordings were in inside-out macropatches with 10 µM Ca²⁺ in intracellular solution. (A) Macroscopic currents in response to step depolarizations from −100 to +140 mV conducted by pWT α coexpressed without β1 or with the indicated pWT or mutant β1. (B) V₅₀ of single-Cys β1 mutants. Mean ± SEM. The mean V₅₀ of pWT α + pWT β1 is shown as a dashed red line. (C) tau_act and tau_deact. Mean ± SEM; n = 3–5 for each. (D–G) Normalized G-V curves of untreated macropatches (control) and after treatment with 40 µM QPD for 5–7 min. The G-V curve of pWT α + pWT β1 is shown as a dashed red line. (H) V₅₀s before and after treatment with QPD for selected single- and double-Cys–substituted mutants. Mean ± SEM. The mean V₅₀ of pWT α + pWT β1 is shown as a dashed red line.