Neutralization of R20 alters channel activation but does not prevent conformational rearrangements reported from the S0 region. (A) Putative topology of the BK_Ca channel α subunit voltage sensor domain. A unique cysteine was substituted at position 19 at the putative N-terminal flank of S0, bearing mutation R20A and covalently labeled with the fluorophore TMRM to resolve conformational rearrangements from this region. (B) Voltage pulses and characteristic evoked K⁺ currents from R20A BK_Ca channels labeled with TMRM at position 19. (C) TMRM fluorescence traces recorded during the voltage pulses in B. (D) Normalized K⁺ conductance from channels with mutation R20A (black circles) and without (gray triangles) plotted against membrane potential and fitted with Boltzmann distributions (black and gray curves, respectively). Boltzmann parameters are listed in Table I. Error bars represent SEM. (E) Normalized TMRM fluorescence from channels with mutation R20A (red squares) and without (pink diamonds), plotted against membrane potential and fitted with Boltzmann distributions (red and pink curves, respectively). Boltzmann parameters are listed in Table I. Error bars represent SEM. (F–J) As in A–E, respectively, for BK_Ca channels labeled with TMRM at position 145 at the extracellular tip of S2. (K–O) As in A–E, respectively, for BK_Ca channels labeled with TMRM at position 202 at the short extracellular linker between segments S3 and S4. Mutation W203V was included to enhance ΔF/F signals from position 202 (Savalli et al., 2006). Mutation R20A impaired the activation of all TMRM-labeled BK_Ca channels investigated, as it shifted the midpoint of activation (V_half) toward depolarized potentials by ≈30 mV (see Table I).