W203, outside S4, is the principal quencher of ΔF reported from S2. (A) Illustration of the BK channel construct used; only the VSD transmembrane helices of one subunit are shown. A unique cysteine was substituted at the extracellular flank of S2 (Y145C) and covalently labeled with fluorophore TMRM to resolve conformational rearrangements from this region. (B) Voltage-pulse protocol, characteristic evoked K⁺ currents (black), and simultaneously recorded TMRM fluorescence (red) from the BK channel construct illustrated above. ΔF voltage dependence: V_0.5 = −82 ± 5 mV and z = 0.74 ± 0.05 e⁰. Conductance–voltage dependence: V_0.5 = −26 ± 10 mV and z = 1.2 ± 0.11 e⁰; n = 14. (C and D) As in A and B, for channels with the additional mutation W203V to remove the Trp residue at the extracellular portion of S4. TMRM fluorescence traces are in blue. Note that the fluorescence scale is 10 times that for WT channels. ΔF voltage dependence: V_0.5 = −58 ± 9 mV and z = 0.57 ± 0.05 e⁰. Conductance–voltage dependence: V_0.5 = 10 ± 2 mV and z = 0.86 ± 0.1 e⁰; n = 8. (E) Mean fitted ΔF_total/F₀ signal, normalized for fitted maximum conductance to normalize for channel expression for WT (red; ΔF_total/F₀/G_max = 0.51 ± 0.17%/mS) and W203V (blue; ΔF_total/F₀/G_max = 0.05 ± 0.007%/mS) channels. Mutation W203V attenuated the ΔF reported by TMRM from position 145 by ≈90%. (F) Mean, normalized ΔF from WT (red squares) or W203V (blue diamonds) BK channels labeled at position 145 with TMRM. Error bars represent SEM.