Voltage-dependent ΔF reported from the S1 extracellular flank is abolished by substitution of W203 at the extracellular flank of S4. (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 S1 (S135C) and covalently labeled with fluorophore MTS-TAMRA to resolve conformational rearrangements from this region. (B) Voltage-pulse protocol, characteristic evoked K⁺ currents (black), and simultaneously recorded MTS-TAMRA fluorescence (red) from the BK channel construct illustrated above. ΔF voltage dependence: V_0.5 = −81 ± 4 mV and z = 0.68 ± 0.04 e⁰. Conductance–voltage dependence: V_0.5 = −29 ± 7 mV and z = 0.86 ± 0.03 e⁰; n = 11. (C and D) As in A and B, for channels with the additional mutation W203V to remove the native Trp at the extracellular portion of S4. MTS-TAMRA fluorescence traces are in blue. Note that the fluorescence scale is 10 times that for wild-type (WT) channels. ΔF voltage dependence: V_0.5 = −97 ± 2 mV and z = 0.84 ± 0.04 e⁰. Conductance–voltage dependence: V_0.5 = 32 ± 2 mV and z = 0.99 ± 0.06 e⁰; n = 5. (E) Mean fitted ΔF_total/F₀ signal, normalized for fitted maximal conductance for WT (red; ΔF_total/F₀/G_max = 1.8 ± 0.42%/mS) and W203V (blue; ΔF_total/F₀/G_max = 0.41 ± 0.09%/mS) channels. Mutation W203V attenuated the ΔF reported by MTS-TAMRA from position 135 by ≈85%. (F) Mean, normalized ΔF from WT (red squares) or W203V (blue diamonds) BK channels labeled at position 135 with MTS-TAMRA. Error bars represent SEM.