The Activation Transition of the Human BK (Slo1) Voltage Sensor Involves a Complex Rearrangement of its Helix Bundle as Resolved by Site-Directed Fluorophore/Quencher Pairs

Abstract

The typical voltage sensor domain (VSD) consists of four transmembrane helices (S1-S4). S4, which bears voltage-sensing charged residues, transitions into an active state upon membrane depolarization, conferring voltage dependence to some classes of ion channels and phosphatases. The VSD of voltage- and Ca2+-activated K+ (BK) channels exhibits salient differences from the current 4-helix, S4-central model of VSD activation, as it possesses additional helix S0, and most voltage-sensing charge is borne by S2 and S3. Moreover, the BK VSD activation properties are tuned by its tissue-specific association with modulatory β subunits. To resolve the structural basis of BK VSD operation, we used an improved implementation of voltage-clamp fluorometry, enhanced with site-directed collisional quenching, exploiting the photochemical property of Tryptophan to quench fluorophores by collision-dependent electron exchange. Thus, fluorescence deflections (ΔF) are interpreted as the relative rearrangement of fluorescent label and the Trp quencher. Upon depolarization, positive ΔF is reported from S0 (TMRM at positions 17,18,19), S1 (MTS-TAMRA at 134,135) and S2 (TMRM at 145). Substitution of the native Trp at S4 (W203V) resulted in ΔF attenuation by ≈90%, suggesting that the voltage-dependent ΔF reports the divergence of S4 from helices S0, S1, and S2. Furthermore, VSD activation likely involves a movement of S2 towards S1: MTS-TAMRA labeling S1 (position 135) is quenched by a Tryptophan introduced near S2 (C141W) upon depolarization. This voltage-dependent fluorescence quenching is not present when BK channels are associated with β2 subunits, suggesting that the β2-mediated BK modification involves a perturbation of the S1-S2 rearrangement. The information provided by these studies was used to construct a model-independent map of conformational changes in the BK VSD, revealing how it responds to depolarization.