| Gating of large conductance Ca2+-activated K+ channels (BK channels) is controlled by a Ca2+-sensor, formed by the channel's cytoplasmic carboxy-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. While we now have a detailed functional understanding of the relation between voltage, Ca2+, and gating of BK channels, our understanding of the structure that underlies this mechanism is not clear.;Recent studies identified several charged residues in the BK channel voltage sensing domain that, being mutated, can change the equilibrium constant for voltage sensor movement. These studies have provided a working hypothesis for BK channel voltage sensor activation; however, we do not yet have a clear picture of the structural correlates of these functional measures for BK channels. Using site-specific labeling with bimane as a fluorescent probe, we analyzed structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker. Bimane fluorescence is quenched by tryptophan sidechains, and we used this property to gauge intermolecular distances in the S3-S4 and thus probe its structure. We found that fluorescence in bimane-labeled BK channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the fluorescence decay. Based on these results, we present a working hypothesis for the secondary structure of the BK channel S3-S4 region, which places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix.;The specific arrangement Ca2+- and voltage-sensing domains in the BK channel is not clear, and this arrangement may be relevant to our understanding of possible functional interactions of between Ca2+ and voltage that may underlie gating. We have thus probed the relative arrangement of Ca2+- and voltage-sensing domains and the pore-forming domain in the BK channel using covalent sulfhydryl crosslinking. We observed that adjacent BK channel subunits can be covalently crosslinked at native cysteine sidechains, using the bifunctional reagent 1,11-bis-maleimidotriethyleneglycol (BM(PEO)3), which has an extended spacer arm length of 17.8 A. Our results suggest that adjacent subunits are not crosslinked to one another by BM(PEO)3 at any of the three native extracellular cysteines (C14, C141, or C277). However, cysteines in the intracellular S0-S1 loop (C54, C55, C57) are crosslinked to cysteines in the RCK1 domain of an adjacent subunit. These results support the hypothesis that portions of the voltage-sensor domain may lie in close opposition to the cytoplasmic Ca 2+-sensing domain, which may underlie a functional interaction between these domains. Together, these new observations of structural features of the BK channel voltage-sensing domain reveal structural underpinnings of the BK channel gating. |
