Molecular determinants of cholesterol binding to BK channel-forming slo1 subunits

The BK (big potassium, MaxiK) channel is a potassium channel of large conductance gated by calcium (Ca²⁺) and voltage. Formed by a homotetramer of alpha (slo1) subunits, this channel plays a major role in numerous physiological systems and processes. Consistently, BK channel expression and function are regulated by a variety of endogenous ligands. In particular, the activity of homotetrameric channels made of slo1 subunits is inhibited by cholesterol (CLR). However, it remains unknown whether BK channel inhibition involves direct chemical binding of CLR molecules to the slo1 protein, or results from allosteric coupling between slo1 and CLR binding elsewhere, such as the lipid bilayer. Here, we demonstrate by equilibrium dialysis that CLR binds slo1 proteins cloned from rat cerebral artery myocytes (cbv1) in both the absence and presence of activating Ca²⁺. This binding is saturable with a K_D of 1.0-1.2 mM and requires the physical association between the cbv1 transmembrane core and its cytosolic tail domain (CTD). Moreover, F substitution of Y450, a CTD residue located nearby the membrane inner leaflet, abolishes CLR binding independent of Ca²⁺. Remarkably, cbv1Y450F protein intrinsic fluorescence is unaffected by Ca²⁺, suggesting that Y450 contributes to Ca²⁺ sensing by cbv1. In summary, the present study demonstrates for the first time direct binding of CLR to slo1 channels and underscores the critical role of Y450 in such binding and in Ca²⁺ sensing of slo1 channels.