Strict Structural Requirements for Cholesterol to Inhibit BK Channels Point to Specific Steroid-Protein Interactions

Abstract

Cholesterol decreases large conductance, voltage/calcium-gated potassium (BK) channel activity (NPo), an action that solely requires the channel pore-forming (α) subunit and a minimum phospholipid environment (Crowley et al., 2003). This cholesterol action is attributed to cholesterol-induced tight packing of bilayer lipids (Chang et al., 1995). Cholesterol modulation of ion channels via direct protein-steroid interactions, however, is increasingly recognized (Epshtein et al., 2009). Cholesterol analogs have been widely used to distinguish between lipid bilayer-mediated and specific protein recognition mechanisms. Thus, we probed cholesterol analogs on BK α subunits cloned from rat cerebral artery myocytes (“cbv1”; AY330293) after channel reconstitution into 3:1 (w/w) POPE:POPS bilayers. Cholesterol (33 mol%) decreased cbv1 NPo by ≈25%. In contrast, 5-cholenic acid-3β-ol, having a carboxyl group at the lateral chain end, failed to decrease NPo, underscoring the importance of a hydrophobic chain for sterol insertion into the bilayer hydrophobic core and channel inhibition. Coprostanol and cholestanol having the A/B junction in cis and trans, respectively, also decreased NPo (≤25%). In contrast, cholesterol, coprostanol and cholestanol epimers, having the C3-hydroxyl group in α-configuration, failed to decrease NPo. Therefore, a β-conformation in the hydroxyl is necessary for these monohydroxy-sterols to inhibit BK channels, strongly suggesting specific, steroid-protein interactions. Moreover, we probed the cbv1 channel with enantiomeric cholesterol (ent-cholesterol), which has physico-chemical properties similar to those of cholesterol yet can be differentially sensed by protein sites, as demonstrated by the lack of viability of C. Elegans when only ent-cholesterol is present (Crowder et al., 2001). Remarkably, ent-cholesterol repeatedly failed to reduce cbv1 NPo, buttressing the idea that cholesterol inhibition of BK channels requires steroid recognition by protein site(s), likely present in the cbv1 subunit itself.Support: AA11560 and HL077424 (AMD), UTHSC Neuroscience Postdoctoral Fellowship (AB).