Structural determinants of monohydroxylated bile acids to activate β1 subunit-containing BK channels

Lithocholate (LC) (10-300 mu M) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel beta(1) accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1 + beta(1) subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5 beta-cholanic acid-3 alpha-ol), 5 alpha-cholanic acid-3 alpha-ol, and 5 beta-cholanic acid-3 beta-ol increased NPo (EC50 similar to 45 mM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5 alpha-cholanic acid-3 alpha-ol and 5 beta-cholanic acid-3 beta-ol raised NPo by 40%. Thus, the alpha-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5 alpha-cholanic acid-3 beta-ol and 5-cholenic acid-3 beta-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5 alpha-cholanic acid-3 alpha-ol and 5 beta-cholanic acid-3b-ol partially matched LC shape, and 5 alpha-cholanic acid-3 beta-ol and 5-cholenic acid-3 beta-ol did not. Increasing polarity in steroid rings (5 beta-cholanic acid-3 alpha-sulfate) or reducing polarity in lateral chain (5 beta-cholanic acid 3 alpha-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between beta(1) and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in beta(1) transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.