The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory β1 subunit

Large conductance, voltage-and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2x increase in vascular myocyte BK activity (EC(50) = 45 mu M), an action that requires beta 1 but not other (beta 2-beta 4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK alpha-(cbv1) and beta 1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK beta 1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+r beta 1T165A, T169A channels were LC-unresponsive. However, cbv1 + r beta 1T165A and cbv1 + r beta 1T165A, L157A, L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + r beta 1T169A was LC-insensitive, with r beta 1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + r beta 1L172A, and cbv1 + r beta 1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid alpha-hydroxyl, and hydrophobic interactions between L172, L173 and the steroid rings are both necessary for LC action. Therefore, beta 1 TM2 T169, L172, L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK beta 1, our study provides a structural basis for advancing beta 1 subunit-specific pharmacology of BK channels.