The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory ?1 subunit.

ABSTRACT: 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 2× increase in vascular myocyte BK activity (EC(50) = 45 ?M), an action that requires ?1 but not other (?2-?4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK ?- (cbv1) and ?1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK ?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?1T165A,T169A channels were LC-unresponsive. However, cbv1 + r?1T165A and cbv1 + r?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?1T169A was LC-insensitive, with r?1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + r?1L172A, and cbv1 + r?1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid ?-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, ?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 ?1, our study provides a structural basis for advancing ?1 subunit-specific pharmacology of BK channels.

SUBMITTER: Bukiya AN 

PROVIDER: S-EPMC3250185 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature