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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2011-12, Vol.108 (50), p.20207-20212
• Main Authors: Bukiya, Anna N, Singh, Aditya K, Parrill, Abby L, Dopico, Alejandro M
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Amino Acid Substitution - drug effects; Amino acids; Animals; Bile acids; Binding Sites; bioinformatics; Biological Sciences; calcium; Cell Membrane - chemistry; Cell Membrane - drug effects; Cerebral arteries; Cholanes; Cholanes - pharmacology; Drug interactions; Hydrogen; hydrogen bonding; hydrophobic bonding; Hydroxyls; Large conductance calcium activated potassium channels; Large-Conductance Calcium-Activated Potassium Channel beta Subunits - chemistry; Large-Conductance Calcium-Activated Potassium Channel beta Subunits - metabolism; Lithocholic Acid - chemistry; Lithocholic Acid - pharmacology; Models, Molecular; mutagenesis; myocytes; pharmacology; potassium; Protein Structure, Tertiary; Rats; Steroids; Structure-Activity Relationship; Vehicles
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1112901108

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 (EC50 = 45 μM), an action that requires β1 but not other (β2–β4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and β1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK β1 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+rβ1T165A,T169A channels were LC-unresponsive. However, cbv1 + rβ1T165A and cbv1 + rβ1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rβ1T169A was LC-insensitive, with rβ1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rβ1L172A, and cbv1 + rβ1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, β1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK β1, our study provides a structural basis for advancing β1 subunit–specific pharmacology of BK channels.