Identification of an Ethanol Recognition Site in BK Beta1 Subunit that Mediates Ethanol-Induced Cerebral Artery Myocyte BK Channel Inhibition and the Resulting Artery Constriction

Abstract

Ethanol at concentrations obtained in circulation during moderate-heavy episodic drinking (30-80 mM) decreases the activity of voltage/calcium-gated, large conductance potassium (BK) channels in smooth muscle (SM), which leads to cerebral artery constriction (Liu et al., 2004). The SM-abundant BK beta1 subunit (encoded by KCNMB1) is necessary for ethanol to inhibit cerebral artery SM BK channels under physiological voltages and calcium (Bukiya et al., 2009, Kuntamallappanavar and Dopico, 2016). Moreover, beta1 transmembrane domain 2 (TM2) is required for such action of ethanol (Kuntamallappanavar and Dopico, 2017). The precise ethanol recognition site(s) in beta1-TM2 that mediates ethanol-induced BK channel inhibition and associated cerebral artery constriction has remained unknown. Here, we address this question by combining computational modeling, mutagenesis, patch-clamp electrophysiology, in vitro artery electroporation and pressurization. Computational dynamics revealed two potential ethanol-recognition residues in beta1-TM2: Ser160 and Thr165. Macroscopic currents evoked from BK channel-forming cbv1 subunits co-expressed with beta1T165A displayed an ethanol response similar to that of cbv1+beta1, allowing ethanol inhibition of current at physiological, internal calcium>2 microM. In contrast, currents from cbv1+beta1S160A,T165A and cbv1+beta1S160A channels displayed an ethanol response identical to that of cbv1 alone: ethanol inhibition was blunted. Collectively, our data suggest that hydrogen bonding between Ser160 and ethanol's hydroxyl group is essential for ethanol-mediated inhibition of slo1+beta1 current. Consistent with in vitro electrophysiology findings, KCNMB1 K/O mouse arteries electroporated with beta1S160A cDNA failed to constrict in response to ethanol whereas electroporation with wt beta1 cDNA evoked ethanol-induced vasoconstriction. In conclusion, Ser160 in the beta1-TM2 region is required for ethanol-induced inhibition of beta1-containing BK channels and the resulting cerebral artery constriction. Support: R37-AA11560 (AMD) and AHA Predoctoral Fellowship (GK).