The Slo1 C-Tail Domain Confers Cholesterol-Sensitivity to Arterial Smooth Muscle BK Channels

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Large conductance, voltage- and calcium-gated potassium (BK) channels are known to cluster in cholesterol-rich cell membrane domains. Furthermore, cholesterol-BK channel interaction usually results in reduced channel activity (Po), as reported with native (Bolotina et al., 1989) and recombinant (Crowley et al., 2003) channels. We previously communicated that cholesterol-inhibition of BK channels cloned from rat cerebral artery myocytes (cbv1; AY330293) and reconstituted into POPE:POPS (3:1) bilayers displayed enantiospecificity and stereoselectivity, strongly suggesting that the decrease in Po involved cholesterol-recognition by a protein surface (Bukiya et al., Biophys. Soc. 2010). Using a similar system, we now demonstrate that cholesterol (16-33 mol%) inhibition of cbv1 channels is similar whether the channel is open by positive voltage or increased intracellular calcium, suggesting that the cholesterol-protein(s) interaction leads to altered channel function independently of the signal that gates the channel. Because the channel phenotype was characteristic of homomeric slo1, we hypothesize that regulation of BK gating by cholesterol involves an interaction between the steroid and the cbv1 subunit.One of the motifs that favors cholesterol-protein interactions is CRAC: -L/V-(X)(1-5)-Y-(X)(1-5)-R/K-, where X denotes any residue (Epand, 2008). We used CRAC sequence patterning and found ten CRAC motifs in cbv1, three in the core (S0-S6) and seven in the C-tail (S7-C end) domain. Thus, we next evaluated the cholesterol sensitivity of cbv1 channels truncated immediately after S6 (trS6cbv1). Remarkably, trS6cbv1 was consistently resistant to cholesterol-induced modulation (n=11) under conditions where wt cbv1 remained sensitive. Therefore, the C-tail domain confers cholesterol-sensitivity to cbv1 channels. We are currently using a combination of computational dynamics, sequential cbv1 truncation, and point mutagenesis in CRAC motifs to determine the relative contribution, if any, of these motifs to the cholesterol-sensitivity of BK channels.Support: HL104631 (AMD), UTHSC Neurosci. Postdoc. Fellowship (AKS).