Role of the slo1 CRAC4 Motif in BK Channel's Ethanol Sensitivity

Abstract

Cholesterol (CLR) has been recognized for its ability to interact with ion channels. Among ion channels that sense CLR presence are large conductance, voltage/Ca2+-gated K+ (BK) channels. Functional BK channels result from tetrameric association of pore-forming slo1 subunits. CLR inhibition of slo1 current may result from CLR-sensing by the slo1 cytosolic tail domain (CTD). CTD contains seven CLR-recognition amino acid consensus (CRAC) motifs that contribute to the overall CLR sensitivity of the channel, with the contribution by CRAC4 being studied (Singh et al., 2012). Alcohol (ethanol) also modifies BK channel function, this effect resulting from an orchestration of several factors that include [Ca2+]i. In native membranes, ethanol increases slo1 channel open probability (Po) at [Ca2+]i<20-30 µM whereas reduces Po at [Ca2+]i<30 µM. Both CLR-enrichment and CLR-depletion of native membranes modify ethanol action on BK channels. Thus, we set to determine the role of CLR-sensing by the CRAC4 motif in the slo1 channel's ethanol sensitivity. Slo1 cloned from rat cerebral artery myocytes (cbv1) and the cbv1CRAC4T450F were incorporated into POPE:POPS (3:1 w/w) bilayers in 300/30 mM K+i/K+o gradient. At 0.3 μM Ca2+i, ethanol increased cbv1 Po by ≈3-fold. At 300 μM Ca2+i, ethanol failed to modify cbv1 Po in CLR-free bilayers but increased cbv1 Po in CLR-containing (33 mol%) bilayers. The discrepancy in ethanol effect between native membranes and planar bilayers may likely arise from their difference in proteo-lipid, including CLR, composition. Cbv1CRAC4T450F did not respond to ethanol in CLR-free bilayers, but was activated by ethanol in presence of 33 mol% CLR. Collectively, the similarity of ethanol modulation of BK channel activity studied with cbv1 vs. cbv1CRAC4T450F suggests that CRAC4 does not contribute to CLR modulation of ethanol action on slo1 at high [Ca2+]i. Support: R01AA023764 (AB), R37AA11560 (AD).