Cholesterol binding to BK channel-forming proteins involves the central tyrosine of a CRAC domain located at the channel core-cytosolic tail interface.

Abstract

Cholesterol (CLR) decreases the open probability of voltage/Ca2+-gated, large conductance K+ (BK, cbv1) channels. Previous studies strongly favored the possibility of cholesterol-cbv1 direct interactions, yet CLR binding to cbv1 has not been tested. The Tyr450Phe substitution in the CLR recognition amino acid consensus motif 4 (CRAC4) blunts CLR inhibition of channel activity. CRAC4 is located at the interface of two structural domains of cbv1: the core and the cytosolic tail domain (CTD). Here, we set out to determine whether CLR binds to cbv1, and the importance of CRAC4 for this binding. We performed equilibrium dialysis binding experiments with recombinant cbv1 protein from Chinese hamster ovary (CHO) cell cultures at 5.8, 3, 1.5, 0.65, 0.3, 0.03 and zero mM CLR. CLR was delivered in a 1:8 molar complex with methyl-β-cyclodextrin. The protein constructs of study included full-length wild-type cbv1, cbv1 core following truncation at Ile327, cbv1 CTD alone, and full-length cbv1Tyr450Phe. Cbv1 denatured by heating to 75oC for 5 minutes prior to loading the dialysis tubes (denatured cbv1) was used as a negative control. Proper folding of cbv1Tyr450Phe was confirmed by nanoscale differential scanning fluorimetry. CLR concentrations present in the protein sample after 20-24 hours were measured by fluorimetry. Results showed that CLR affinity was greatly diminished in all experimental constructs. This suggests that Tyr450 located at the core-CTD interface either participates directly in CLR-cbv1 interaction or transduces allosteric communication between the core and CTD to enable CLR binding elsewhere. In summary, we detected CLR binding to cbv1 and established a critical role for Tyr450 within CRAC4 in this binding. Support: NIH grant HL148941 (ANB and AMD) and the UTHSC Frank W. Dugan Fellowship (EHS).