The CRAC Motif Nearest to the N-End of Slo1 Cytosolic C-Tail is the Main Determinant of BK Channel Sensitivity to Membrane Cholesterol

Abstract

Increased membrane cholesterol (CLR) decreases BK channel activity (Po) (reviewed by Levitan et al., 2010). Electrophysiological data from a recent structure-activity study of CLR and several analogs on BK channel-forming slo1 proteins (cbv1) reconstituted into phospholipid bilayers led us to hypothesize that CLR action resulted from CLR selective and direct recognition by a site(s) in cbv1 (Bukiya et al., 2011).We identified ten Cholesterol Recognition/Interacting Amino acid Consensus (CRAC) domains in cbv1: CRACs 1-3 in the transmembrane core (S0-S6), and CRACs 4-10 in the cytosolic C tail (CTD). Bilayer electrophysiology demonstrated that construct trcbv1S6, which lacked CTD, failed to respond to CLR. However, an extended construct that included CRAC4 (trcbv1-CRAC4) was CLR-sensitive, indicating that a major determinant of CLR sensitivity lies in CRAC4 and/or the linker between this motif and S6. We next removed two CRACs at a time: immediately distal to CRAC6 (trcbv1-CRAC6) and to CRAC8 (trcbv1-CRAC8). Trcbv1-CRAC6 and trcbv1-CRAC8 were CLR-sensitive, further supporting the hypothesis that the S6-CRAC4 linker and/or CRAC4 itself is essential for CLR action. However, elimination of CRACs 7-10 or CRACs 9-10 somewhat decreased CLR action. To determine whether CTD CRACs themselves contributed to CLR responses, we introduced progressive Phe substitutions of signature tyrosines in CTD CRACs. The most drastic reduction of CLR action occurred with Y450F in CRAC4. However, cumulative Tyr to Phe substitutions in CRACs 5-10 gradually reduced CLR action. In conclusion: 1) CRAC4 in BK CTD is the main region contributing to CLR action; 2) the signature CRAC residue Y450 is essential for CLR sensitivity; 3) tyrosines in CRACs distal to CRAC4 also contribute to overall CLR sensitivity of BK channels. Support: R01-HL104631;R37-AA011560 (AMD); UTHSC NI Fellowship (AKS).