Abstract
We demonstrated that the CRAC domain nearest the N-end of BK channel-forming cbv1 subunits (CRAC4) is the main region that provides cholesterol (CLR) sensitivity to cbv1, the signature CRAC4 Y450 being critical. Here we combined amino acid substitutions in CRAC4, bilayer electrophysiology, and computational dynamics to advance structural bases of CRAC4-CLR interactions. First, trcbv1-CRAC4 constructs including V444A or K453A were insensitive to CLR. Second, trcbv1-CRAC4 K453R retained CLR sensitivity. Third, trcbv1-CRAC4 Y450F was CLR-resistant. However, Y429F in the S6-CRAC4 linker rendered CLR-sensitive channels. Therefore, CLR sensitivity of trcbv1-CRAC4 is determined by the key residues that define a CRAC motif (Epand, 2006). Moreover, the substitution Y450F in CRAC4 was sufficient to blunt the CLR sensitivity of full-length BK channels. Computational simulations of full-length cbv1 CTD and trcbv1 CTD-CRAC4 showed CLR remaining in CRAC4 for the majority of the simulation with CLR D ring near Y450 for over one-third of the simulation. CLR A ring resided predominantly near E417, which hydrogen bonded with CRAC4 through the Y450 hydroxyl, also indicating that CLR stayed close to CRAC4. Conversely, the highest residence for trcbv1 CTD-CRAC4 Y450F was for CLR D ring and tail, which remained near H409 and Y336, respectively, neither of which are part of CRAC4. Notably higher mobility of positions 450 and K453 in CRAC4 were also noted in the simulations of trcbv1-CRAC4 Y450F, indicating that a stable interaction site for CLR is lacking when the Y450 hydroxyl is not present to hydrogen bond to E417. Data provide a structural interpretation for the prominence of CRAC4 and its Y450 in providing CLR-sensitivity to trcbv1-CRAC4 and full-length BK channels. Support: R01-HL104631;R37-AA011560 (AMD); UTHSC NI Fellowship (AKS).
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