RT Journal Article SR Electronic T1 Cholesterol inhibition of slo1 channels is Ca2+-dependent and can be mediated by either high-affinity Ca2+-sensing site in the slo1 cytosolic tail JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP MOLPHARM-AR-2021-000392 DO 10.1124/molpharm.121.000392 A1 North, Kelsey Cleland A1 Zhang, Man A1 Singh, Aditya K A1 Zaytseva, Dasha A1 Slayden, Alexandria V. A1 Bukiya, Anna N. A1 Dopico, Alex M. YR 2021 UL http://molpharm.aspetjournals.org/content/early/2021/12/30/molpharm.121.000392.abstract AB Ca2+-/voltage-gated K+ channels of large conductance (BK) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in Ca2+ nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity sites locate to the Regulator of Conductance for K+ (RCK) 1 domain, while another high-affinity site locates within the RCK2 domain. Here we first evaluated the cross-talking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+. CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of Mg2+ (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A, and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. Significance Statement Results reveal that the widely reported inhibition of BK (slo1) channels by membrane cholesterol requires a physiologically range of internal Ca2+ and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain of slo1 proteins, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.