Specificity of cholesterol and analogs to modulate BK channels points to direct sterol–channel protein interactions

Abstract

The activity (Po) of large-conductance voltage/Ca2+-gated K+ (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel–forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol’s mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself.

Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel

“…4), indicating that the effect of cholesterol is not due to a nonspecific membrane effect, but rather to a stereospecific binding site. Similar results with other channels, including inward-rectifier K ϩ channels and large-conductance voltage/Ca 2ϩ -gated K ϩ channels, have also been interpreted to mean that the effect of cholesterol are not due to an indirect modification of lipid bilayer properties, but rather to a stereospecific interaction at the surface of the protein (19,32). We consequently explored rTRPV1 for possible cholesterolbinding sites.…”

“…Epicholesterol (Fig. 1B) (Steraloids Inc., Newport, RI) was added to the membrane by incubating the patch with 10:1 M␤CD:epicholesterol (0.08 mg/ml) (31,32).…”

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

“…Consistent with these observations, it was shown more recently that ent-cholesterol, an enantiomer of cholesterol (17), has no effect either on KirBac1.1 or on Kir2.1 function (18). Differential effects of cholesterol analogues on channel function were also demonstrated in several other types of ion channels, including the nicotinic acetylcholine receptor (19), BK channels (20), and TRPV1 channels (21). Most recently, our laboratory has shown that cholesterol binds to purified KirBac1.1 channels and that cholesterol-KirBac1.1 binding is essential for the inhibitory effect of cholesterol on channel activity (22).…”

Multiple Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs in Cytosolic C Tail of Slo1 Subunit Determine Cholesterol Sensitivity of Ca2+- and Voltage-gated K+ (BK) Channels

“…For proper comparisons with previous data obtained by us (17,20,21,25) and others (19,22,31), studies were conducted at room temperature (20 -25°C).…”

“…In a recent structure-activity relationship study of CLR and analogs on recombinant Slo1 channels cloned from rat cerebral artery myocytes (Cbv1) and reconstituted into a bare, two-species phospholipid bilayer, we demonstrated that CLR inhibition of BK P o was defined by strict structural requirements, including the ␤ configuration of a CLR single polar group at C3 and, more importantly, enantiospecificity of the CLR molecule, suggesting CLR recognition by a protein surface. Thus, we hypothesized that the BK channel-forming subunit contained a region(s) that specifically sensed membrane CLR presence, leading to BK P o reduction (25).…”