Abstract
Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K+ (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by 86Rb+ influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.
Highlights
Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth
We have recently shown that Saccharomyces cerevisiae can be used to express and purify human Kir channels that can be functionally reconstituted in artificial membranes of defined composition [24,25,26]
With the use of the enantiomeric cholesterol—which has the same effects on membrane properties as natural cholesterol [27,28,29]—we show that in both the prokaryotic and eukaryotic Kir channels the effects of cholesterol are due to direct enantioselective binding of cholesterol to the channel protein, and not an indirect effect due to changes in lipid bilayer properties
Summary
Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Kir channels are expressed in a wide variety of cells including skeletal, cardiac, and vascular myocytes, neurons, and pancreatic b-cells and play numerous key physiological roles including stabilizing the resting membrane potential, regulating K+ ion flux across cellular membranes, and regulation of cellular excitability [18]. Previous studies have identified cholesterol suppression of inward rectifier K+ channels (‘‘Kir’’ or ‘‘KCNJ’’ channels) in aortic endothelial cells [8] and by heterologous expression in chinese hamster ovary (CHO) cells [7,19,20]. Changes in Kir channel function due to changes in the physical properties of the lipid bilayer rather than direct binding cannot be excluded based on previous studies
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