Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein

Abstract

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a KD of 390 μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

“…[13][14][15][16] However, in CCD cells, our data indicate that ROMK1 is not located in cholesterolrich lipid rafts but mainly, the planar regions of the apical membrane and that the cholesterol-lowering drug, lovastatin, does not alter ROMK1 channel activity in the CCD cells when PI(4)P5K I g expression is significantly reduced. These results suggest that the endogenous cholesterol levels in the planar regions, at least under resting conditions, are unable to reach the threshold that is required to directly inhibit ROMK1 channels but indirectly regulate ROMK1 channels through PI(4)P5K.…”

“…[13][14][15][16] However, the evidence came from either cell overexpression models or artificial bilayer membranes. Because endogenous cholesterol, especially in epithelial cells, is usually strongly associated with sphingolipids through hydrogen bonds in the cell membrane to form lipid rafts, 18 it remains unclear whether the endogenous cholesterol could reach and interact with the cytosolic pore of Kir channels, although the exogenously added cholesterol can.…”

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

“…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).…”

Cholesterol: Biosynthesis, Functional Diversity, Homeostasis and Regulation by Natural Products

“…In general three different mechanisms may be involved in regulation of ion channels by cholesterol: (i) specific interactions (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for protein-protein interactions. Furthermore, our recent data for the first time demonstrate a specific cholesterol-channel binding (Singh et al, 2011). One possibility is that cholesterol may interact directly and specifically with the transmembrane domains of the channels protein.…”

“…Clearly, therefore, more than one mechanism has to be involved in cholesterol-induced regulation of different ion channels. Studies from our laboratory have shown that many major and important ion channels are regulated by changes in the level of membrane cholesterol (Epshtein et al, 2009;Levitan, 2009;Levitan et al, 2010;Rosenhouse-Dantsker et al, 2011;Singh et al, 2009b;Singh et al, 2011). In general three different mechanisms may be involved in regulation of ion channels by cholesterol: (i) specific interactions (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for protein-protein interactions.…”