Cholesterol Regulates Prokaryotic Kir Channel by Direct Binding to Channel Protein

Department of Medicine, University of Illinois, Chicago, IL 60612, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 10/2011; 1808(10):2527-33. DOI: 10.1016/j.bbamem.2011.07.006
Source: PubMed


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 K(D) of 390μM. 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.

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    • "(B) Competition between 3 H-cholesterol and unlabeled cholesterol, epicholesterol, 25-Hydroxycholesterol and, 5-Androsten 3β-17 β-diol. From Singh et al. (2011). * p < 0.05. "
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    ABSTRACT: Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV) are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.
    Frontiers in Physiology 02/2014; 5:65. DOI:10.3389/fphys.2014.00065 · 3.53 Impact Factor
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    • "Ion channels may go through a change in conformation within the viscous medium of the lipid membrane that may cause deformation of the lipid bilayer surrounding the channel [39]. Kir2.x channel regulation by cholesterol is an example of the cholesterol interaction with lipid rafts and the channel protein [55], [56]. "
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    ABSTRACT: Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca(2+)-activated K(+) (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit.
    PLoS ONE 11/2013; 8(11):e79952. DOI:10.1371/journal.pone.0079952 · 3.23 Impact Factor
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    • "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. "

    Biochemistry, 03/2012; , ISBN: 978-953-51-0076-8
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