Cholesterol binding to ion channels

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.

Keywords: cholesterol; cholesterol-binding motifs; ion channels; lipids; membrane proteins.

Figure 1

Substitution of cholesterol by epicholesterol increases Kir current density. (A) Substitution of endogenous cholesterol by epicholesterol using MβCD. Dark bars represent the level of cholesterol, and the lighter portion of the bar, epicholesterol level. (B) Typical current traces recorded from a cell exposed to Mβ CD-epicholesterol and from a control cell. Both cells were maintained in 6 mM extracellular K⁺. (C) Peak current densities of MβCD-epicholesterol treated cells (n = 32) and in control cells (n = 31) recorded in 6 mM extracellular K⁺. All values are means ± SE ^*P < 0.05 vs. control. From Romanenko et al. (2002).

Figure 2

Differential effects of sterol on KirBac1.1-mediated ⁸⁶Rb⁺ uptake. (A) Time-courses of ⁸⁶Rb⁺ uptake in liposomes containing different sterols. All experiments included control liposomes containing no sterol and liposomes containing 50 μg cholesterol/mg PL as a positive control. (B) KirBac1.1 activity vs. membrane anisotropy. The normalized maximal uptake of ⁸⁶Rb⁺ isplotted vs. the anisotropy (r) following incorporation of respective sterols. The correlation coefficient between the ⁸⁶Rb⁺ uptake and the anisotropy value is R = −0.08276, p > 0.05 (a meaningful correlation would require |R| > 0.602) (Abbreviations: 25-HC, 25-Hydroxycholesterol; Desm, Desmosterol; β-Sito, β-Sitosterol; Camp, Campesterol; Fuco, Fucosterol; Chol, Cholesterol; Copro, Coprosterol; 19-HC, 19-Hydroxycholesterol; Epicopro, Epicoprosterol; Epichol, Epicholesterol; Andro, 5-Androsten 3β-17 β-diol; Ergo, Ergosterol; Stigma, Stigmastanol). From Singh et al. (2009).

Figure 3

Specific cholesterol binding to KirBac1.1. (A) Typical elution profiles of [³H]-cholesterol with and without 1.5 μg His₆-KirBac1.1 protein from Ni-NTA-agarose affinity column. The unbound cholesterol is eluted in fractions 1–4 and cholesterol bound to the KirBac1.1 protein is eluted in fractions 5–7 after the addition of imidazole-HCl. (B) Competition between ³H-cholesterol and unlabeled cholesterol, epicholesterol, 25-Hydroxycholesterol and, 5-Androsten 3β-17 β-diol. From Singh et al. (2011). ^*p < 0.05.

Figure 4

Cholesterol recognition residues in the two putative transmembrane binding regions in Kir2.1. Whole-cell basal currents recorded in Xenopus oocytes at −80 mV showing the effect of cholesterol enrichment on Kir2.1 and (A) L69I, A70V, and V77I of the slide helix, (B) L85I, V93I, and S95T of the outer helix, and (C) I166V, V167L, I175L, and M183I of the inner helix (n = 9–90). Significant difference is indicated by an asterisk (^*p ≤ 0.05). A black asterisk indicates significant difference between whole-cell currents obtained for same construct following cholesterol enrichment and in the absence of treatment (control). A blue asterisk indicates significant difference between the effect of cholesterol enrichment on a mutant and the WT Kir2.1 channel. (D,E) Stick and ball presentations of the cholesterol recognition residues that surround the cholesterol molecule in each of the two putative binding regions after 50 ns of all-atom full-membrane molecular dynamics simulations. Two representative poses in region 1 are shown in (D) and one representative pose in region 2 is shown in (E). The relative position of each binding regions in the TM domain of the channel is shown in the ball presentation that includes the two adjacent subunits of the channel that interact with the cholesterol molecule in a cartoon presentation. From Rosenhouse-Dantsker et al. (2013).

Figure 5

Location of the putative transmembrane cholesterol binding regions in Kir2.1. (A) Ribbon presentation of two adjacent subunits of Kir2.1 (pink and gray) showing the TM residues whose mutation affects the sensitivity of the channel to cholesterol (in red balls) relative to the location of the five cholesterol sites (in cyan sticks and surface presentation). Also shown are the continuous chains of residues that border the cholesterol binding groove in the channel (in blue balls). (B) Schematic model illustrating the location of the two cholesterol binding regions along with labeling of the channel regions. Note that for clarity purposes, the model shows the cholesterol molecules next to one of the two adjacent channel subunits with which they are predicted to interact. From Rosenhouse-Dantsker et al. (2013).