Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by ⁸⁶Rb⁺ uptake. Our results show that ⁸⁶Rb⁺ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses ⁸⁶Rb⁺ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated ⁸⁶Rb⁺ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.Inwardly rectifying potassium channels (Kir) are known to play critical roles in the regulation of multiple cellular functions including membrane excitability, heart rate, and vascular tone (1–3). Kir channels are classified into seven subfamilies (Kir1–7) identified by distinct biophysical properties and sensitivities to different regulators (2). Our earlier studies have shown that Kir2 channels, one of the major subfamilies of Kir that are responsible for maintaining membrane potential in a variety of cell types, are strongly suppressed by the elevation of membrane cholesterol (4, 5). Cholesterol-induced suppression of Kir2 was first observed in aortic endothelial cells (4), in which resting K⁺ conductance is dominated by Kir2.1 and Kir2.2 channels (6), and then when channels were heterologously expressed in Chinese hamster ovary cells (5, 7). Furthermore, the same effect was observed ex vivo in endothelial cells and bone marrow-derived progenitor cells isolated from hypercholesterolemic pigs (8, 9).In terms of the mechanism, the first insights came from comparing the effects of cholesterol and of its chiral analogue, epicholesterol. Although the two sterols are known to have almost identical effects on the biophysical properties of the lipid bilayer (10, 11), their impact on Kir activity is completely different; partial substitution of endogenous cholesterol with epicholesterol resulted in significant increase in Kir current in endothelial cells (4). These observations suggest that specific sterol-protein interactions may be involved in the cholesterol sensitivity of Kir2 channels. However, in the complex environment of the plasma membrane, cholesterol may interact not only with the channels themselves but also with other proteins, which in turn may regulate the activity of the channels. In the cellular environment, therefore, it is impossible to discriminate between direct channel-cholesterol interactions and indirect effects. Moreover, it is impossible to define the actual concentrations of cholesterol in any given membrane compartment. To quantitatively test direct cholesterol-protein interactions, it is necessary to examine sensitivity of pure Kir channels to membrane cholesterol in a membrane of defined lipid composition. To date, only the cytoplasmic domains of several mammalian Kir channels have been purified (Kir2.1, Kir3.1, and Kir3.2) (12–15). We therefore concentrate in this study on the effect of cholesterol on two bacterial K⁺ channels that differ in the level of their homology to mammalian Kir channels, KirBac1.1 and KcsA. KirBac channels have high sequence homology with mammalian Kirs (e.g. 52% homology between KirBac1.1 and Kir2.1; see Fig. 7A) and have now been extensively used as structural models of mammalian Kir channels (3, 16, 17). The sequence similarity between KcsA and mammalian K channels lies mainly in the transmembrane domain (18). The overall sequence homology of KcsA to mammalian Kir channels is relatively low (e.g. 22% homology between KcsA and Kir2.1; see Fig. 7A), with an entirely different cytoplasmic domain structure.Open in a separate windowFIGURE 7.Cholesterol has no effect on KcsA-mediated ⁸⁶Rb⁺ uptake. A, time courses of ⁸⁶Rb⁺ uptake into liposomes reconstituted with 50 μg of cholesterol/mg of PL and as compared with liposomes containing no cholesterol (control). Both batches of liposomes contained 5 μg of KcsA/mg of PL. Blank liposomes contain no protein. The points represent averages of three independent experiments (means ± S.D.). B, normalized time courses of ⁸⁶Rb⁺ uptake in liposomes incorporating 50, 150, and 250 μg of cholesterol/mg of PL. C, maximal uptake of ⁸⁶Rb⁺ after 240 s in liposomes containing 10, 25, 50, 100, 150, 200, and 250 μg of cholesterol/mg of PL normalized to control (means ± S.D. of 3–5 independent experiments; *, p < 0.05). DPM, disintegrations per minute.Here we show that, similarly to Kir2 channels, prokaryotic Kir channels incorporated into liposomes are strongly suppressed by an increase in membrane cholesterol. Furthermore, the sensitivity of prokaryotic Kir to cholesterol is stereo-selective to cholesterol optical analogues. In contrast, KscA channels are insensitive to membrane cholesterol. These observations suggest that cholesterol directly suppresses Kir channels.