Modulation of Endothelial Inward-Rectifier K+ Current by Optical Isomers of Cholesterol

Abstract

Membrane potential of aortic endothelial cells under resting conditions is dominated by inward-rectifier K(+) channels belonging to the Kir 2 family. Regulation of endothelial Kir by membrane cholesterol was studied in bovine aortic endothelial cells by altering the sterol composition of the cell membrane. Our results show that enriching the cells with cholesterol decreases the Kir current density, whereas depleting the cells of cholesterol increases the density of the current. The dependence of the Kir current density on the level of cellular cholesterol fits a sigmoid curve with the highest sensitivity of the Kir current at normal physiological levels of cholesterol. To investigate the mechanism of Kir regulation by cholesterol, endogenous cholesterol was substituted by its optical isomer, epicholesterol. Substitution of approximately 50% of cholesterol by epicholesterol results in an early and significant increase in the Kir current density. Furthermore, substitution of cholesterol by epicholesterol has a stronger facilitative effect on the current than cholesterol depletion. Neither single channel properties nor membrane capacitance were significantly affected by the changes in the membrane sterol composition. These results suggest that 1) cholesterol modulates cellular K(+) conductance by changing the number of the active channels and 2) that specific cholesterol-protein interactions are critical for the regulation of endothelial Kir.

Cholesterol Inhibits M-type K+ Channels via Protein Kinase C-dependent Phosphorylation in Sympathetic Neurons

“…Treating cells with cholesterol-free or cholesterol-loaded M␤CD (M␤CD/Chol) has been widely used to selectively and efficiently reduce or enhance the cholesterol content of the membrane (22)(23)(24). We applied an M␤CD/Chol complex to increase the cholesterol content of the cell membrane.…”

“…Even though cholesterol has multiple roles, however, it is unclear whether it also acts as a signaling molecule. Recently, it was shown that cholesterol, itself, modulated inwardly rectifying K ϩ (IRK) channel activity in vascular endothelial cells (21,22) and delayed rectifier K ϩ channels in cultured hippocampal neurons (23). These observations raised these questions: how does cholesterol regulate channel activity and does it also regulate other ion channels?…”

Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel

“…Epicholesterol (Fig. 1B) (Steraloids Inc., Newport, RI) was added to the membrane by incubating the patch with 10:1 M␤CD:epicholesterol (0.08 mg/ml) (31,32).…”

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

“…Our studies demonstrated that specific sterol-protein interactions are responsible for the suppression of Kir channels. First, we showed that cholesterol and its optical stereoisomer, epicholesterol, have opposite effects on endothelial Kir current (5) and that there is no correlation between the effects of different sterols on membrane fluidity and on the activity of a purified bacterial analog of Kir channels, KirBac1.1, incorporated into a pure lipid environment (liposomes) (16). Consistent with these observations, it was shown more recently that ent-cholesterol, an enantiomer of cholesterol (17), has no effect either on KirBac1.1 or on Kir2.1 function (18).…”

“…Here we study the mechanism underlying cholesterol regulation of Kir2 channels, which we have shown earlier to be suppressed by increased levels of membrane cholesterol (5)(6)(7)(8). The members of this subfamily of constitutively active, strongly inwardly rectifying K ϩ (Kir) 4 channels are critically involved in regulation of the excitability and contraction in a variety of cell types and in maintaining the membrane potential in several types of non-excitable cells (9 -11).…”