Comparative analysis of cholesterol sensitivity of Kir channels: Role of the CD loop

Abstract

Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir2 channels and several other ion channels that has emerged in recent years is that they are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. Yet, the mechanism by which cholesterol affects channel function is not clear. We have recently shown that the sensitivity of Kir2 channels to cholesterol depends on residues in the CD loop of the cytosolic domain of the channels with one of the mutations, L222I, abrogating cholesterol sensitivity of the channels completely. Here we show that in addition to Kir2 channels, members of other Kir subfamilies are also regulated by cholesterol. Interestingly, while similarly to Kir2 channels, several Kir channels, Kir1.1, Kir4.1 and Kir6.2Δ36 were suppressed by an increase in membrane cholesterol, the function of Kir3.4* and Kir7.1 was enhanced following cholesterol enrichment. Furthermore, we show that independent of the impact of cholesterol on channel function, mutating residues in the corresponding positions of the CD loop in Kir2.1 and Kir3.4*, inhibits cholesterol sensitivity of Kir channels, thus extending the critical role of the CD loop beyond Kir2 channels.

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.…”

“…16 Because cholesterol is a component of lipid rafts, 18,29 we originally hypothesized that ROMK1 might be in lipid rafts and that endogenous cholesterol could directly regulate the channel. Surprisingly, our data from sucrose gradient experiments showed that the ROMK1 channel was detected mainly in nonlipid raft membranes ( Figure 3, A and B).…”

“…DIC, differential interference contrast. 16 [control]; P=0.10; n=10). Knocking down PI(4)P5K I g expression with its siRNA was confirmed with Western blot experiments, which are shown in Figure 8C.…”

“…12 Interestingly, cholesterol inhibits Kir channels, 1 3 -1 5 including the Kir 1.1 (ROMK1) channel. 16 Therefore, CsA-induced elevation of cholesterol in CCD cells may account, at least in part, for CsA inhibition of ROMK channels. Recently, we have shown that CsA causes damage to CCD cells by elevating intracellular cholesterol and that lovastatin antagonizes the CsA-induced damage by reducing intracellular cholesterol.…”

“…[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.…”