A variety of ion channels are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. However, the mechanism underlying cholesterol sensitivity of ion channels is unknown. We have recently shown that an increase in membrane cholesterol suppresses inwardly rectifying K ؉ (Kir2) channels that are responsible for maintaining membrane potential in a variety of cell types. Here we show that cholesterol sensitivity of Kir2 channels depends on a specific region of the C terminus of the cytosolic domain of the channel, the CD loop. Within this loop, the L222I mutation in Kir2.1 abrogates the sensitivity of the channel to cholesterol whereas a reverse mutation in the corresponding position in Kir2.3, I214L, has the opposite effect, increasing cholesterol sensitivity. Furthermore, the L222I mutation has a dominant negative effect on cholesterol sensitivity of Kir2.1 WT. Mutations of 2 additional residues in the CD loop in Kir2.1, N216D and K219Q, partially affect the sensitivity of the channel to cholesterol. Yet, whereas these mutations have been shown to affect activation of the channel by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2], other mutations outside the CD loop that have been previously shown to affect activation of the channel by PI(4,5)P2 had no effect on cholesterol sensitivity. Mutations of the lipid-facing residues of the outer transmembrane helix also had no effect. These findings provide insights into the structural determinants of the sensitivity of Kir2 channels to cholesterol, and introduce the critical role of the cytosolic domain in cholesterol dependent channel regulation.

Background: Cholesterol modulates inwardly rectifying potassium (Kir) channels. Results: A two-way molecular cytosolic switch controls channel modulation by cholesterol and PI(4,5)P 2 . Conclusion: Cholesterol and PI(4,5)P 2 induce a common gating pathway of Kir2.1 despite their opposite impact on channel function. Significance: These findings provide insights into structure-function relationship of ion channels and contribute to understanding of the mechanisms underlying their regulation by lipids.

(ECs) in the direction of the flow is considered a key factor in maintaining endothelial integrity in an active hemodynamic environment. Our recent studies showed that exposure to oxidized LDL (oxLDL), one of the major proatherogenic lipoproteins, significantly increases the stiffness of human aortic ECs, suggesting that oxLDL may have a significant impact on the sensitivity of ECs to mechanical stimuli. In this study, we show that oxLDL strongly enhances the ability of ECs to realign in the direction of the flow and facilitates the formation of F-actin stress fibers under static and flow conditions. The impact of oxLDL on the flow-induced realignment is observed on whole cell and single-fiber levels. We also show that, similar to the effect of oxLDL on endothelial stiffness, the impact of oxLDL on flow-induced realignment can be simulated by methyl-␤-cyclodextrininduced cholesterol depletion, supporting the hypothesis that oxLDL acts as cholesterol acceptor, rather than cholesterol donor, for ECs. Finally, we propose that oxLDL/cholesterol depletion-induced sensitization of ECs to flow may be a result of an increase in cellular stiffness and a respective increase in membrane-cytoskeleton tension.cholesterol; lipid rafts HEMODYNAMIC FORCES GENERATED by blood flow are known to play prominent roles in the acute control of vascular tone, regulation of arterial structure, and localization of atherosclerotic lesions (12,19,31). The primary tissue that is affected by the hemodynamic environment is vascular endothelium, a single-cell layer that constitutes the inner lining of the blood vessels and exists on the blood-vascular wall interface. Multiple studies have shown that endothelial cells (ECs) respond to flow by a variety of mechanisms, including cytoskeleton rearrangement, activation of multiple signaling pathways, and changes in gene expression (for review see 24,25,34). Although the exact mechanisms responsible for conversion of mechanical stimuli to the biological responses are not fully understood, it is generally accepted that the cytoskeleton provides the structural framework for transmission of mechanical signals throughout the cell. Flow-induced changes of endothelial morphology, such as realignment in the direction of flow and cell elongation, are suggested to be important for maintaining endothelial integrity in active flow environments (9,20,25).Our recent studies showed that endothelial mechanical properties are strongly affected by exposure of cells to the oxidized form of low-density lipoproteins (oxLDL) (6). More specifically, exposure to oxLDL, but not the unoxidized form of LDL, resulted in a significant decrease in the deformability/increase in stiffness of aortic ECs, as measured by micropipette aspiration (microaspiration), as well as an increase in the ability of ECs to generate force on the cell-substrate interface (6). Depletion of cellular cholesterol had the same effect (6) and also increased membrane-cytoskeleton adhesion (37). Multiple studies have shown that deformability of the cellu...

Cholesterol is one of the major lipid components of membranes in mammalian cells. In recent years, cholesterol has emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels in general and on inwardly rectifying potassium (Kir) channels in particular is a decrease in activity. In contrast, we have recently shown that native G-protein gated Kir (GIRK or Kir3) channels that underlie atrial KACh currents are up-regulated by cholesterol. Here we unveil the biophysical basis of cholesterol-induced increase in KACh activity. Using planar lipid bilayers we show that cholesterol significantly enhances the channel open frequency of the Kir3.1/Kir3.4 channels, which underlie KACh currents. In contrast, our data indicate that cholesterol does not affect their unitary conductance. Furthermore, using fluorescent and TIRF microscopy as well as surface protein biotinylation, we also show that cholesterol enrichment in vitro has no effect on surface expression of GFP-tagged channels expressed in Xenopus oocytes or transfected into HEK293 cells. Together, these data demonstrate for the first time that cholesterol enhances Kir3-mediated current by increasing the channel open probability.

The maintenance of cell volume homeostasis is critical for preventing pathological cell swelling that may lead to severe cellular dysfunction or cell death. Our earlier studies have shown that volume-regulated anion channels that play a major role in the regulation of cell volume are facilitated by a decrease in cellular cholesterol suggesting that cholesterol depletion should also facilitate regulatory volume decrease (RVD), the ability of cells to recover from hypotonic swelling. In this study, we test this hypothesis using a novel methodology developed to measure changes in cell volume using a microfluidics chamber. Our data show that cholesterol depletion of Chinese Hamster Ovary (CHO) significantly facilitates the recovery process, as is apparent from a faster onset of the RVD (162±10 s. vs. 114±5 s. in control and cholesterol depleted cells respectively) and a higher degree of volume recovery after 10 min of the hypotonic challenge (41%±6% vs. 65%±6% in control and cholesterol depleted cells respectively). In contrast, enriching cells with cholesterol had no effect on the RVD process. We also show here that similarly to our previous observations in endothelial cells, cholesterol depletion significantly increases the stiffness of CHO cells suggesting that facilitation of RVD may be associated with cell stiffening. Furthermore, we also show that increasing cell stiffness by stabilizing Factin with jasplakinolide also facilitates RVD development. We propose that cell stiffening enhances cell mechano-sensitivity, which in turn facilitates the RVD process.