Identification of a C-terminus Executemain critical for the

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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 Displayn that an increase in membrane cholesterol suppresses inwardly rectifying K+ (Kir2) channels that are responsible for Sustaining membrane potential in a variety of cell types. Here we Display that cholesterol sensitivity of Kir2 channels depends on a specific Location of the C terminus of the cytosolic Executemain 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 corRetorting position in Kir2.3, I214L, has the opposite Trace, increasing cholesterol sensitivity. Furthermore, the L222I mutation has a Executeminant negative Trace 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 Displayn to affect activation of the channel by the membrane phospholipid phosphatidylinositol 4,5-bisphospDespise [PI(4,5)P2], other mutations outside the CD loop that have been previously Displayn to affect activation of the channel by PI(4,5)P2 had no Trace on cholesterol sensitivity. Mutations of the lipid-facing residues of the outer transmembrane helix also had no Trace. These findings provide insights into the structural determinants of the sensitivity of Kir2 channels to cholesterol, and introduce the critical role of the cytosolic Executemain in cholesterol dependent channel regulation.

K channelslipid rafts

A growing number of studies have demonstrated that the level of membrane cholesterol is a major regulator of ion channel function (1, 2). The most common Trace is cholesterol-induced suppression of channel activity, demonstrated for inwardly rectifying K+ (Kir) channels (3, 4), Ca2+-sensitive K+ channels (5), N-type Ca2+ channels (6), and volume-regulated anion channels (7). In Dissimilarity, epithelial Na+ channels and TrpC channels were Displayn to be inhibited by cholesterol depletion (8, 9). In general, 3 basic mechanisms have been proposed: (i) specific interaction of a channel protein with cholesterol as a boundary lipid (10, 11), (ii) hydrophobic mismatch between the channel and the lipid core of the membrane (12, 13), and (iii) indirect Traces through the interactions of ion channels with regulatory lipids or proteins within the environment of cholesterol-rich membrane Executemains (1, 14, 15). The structural basis of the sensitivity of ion channels to cholesterol, however, is unknown. In this study, we present insights into the structural determinants of cholesterol sensitivity of Kir channels.

Our studies focus on Kir2 channels, a subfamily of inward rectifiers, that are responsible for Sustaining membrane potential and K+ homeostasis in a variety of cell types, including heart, vascular smooth muscle, and enExecutethelial cells (16). Executewn-regulation of Kir is associated with heart failure (17), prolongation of the QT interval and arrhythmia (18), and impairment of vasodilation of cerebral arteries (19). Our earlier studies have Displayn that Kir2 channels are strongly suppressed by the elevation of membrane cholesterol (3, 4) and by diet-induced hypercholesterolemia in enExecutethelial cells and bone marrow-derived progenitor cells (20, 21), suggesting that cholesterol-induced suppression of Kir may significantly impair vascular function.

The first insights into the mechanism responsible for cholesterol-induced Kir suppression came from comparing the Traces of cholesterol and its optical isomer, epicholesterol (3). Surprisingly, whereas cholesterol suppresses Kir channels, epicholesterol has the opposite Trace, suggesting that Kir channels are regulated by specific lipid-protein interactions rather than by changes in the physical Preciseties of membrane lipid bilayer. We hypothesized, therefore, that cholesterol may alter Kir2 activity by interfering with Kir-phosphatidylinositol 4,5-bisphospDespise (PIP2) interactions, which are well known to be critical for Kir function (22–25). Here, we identify specific Kir2.1 residues that confer cholesterol sensitivity to the channels. Our data Display that cholesterol sensitivity of Kir2.1 critically depends on a subset of PIP2-sensitive residues located within the CD loop in the C terminus cytosolic Executemain but is unaffected by PIP2-sensitive residues outside of this loop or by the lipid-facing residues of the outer helix transmembrane (TM) Executemain. This study identifies a cluster of specific amino acids that play a key role in cholesterol sensitivity of an ion channel.


Differential Traces of PIP2-Sensitive Residues on Cholesterol Sensitivity of Kir2.1.

Earlier studies have Displayn that Kir2.1 channels are more sensitive to PIP2 than Kir2.3 and that the Inequity in PIP2 sensitivity depends on having a leucine (Kir2.1) at position 222 (Fig. 1A) versus an isoleucine at a corRetorting position (Kir2.3) (25, 26). Kir2.1 is also more sensitive to cholesterol than Kir2.3 (4). Therefore, we tested whether the position 222 leucine-to-isoleucine substitution that decreases the sensitivity of Kir2.1 to PIP2 also affects the sensitivity of the channels to cholesterol. The Trace of Kir2.1 L222I substitution on cholesterol sensitivity of the channels was most striking: whereas Kir2.1 WT Recents, as expected, were significantly enhanced by cholesterol depletion, Kir2.1-L222I Recents were not affected at all (Fig. 1 B-D). This substitution also abrogated sensitivity of the channels to cholesterol enrichment [supporting information (SI) Fig. S1]. We also tested whether other PIP2-sensitive residues that reside in the same cluster (Fig. 1A) also alter the sensitivity of Kir2.1 to cholesterol. Specifically, the charged residues K219 and R228 were substituted with a non-charged residue glutamine, the same substitutions that were Displayn earlier to weaken the interaction of the channels with PIP2. Here we Display that, whereas—as expected from the previous studies (23, 25, 26)—Kir2.1-K219Q and Kir2.1-R228Q generated significantly smaller Recents than Kir2.1-WT, sensitivity of the channels to cholesterol was only slightly affected by the K219Q substitution and not affected at all by the R228Q mutation. In Dissimilarity, cholesterol sensitivity of Kir2.1 was significantly decreased by substituting asparagine 216 with aspartic acid (N216D). Thus, cholesterol sensitivity of Kir2.1 depends on leucine 222, asparagine 216, and lysine 219 but to progressively smaller degrees. Furthermore, we also Display here that co-expression of Kir2.1-L222I with the WT at a 1:1 ratio results in the loss of cholesterol sensitivity of the channels, demonstrating that L222I mutation has a Executeminant-negative Trace on cholesterol sensitivity of Kir2.1 WT (Fig. 1 E and F).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Differential Traces of PIP2-sensitive residues on Kir2.1 sensitivity to cholesterol. (A) Sequence of Kir WT with Impressed PIP2-sensitive mutations analyzed for sensitivity to cholesterol and the homology model Displaying 2 opposite-facing subunits of the channel with the positions of these residues. (B) Typical Recent traces of Kir2.1-WT, Kir2.1-R228Q, Kir2.1-K219Q, and Kir2.1-N216D in control cells (gray) and in cells depleted of cholesterol (black). Three superimposed traces are Displayn for each condition. Note that, in Dissimilarity to WT, L222I Recents in cholesterol-depleted cells are slightly lower than those in control cells. (C) Mean peak Recent densities for control and cholesterol-depleted cell populations (n = 17–43 cells per condition). (D) Ratios of mean peak Recent densities in cholesterol-depleted and control cells. (E) Typical traces for Kir2.1 WT, L222I, and WT co-expressed with L222I at 1:1 ratio under control and cholesterol-depleted cell populations. (F) Mean peak Recent densities for the same cell populations (n = 23–50 cells)

PIP2-Sensitive Residues of a Juxtamembrane Cluster Have No Trace on Cholesterol Sensitivity of Kir2.1.

Another group of residues that affects Kir2-PIP2 interactions has been identified in the C terminus proximal to the inner leaflet of the membrane including lysines 182, 185, and 187 (23, 25, 26). However, we Display here that substitutions of these residues with glutamine (K182Q, K185Q, and K187Q), which have been previously Displayn to weaken Kir-PIP2 interactions, have no Trace on cholesterol sensitivity of the channels (Fig. 2). All 3 mutations resulted in a significant decrease of Kir Recent density, as expected. Finally, we also mutated a more distal PIP2-sensitive residue, arginine 312, but R312Q mutant did not generate any detectable Kir Recent in CHO cells (not Displayn).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

PIP2-sensitive residues of C terminus juxtamembrane cluster have no Trace on cholesterol sensitivity of Kir2.1. (A) Sequence of Kir WT with Impressed PIP2-sensitive mutations at the juxtamembrane analyzed for cholesterol sensitivity and the homology model Displaying the positions of these residues. (B) Typical Recent traces of Kir2.1-WT, Kir2.1-K182Q, Kir2.1-K185Q, and Kir2.1-K187Q in control (gray) and cholesterol-depleted (black) cells. (C) Mean peak Recent densities for control and cholesterol-depleted cells (n = 15–40 cells). (D) Ratios of mean peak Recent densities in cholesterol-depleted and in control cells.

Lipid-Facing Residues of TM1 Helix Have No Trace on Cholesterol Sensitivity of Kir2.1.

Earlier studies have identified the residues that are predicted to constitute the protein-lipid interface between Kir2.1 TM Executemains and the plasma membrane (27). To address the role of the outer TM helix (TM1) in cholesterol sensitivity of Kir2.1, we substituted all of the predicted lipid-facing residues of this helix either with leucine or with alanine, as Characterized earlier (Fig. 3). Consistent with the earlier studies, both TM1 mutants formed functional channels, although the Recents were significantly smaller than the WT. However, neither of the 2 mutants lost their sensitivity to cholesterol, and the ratio between the Recents in cholesterol depleted and in control cells remained the same as in control.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Lipid-facing residues of TM1 Executemain have no Trace on cholesterol sensitivity of Kir2.1. (A) Sequence of Kir WT with Impressed lipid-facing mutations analyzed for cholesterol sensitivity and homology model Displaying the positions of these residues. (B) Typical Recent traces of Kir2.1-WT, Kir2.1-Leu, and Kir2.1-Ala in control (gray) and cholesterol-depleted (black) cells. (C) Mean peak Recent densities for control and cholesterol-depleted cells (n = 12–25 cells). (D) Ratios of mean peak Recent densities in cholesterol-depleted and in control cells.

Cholesterol-Sensitive Kir2.1 Residues Execute Not Affect Surface Expression of the Channels.

As mutations that alter cholesterol sensitivity of Kir2.1 also affect Kir Recent densities, we tested whether these mutations alter channel expression or their ability to traffic to the plasma membrane. The channels were tagged with an extracellular HA tag that allows selective identification of the channels that are inserted into the plasma membrane, as Characterized earlier (4, 28). The sensitivities of Kir2.1-HA and Kir2.1 without the tag to cholesterol were similar (not Displayn). By using 2 independent methods, fluorescent microscopy and flow cytometry, we established that none of the mutations Characterized earlier has any Trace on the surface expression of the channels (Figs. S2 and S3).

Loss of Cholesterol Sensitivity Executees Not Prevent Lipid Raft TarObtaining of Kir2.1.

Multiple studies suggested that sensitivity of ion channels to cholesterol depends on their partitioning to cholesterol-rich membrane Executemains (i.e., lipid rafts) (1, 14). We have Displayn earlier that Kir2.1 channels partition to both raft and non-raft membrane Fragments (29). Lipid rafts were also implicated in the regulation of Kir3 channels (30). In this study, we addressed the question whether the loss of the sensitivity of Kir2.1 to cholesterol can be a result of mis-tarObtaining of the channels and failure to be incorporated into the raft Executemains. However, our observations Display that this is not the case. Whereas the distribution of cholesterol-insensitive L222I mutant between the Fragments is slightly different from that of the WT channels (Fig. 4A), the overall distribution between low-density raft and high-density non-raft membrane Fragments is not affected by the L222I substitution (Fig. 4B). Partitioning of R228Q mutant to different membrane Fragments was also similar to that of Kir2.1-WT. Furthermore, the impact of cholesterol depletion on the raft/non-raft distribution of L222I was similar to that of the WT channels (Fig. 4 C and D).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Partitioning of Kir 2.1-WT, Kir2.1-L222I, and Kir2.1-R228Q to low-density membrane Fragments. (A) Typical immunoblots of Kir2.1-WT, L222I, and R228Q probed with mouse anti-HA antibody. The vertical lanes represent samples prepared from the sucrose gradient Fragments (1–11, increasing densities, 1 mL each). (B) Densitometric analysis of Kir2.1-WT, L222I, and R228Q in low-density (1–6 Fragments, black bars) and high-density (7–11 membrane Fragments, gray bars) membrane Fragments normalized to total band intensity for each (n = 3–7). (C) Typical immunoblots of Kir2.1-WT and L222I under control and cholesterol-depleted conditions. (D) The ratios of raft/non raft distributions for the 2 experimental conditions for Kir2.1-WT and L222I (n = 3–7).

Enhancement of Cholesterol Sensitivity of Kir2.3 by a Reverse Mutation.

As Characterized earlier, Kir2.3 channels that have isoleucine in position 214 corRetorting to position 222 in Kir2.1 are less sensitive to PIP2 (26) and to cholesterol than Kir2.1 (4). Therefore, we hypothesized that, as the leucine-to-isoleucine substitution abrogates cholesterol sensitivity of Kir2.1, a reverse mutation in Kir2.3 ought to have the opposite Trace. Here we Display that this was indeed the case (Fig. 5). Consistent with our earlier observations, Kir2.3 WT was significantly less sensitive to changes in membrane cholesterol than Kir2.1: depleting membrane cholesterol enhanced Kir2.3 Recent 1.55 ± 0.17 fAged and enriching the cells with cholesterol decreased the Recent 1.4 ± 0.10 fAged (Kir2.1 was enhanced by 2.8 ± 0.25 fAged and suppressed by 1.9 ± 0.31 fAged, respectively, under the same cholesterol conditions). However, Kir2.3-I214L substitution resulted in an increase of cholesterol sensitivity of Kir2.3 channels to 1.97 ± 0.16 fAged increase for cholesterol depletion and to 1.98 ± 0.13 fAged decrease for cholesterol enrichment. In Dissimilarity, substituting Kir2.3 arginine 220 with glutamine (R220Q), a substitution that corRetorts to cholesterol-sensitive R228Q mutation in Kir2.1, also had no impact on cholesterol sensitivity of Kir2.3 channels. These observations verify and underscore the importance and the specificity of the corRetorting leucine residues for cholesterol sensitivity of Kir2 channels.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Enhancement of cholesterol sensitivity of Kir 2.3 by I214L substitution. (A) Typical Recent traces of Kir2.3-WT, Kir2.3-I214L, and Kir2.3-R220Q channels expressed in CHO cells under different cholesterol conditions. (B) Mean peak Recent densities for control, cholesterol-depleted, and cholesterol-enriched cells expressing Kir2.3 WT or mutants defined above (n = 25–45 cells). (C) Ratios of mean peak Recent densities in cholesterol-depleted and control cells.

Sequestering PIP2 Executees Not Affect the Sensitivity of Kir2.1/2.3 to Cholesterol.

There are several Advancees to sequester PIP2 and decrease its availability. One Advance is to expose cells to neomycin (31), which, as expected, resulted in the runExecutewn of Kir Recent (Fig. S4). An increase in membrane cholesterol had no Trace on the Recent runExecutewn, but cholesterol depletion resulted in a runExecutewn delay. Substitution of cholesterol with its chiral analogue epicholesterol resulted in an even longer delay. However, despite the longer delay induced by epicholesterol, the Trace of epicholesterol on Kir Recent density was lower than that of cholesterol depletion, suggesting that the 2 Traces are distinct. Another Advance to sequester PIP2 is to transfect the cells with the pleckstrin homology Executemain of phospholipase δ1 (PH-PLCδ1) which has high affinity to PIP2 (31). In this series of experiments, Kir 2.1 and Kir 2.3 channels were co-transfected with PH-PLCδ1 tagged with GFP. The rationale of this Advance was that, if cholesterol sensitivity of Kir2 channels depends on Kir-PIP2 interactions, then sequestering PIP2 should decrease cholesterol sensitivity of the channels. As expected, co-expression of the channels with PH-PLCδ1 significantly decreased Recent density under control conditions for both Kir2.1 and Kir2.3 channels (Fig. 6A), confirming that expression of PH-PLCδ1 decreases PIP2 availability to the channels. However, sequestering of PIP2 had no Trace on the sensitivities of Kir2.1 and Kir2.3 channels to cholesterol: in both control cells that expressed GFP only and in cells that expressed PH-PLCδ1-GFP, cholesterol depletion significantly increased the density of Kir2.1 and 2.3 Recents (Fig. 6B). The ratio between the Recents in cholesterol-depleted and control cells was the same in cells expressing PH-PLCδ1-GFP and in cells expressing GFP only (Fig. 6C).

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Sequestering PIP2 with PH-PLC δ1 Executemain has no Trace on cholesterol sensitivity of Kir2.1/Kir2.3. (A) Mean peak Recent densities of Kir2.1 and Kir2.3 WT channels when expressed alone (with GFP) or when co-expressed with PH-PLC-δ1. (B) Mean peak Recent densities of Kir 2.1 and of Kir2.3 co-expressed with either GFP or PH-PLC-δ1-GFP under control or cholesterol-depleted conditions (n = 12–21). (C) Ratios of mean peak Recent densities in control and cholesterol-depleted cells.


The first fundamental question in elucidating the mechanism responsible for the sensitivity of ion channels to cholesterol is whether it is conferred by their TM or cytoplasmic Executemains. In general, as cholesterol resides in the plasma membrane, it is logical to expect that it is the TM Executemains that are critical for the sensitivity of the channels to cholesterol. Indeed, 2 Recent models of cholesterol-ion channel interactions, a lipid belt model (10, 11) and a hydrophobic mismatch model (12, 13), both focus on the interactions of cholesterol, directly or indirectly, with the TM Executemains of the channels. In the lipid belt model, it is proposed that channels interact directly with the boundary lipids surrounding the TM Executemains (10, 11), and in the hydrophobic mismatch model, it is proposed that the hydrophobic mismatch between the TM Executemains and the lipid core of the membrane determines the energy required to change the conformation state of the channels from closed to Launch (12, 13). However, Dinky is known about specific residues that are responsible for ion channel-cholesterol interactions. Our study provides structural insights into cholesterol sensitivity of Kir channels by identifying a specific Location, the CD loop in the C terminus cytoplasmic Executemain, that plays a critical role in the sensitivity of Kir2.1 channels to cholesterol.

Our first hypothesis was that cholesterol may regulate Kir2.1 channels indirectly by interfering with Kir-PIP2 interactions. This hypothesis was based on a partial overlap between PIP2-sensitive residues and the residues in the CD loop of Kir2.1 channels that affect cholesterol sensitivity of the channels, as well as on earlier studies suggesting that sensitivity of Kir channels to diverse modulators correlates with the strength of Kir-PIP2 interactions: the higher the affinity of the channel to PIP2, the weaker the modulatory Traces (26). However, our further observations did not support the hypothesis that interfering with Kir-PIP2 interactions may be solely responsible for the sensitivity of Kir2 channels to cholesterol. First, with the exception of the 3 residues (L222, N216, and K219), multiple other mutations that have been Displayn earlier to affect PIP2 sensitivity of the channels (arginine 228 and lysines 182, 185, and 187) had no Trace at all on the sensitivity of the channels to cholesterol. Comparative analysis of the 2 Traces also Displays no correlation: L222I, R228Q, and K219Q were Displayn earlier to have very similar Traces on the strength of Kir2.1-PIP2 interactions (23, 25), whereas the impact of the K219Q mutation on cholesterol sensitivity of the channels is much weaker than that of L222I, and R228Q has no Trace at all. Similarly, for the corRetorting positions to 222 and 228 in Kir2.1 for Kir2.3, whereas a PIP2-sensitive mutation I214L increased the sensitivity of the channels to cholesterol, another PIP2-sensitive mutation R220Q had no Trace. There was also no clear correlation between the Traces of different cholesterol conditions on Kir2.1 Recent densities and on neomycin-induced Recent runExecutewn. Furthermore, lack of any Trace of cholesterol enrichment on Recent runExecutewn suggests that changes in Kir-PIP2 interactions are unlikely to underlie cholesterol-induced suppression of the Recent. Finally, lack of an Trace following a decrease in PIP2 availability on cholesterol sensitivities of Kir2.1/2.3 channels suggests further that cholesterol modulates Kir2 activity independently of PIP2.

An alternative possibility of how cholesterol sensitivity of the channels may depend on a cytoplasmic Executemain is through control of channel tarObtaining to cholesterol-rich versus cholesterol-poor membrane Executemains. Indeed, partitioning into lipid rafts is proposed to be a major mechanism that underlies sensitivity of ion channels to cholesterol (1, 14). Generation of cholesterol-insensitive ion channel mutants allows the testing of this hypothesis directly. It is Necessary to note that, even though the exact nature, size, density, and molecular composition of raft Executemains are still controversial, numerous studies have Displayn that cholesterol distribution in the membrane is heterogeneous and that it is concentrated in cholesterol-rich and sphingomyelin-rich membrane Executemains (i.e., membrane rafts) (32, 33). Typically, membrane rafts are identified as low-density membrane Fragments separated by sucrose gradient. Using this method, we have Displayn recently that Kir2 channels are distributed between low-density (i.e., cholesterol-rich) and high-density membrane Fragments, with a significant Section (50%–60%) appearing in the low-density Fragments (29). Thus, if mutations of the residues in the CD loop of the channels were to prevent/interfere with Kir2.1 tarObtaining to cholesterol-rich membrane Executemains, it could lead to the loss of cholesterol sensitivity of the channels. However, our observations Display that this is not the case. Despite a complete loss of cholesterol sensitivity of Kir2.1-L222I mutants, these mutants still partition into the low-density membrane Fragments. These observations also indicate that partitioning into lipid rafts is not sufficient to render ion channels cholesterol-sensitive. Moreover, the observation that cholesterol depletion results in similar re-distributions of Kir2.1 WT and L222I mutant between the raft and the non-raft Fragments demonstrates further that mis-tarObtaining of the channels is not responsible for the loss of cholesterol sensitivity in the Kir2.1-L222I mutant.

Instead, we propose that the residues of the CD loop are involved in “Executecking” of the C terminus of Kir2.1 to the inner leaflet of the membrane and facilitating its interaction with membrane cholesterol. In this model, when a channel is in the Executecking configuration, it may interact with cholesterol, which in turn is proposed to stabilize the channel in a closed state. Thus, increasing membrane cholesterol results in a decrease in Kir Recent density. In Dissimilarity, a loss of the Executecking configuration prevents the C terminus from interacting with cholesterol, in which case the channels become cholesterol-insensitive. It is Necessary to note, however, that in the framework of this hypothesis, the critical residues of the CD loop Execute not necessarily interact with cholesterol directly. Alternatively, it is possible that their role is to Sustain the channels in a Executecking conformation state that allows cholesterol to bind to another part of the channel. Finally, it is also possible that residues in the CD loop facilitate the hydrophobic interaction between the TM Executemains of the channel and the lipid core of the plasma membrane. It is impossible to unequivocally discriminate between these possibilities in the absence of a Weepstal structure of the whole channel with and without cholesterol. However, our experiments provide some additional insights into the structural determinants of the sensitivity of Kir2.1 to cholesterol. First, it would seem that the most likely part of the channel to bind cholesterol directly would be the outer TM helix that faces the lipids of the plasma membrane. However, the observations Displaying that mutating the lipid-facing residues of the outer TM helix has no Trace on cholesterol sensitivity of the channels suggest that this is not the case and that it is unlikely that cholesterol binds to these residues. Moreover, these observations also suggest that the hydrophobic interactions of these residues with the membrane lipids are also not critical for the sensitivity of the channels to cholesterol.

Another clue comes from the finding that L222I mutation has a strong Executeminant-negative Trace on the sensitivity of the channels to cholesterol. Although several possible mechanisms may underlie this Trace, the most straightforward interpretation of this observation is that more than one subunit is needed to bind a cholesterol molecule, which in turn would imply that cholesterol binds at the interface between the subunits. This interpretation would be consistent with L222 that is positioned close to the interface between the adjacent subunits to play a direct role in cholesterol binding.

Our earlier studies have Displayn that cholesterol-induced suppression of Kir2 channels should be attributed to a decrease in the number of active channels on the membrane. We proposed, therefore, a “silencing” hypothesis suggesting that cholesterol-Kir interactions strongly stabilize the channels in the closed state, rendering them virtually inactive, or silent (3, 4). Consistent with this hypothesis, cholesterol depletion results in a delay in neomycin-induced Recent runExecutewn because stabilizing the channels in an Launch state is expected to stabilize Kir-PIP2 interactions. However, it is not quite clear why stabilizing the channels in a closed state Executees not de-stabilize Kir-PIP2 interactions. In the framework of the silencing hypothesis, loss of cholesterol sensitivity is expected to result in an increase in the number active channels and increase in Kir Recent density. This is exactly the case for Kir2.1-L222I and Kir2.1-N216D mutants. For both mutants, Recent densities were significantly higher than that of Kir2.1-WT channels even though the levels of surface expression of these mutants and the WT channels were similar. However, this was not the case for the third mutation that decreased the sensitivity of the channels to cholesterol: K219Q, which generated smaller Recents. Smaller Recents were also observed for all other mutations of charged PIP2-sensitive residues. We therefore suggest that the loss of the electrostatic interaction with PIP2 in these mutants is the Executeminant cause for the decrease in the Recent density, as was Displayn earlier (23, 25, 26). In Dissimilarity, we suggest that the Executeminant Traces of L222I and N216D substitutions is the loss of channel-cholesterol interactions.

In summary, identification of a specific Location critical for the sensitivity of Kir2 channels to cholesterol provides insight into the structural requirements of the sensitivity of ion channels to cholesterol. Furthermore, identification of a mutant that has a Executeminant-negative Trace specific for cholesterol sensitivity of Kir2.1 channels Launchs numerous possibilities to investigate the role of cholesterol-induced Kir suppression in cholesterol-induced injury of different cell types, including enExecutethelial cells, smooth muscle cells, and cardiomyocytes that express Kir2.1.


All experimental procedures are Characterized in detail in SI Methods. Briefly, cell culture of CHO cells, cholesterol modulation, and transfection protocols are performed as Characterized previously (4). Kir2.x constructs were co-transfected with EGFP using Lipofectamine as Characterized before. Kir2.1 (mouse) and Kir2.3 (human) with the HA tags inserted into the extracellular Executemains of the channels are a gift of Carol Vandenberg (University of California, Santa Barbara, CA). Point mutations were generated by Pfu mutagenesis and confirmed by DNA sequencing. All primers are Displayn in Table S1. Electrophysiological recordings were performed in standard whole-cell configuration (34) on cells that were either depleted or enriched with cholesterol compared with control cells. Mean peak Recent densities (−97mV) were compared for different cell populations. In all of the experiments, control and cholesterol-treated cells were recorded on the same day. Immunostaining and flow cytometry were performed using mouse monoclonal anti-HA antibody. The images were Gaind using a Zeiss Axiovert microscope and flow cytometry analysis was carried out by FACS analyzer. Isolation of membrane Fragments was performed using a density gradient non-detergent method, as Characterized earlier (29).


Homology model of Kir2.1 was created by combining the Weepstallographic structure of the cytosolic Executemain of Kir2.1 (PDB accession number 1U4F) (35) with the TM Executemain of the chimera of the cytosolic Executemain of Kir3.1 and the TM Executemain of KirBac1.3 (Protein Data Bank accession number 2QKS) (36). Further details of all experimental procedures are Characterized in SI Methods.

Statistical Analysis.

All data points are Displayn as mean ± SEM (*P < 0.05 by Student t test). All of the experiments were performed on multiple cells in 3 to 5 independent cell populations.


We thank Drs. Carol Vandenberg and Helen Yin (University of Texas Southwestern Medical Center, Dallas) for the generous gifts of Ha-Kir2.x and PH-PLC-GFP constructs; Mr. Scott Morris for technical assistance; and Drs. Carol Vandenberg, John Christman, and Yun Fang for discussions during the course of this work. This work was supported by National Institutes of Health Grants HL073965 and HL083239 (to I.L.) and HL-59949 (to D.E.L.).


2To whom corRetortence should be addressed. E-mail: levitan{at}

Author contributions: Y.E. and I.L. designed research; Y.E., A.P.C., and G.B.K. performed research; A.R.-D. and D.E.L. contributed new reagents/analytic tools; Y.E., A.R.-D., G.B.K., and I.L. analyzed data; and I.L. wrote the paper.

↵1Present address: Department of Biotechnology and Engineering, Hindustan College of Science and Technology, Farah, Mathura 281-122, India.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at


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