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VASCULAR BIOLOGY
1Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania; 2Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; 3Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and 4Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York
Submitted 18 September 2006 ; accepted in final form 13 April 2007
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ABSTRACT |
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-cyclodextrin-induced cholesterol depletion removes cholesterol from both raft and non-raft membrane fractions, cholesterol enrichment results in cholesterol increase exclusively in the raft fractions. Kinetics of both depletion-induced Kir2.1 enhancement and enrichment-induced Kir2.1 suppression correlate with the changes in the level of raft cholesterol. Furthermore, we show not only that cholesterol depletion shifts the distribution of the channels from cholesterol-rich to cholesterol-poor membrane fractions but also that cholesterol enrichment has the opposite effect. These observations suggest that change in the level of raft cholesterol alone is sufficient to suppress Kir2 activity and to facilitate partitioning of the channels to cholesterol-rich domains. Therefore, we suggest that partitioning to membrane rafts plays an important role in the sensitivity of Kir2 channels to cholesterol. ion channels; inward rectifiers; inwardly rectifying potassium channels
Our earlier studies have shown that Kir current in vascular endothelium that is mediated by the members of Kir2 family, Kir2.1 and Kir2.2 (7, 15), is strongly suppressed by the elevation of membrane cholesterol (39). Furthermore, we have shown that endothelial Kir currents are suppressed by atherogenic lipoproteins in vitro and by serum hypercholesterolemia in vivo, underscoring the physiological significance of the sensitivity of the channels to membrane cholesterol (6). The mechanism underlying cholesterol-induced suppression of Kir channels, however, is still poorly understood. We have shown that cholesterol enrichment strongly suppresses Kir currents mediated by all four members of Kir2 family (Kir2.1–2.4) (38). However, it has little effect on the single-channel properties and no effect on surface expression of the channels, as determined by visualizing the channels in the plasma membrane using an extracellular tag (38). We have suggested, therefore, that an increase in cellular cholesterol results in "silencing" of the Kir2 channels by changing their local lipid environment. More specifically, we have suggested that enriching the cells with cholesterol facilitates the targeting of Kir2 channels into cholesterol-rich membrane subdomains, commonly called membrane rafts, whereas removing membrane cholesterol results in dissociation of the channels from the rafts. It is important to note that although the exact nature of these domains is still controversial and is likely to vary between cell systems, they are generally defined as "small heterogeneous highly dynamic, sterol- and sphingomyelin-enriched domains that compartmentalize cellular processes" (34). Furthermore, membrane rafts and caveolae (a subpopulation of rafts characterized by flask-shaped membrane invaginations and the presence of the protein caveolin) were suggested to play an important role in the regulation of ion channels in the cardiovascular system (23, 29). In this study, we address this hypothesis by analyzing the distributions of Kir2.1 and Kir2.3 channels between cholesterol-rich and cholesterol-poor membrane fractions under different cholesterol conditions. We show that both Kir2.1 and Kir2.3 have clear double-peak distributions between the two types of membrane fractions and that enriching the cells with cholesterol using methyl--cyclodextrin (MCD)-cholesterol complex results in more channels being targeted to cholesterol-rich domains. Furthermore, we show that cholesterol enrichment results in an increase in the level of cholesterol exclusively in low-density cholesterol-rich fractions with no effect on the level of cholesterol in other membrane fractions. Thus this is the first study to demonstrate that an increase in "raft" cholesterol is sufficient to suppress Kir2 channels.
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METHODS |
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Chinese hamster ovary (CHO) K1 cell line was obtained from the laboratory of Dr. Zhe Lu and maintained at 37°C in a humidified 5% CO2 atmosphere in Ham's F-12 medium (BioWhittaker, San Diego, CA) supplemented with heat-inactivated 10% fetal bovine serum (Gemini BioProducts, Woodland, CA). Cells were fed or split every 2–3 days. Cells were transiently cotransfected with Kir2.x constructs and enhanced green fluorescent protein (cmv-pcDNA3.1-GFP-TOPO; Invitrogen, Carlsbad, CA) using Lipofectamine (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's instructions. The experiments were performed 24–48 h after the transfection.
Isolation of membrane fractions and immunoblotting
Cells were washed three times with ice-cold PBS without Ca2+ and Mg2+. Cells were scraped into buffer A [in mM: 150 NaCl, 20 HEPES, 5 EDTA, pH 7.4, 1x protease inhibitor cocktail (Roche, Indianapolis, IN), 1 µg/ml pepstatin], homogenized in a Dounce tissue grinder (40 strokes), and centrifuged for 10 min at 1,000 g. The pellet was resuspended, dounced, and recentrifuged for 10 min at 1,000 g. The supernatants were combined and centrifuged for 1 h at 200,000 g to obtain the "high-speed pellet" (SW40Ti rotor; Beckman Coulter, Fullerton, CA).
Preparation of Triton-soluble and Triton-insoluble fractions. The high-speed pellet was resuspended in 1 ml of buffer A, sonicated 3 x 10 s on ice, and supplemented with a small volume of concentrated solution of Triton X-100 to a final concentration of 1%. After 15 min of incubation on ice, the suspension was centrifuged for 1 h at 200,000 g in a refrigerated centrifuge. The pellet (Triton-insoluble fraction) was then resuspended in Laemmli buffer.
Preparation of membrane fractions using a nondetergent method.
Total membrane pellet was resuspended in 1 ml of 45% sucrose solution, sonicated, supplemented with 3 ml of 45% sucrose solution, and transferred to 12-ml centrifuge tubes, and then 4 ml of 35% and 4 ml of 5% sucrose solutions were layered sequentially, creating a three-step sucrose gradient (45%, 35%, 5%). Sucrose gradient was then centrifuged for 18 h at 100,000 g. After centrifugation, 11 fractions were collected (1.5 ml of fraction 1, 1 ml of fractions 2–10, and 1.5 ml of fraction 11). Protein was precipitated by TCA and measured using the BCA protein assay kit (Bio-Rad, Hercules, CA). The samples were then resolved on 12% SDS-PAGE under reducing conditions, followed by transfer to polyvinylidene difluoride membranes. The membranes were probed with either affinity-purified rabbit anti-rat Kir2.x antibodies (a generous gift of Dr. Carol Vandenberg), rabbit polyclonal anti-caveolin-1 antibodies, or mouse anti-flotilin-1 antibodies (BD Pharmingen, San Diego, CA). Bound primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase (HRP; Jackson Laboratories, West Grove, PA). Finally, immunoreactivity was visualized with ECL Plus reagent (Amersham, Piscataway, NJ). Detected bands were analyzed densitometrically using the NIH ImageJ image processing program (http://rsb.info.nih.gov/ij/docs/index.html). Cholesterol levels in different fractions were measured using the Amplex red kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Briefly, cells were washed with PBS (without Ca2+/Mg2+), lysed on ice for 30 min with the reaction buffer of Amplex red cholesterol assay kit, and homogenized through a 20-gauge needle. Free cholesterol was measured using cholesterol oxidase (37°C for 30 min), which yields H2O2, which is then detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex red reagent), a highly sensitive and stable probe for H2O2. In the presence of HRP, Amplex red reagent reacts with H2O2 with a 1:1 stoichiometry to produce the highly fluorescent product resorufin. The reaction is performed in the dark. Resorufin fluorescence is detected by Fluoroscan Ascent FL (Thermo Electron, Helsinki, Finland) at 544-nm excitation and 590-nm emission wavelengths according to the manufacturer's conditions and published literature (1). In all of the experiments, the level of cellular cholesterol was modified both by exposing the cells to MCD not complexed with cholesterol and by exposing them to MCD saturated with cholesterol, a treatment that is typically used as a MCD control. The two types of the experiments were performed in parallel. Cholesterol measurements and Western blot analysis were performed on the same samples.
Immunostaining and Imaging
For immunostaining, cells were transfected with hemagglutinin (HA)-tagged Kir2.1 construct as described in Cells and Transfection. HA-Kir2.1-transfected cells were seeded on glass coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton, and then blocked [in PBS containing 1% bovine serum albumin (BSA) and 5% goat serum] for 1 hr, incubated with primary antibodies (1:100 in PBS containing 1% BSA and 5% goat serum) overnight, washed, incubated with Alexa586-conjugated secondary antibodies (1:200 dilution in PBS containing 1% BSA and 1% goat serum for 1 h), washed, mounted, and viewed using a Zeiss Axiovert 100TV microscope. The primary antibody used for Kir2.1-HA was rat monoclonal anti-HA Ab3F10 (Roche Diagnostics), and the secondary antibody used was Alexa568-conjugated goat anti-rat IgG (Molecular Probes).
Electrophysiology
Ionic currents were measured using whole cell configurations of the standard patch-clamp technique (11). Pipettes were pulled (SG10 glass, 1.20-mm inner diameter, 1.60 mm; part no. FPENNU1.20ID1.60OD; Richland Glass, Richland, NJ) to give a final resistance of 2–6 M
. These pipettes generated high-resistance seals without fire polishing. A saturated salt agar bridge was used as reference electrode. Currents were recorded using an EPC9 amplifier (HEKA Electronik, Lambrecht, Germany) and accompanying acquisition and analysis software (Pulse and PulseFit; HEKA Electronik). External recording solution contained (in mM) 150 NaCl, 6 KCl, 10 HEPES, 1.5 CaCl2, 1 MgCl2, and 1 EGTA, pH 7.3. Pipette solution contained (in mM) 145 KCl, 10 HEPES, 1 MgCl2, 4 ATP, and 1 EGTA, pH 7.3. Current was monitored by 500-ms linear voltage ramps from –100 to +40 mV at an interpulse interval of 5 s. The holding potential was –60 mV. Pipette and whole cell capacitances were automatically compensated. Whole cell capacitance and series resistance were compensated and monitored throughout the recording. Series resistance was compensated by 60–90%, with the compensation being limited by the stability of the patch. The experiments are conducted at room temperature.
Statistical Analysis
Statistical analyses of the data were performed using a standard two-sample Student's t-test, assuming unequal variances of the two data sets. Statistical significance was determined using a two-tailed distribution assumption and was set at 5% (P < 0.05).
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RESULTS |
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Multiple studies have shown that low-density membrane fractions are enriched in several protein markers of membrane rafts, such as caveolin and flotilin, but the distribution of membrane cholesterol between the two fractions is still controversial (10, 41, 44). Our first goal, therefore, was to determine the distribution of free (nonesterified) cholesterol in membrane fractions of CHO cells separated using either the detergent-free sucrose density gradient method (44) or the Triton insolubility method (45), the two major methods for membrane raft isolation. Figure 1 shows the distributions of membrane cholesterol (A), total protein (B), and cholesterol/protein ratio (C) in membrane fractions isolated using the detergent-free method. Figure 1 shows that cholesterol has a clear double-peak distribution with a major peak in low-density and a minor peak in high-density membrane fractions, whereas the amount of total membrane protein increases sharply and then steadily as the density increases. Overall, 79 ± 16% of total membrane cholesterol and 19 ± 3% of total membrane protein was found to reside in low-density (fractions 1–5) membrane fractions (Table 1). Thus the most striking difference between the low- and the high-density fractions is the cholesterol/protein ratio (Fig. 1C, Table 1). For the purpose of further discussion, therefore, we will use "high cholesterol/protein ratio" as a working definition of membrane rafts in these cells.
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Partitioning of Kir2.1 and Kir2.3 into Raft and Non-Raft Membrane Fractions
Our earlier study showed that both Kir2.1 and Kir2.3 channels partition virtually exclusively to Triton-insoluble membrane domains (38). However, since exposing cells to cold detergents by itself was shown to induce aggregation artifacts that may lead to false positives, in this study we extended these observations to analyze the distributions of Kir2.1 and Kir2.3 in membrane fractions isolated using the detergent-free method. We have shown presently that both Kir2.1 and Kir2.3 channels have double-peak distributions in the density gradient (the first peak appeared in fractions 3–6 and the second peak in fractions 7–10), demonstrating that the channels partitioned into both cholesterol-rich (raft) and cholesterol-poor (non-raft) membrane fractions (Fig. 2). The appearance of a second peak cannot be attributed just to a general increase in the amount of protein in high-density fractions, because the latter increases over the whole range of fractions tested. Furthermore, it is important to note that although only 52 ± 5% and 36 ± 3% of Kir2.1 and Kir2.3 channels, respectively, were found in the low-density membrane fractions (fractions 1–6), these fractions are enriched with the channels relative to the total protein (if the channels would not partition preferentially into the low-density membrane fractions, only 20% of channel protein would be expected in these fractions, the same as the amount of total protein). Interestingly, the pattern of the distribution of the two channels is not identical: Kir2.1 has a slightly larger peak in low-density membrane fractions, whereas Kir2.3 has a larger peak in high-density fractions, suggesting that Kir2.1 has stronger affinity to cholesterol-rich fractions than Kir2.3.
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To test further whether the appearance of the two proteins in the high-density fractions may be due to the contamination of these fractions with rafts bound to the cytoskeleton, we repeated the experiments using the lysis buffer with high ionic strength (1 M KCl), a procedure that is used to disrupt membrane-cytoskeleton interactions (4, 20). However, an increase in the ionic strength did not significantly reduce the amount of caveolin-1 partitioning into the high-density membrane fractions (not shown), suggesting that association between lipid rafts and cytoskeleton is not a major source for the partial partitioning of lipid raft markers to the high-density fractions. It is also interesting to note that although both Kir2.1 and Kir2.3, as well as caveolin-1 and flotilin-1, preferentially partitioned into fractions 4 and 5, only fraction 4 was strongly cholesterol enriched, whereas fraction 5 was not. The broader peaks of the Kir2/caveolin-1 distributions relative to the cholesterol peak suggest that additional factors are involved in regulating Kir2/caveolin-1 targeting into low-density membrane fractions.
Association of Kir2.1 channels with lipid rafts/caveolae was further investigated by testing the colocalization of Kir2.1 channels with caveolin-1 (Fig. 3). The distribution of the channels was visualized by transfecting the cells with Kir2.1 tagged with hemagglutinin (HA) epitope (YPYDVPDYA) on the extracellular domain. A partial overlap between HA-Kir2.1 and caveolin-1 is consistent with our fractionation analysis showing that the channels are distributed between caveolin-rich and caveolin-poor membrane fractions.
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CD-Induced Depletion of Raft and Non-Raft Cholesterol: Correlation With Kir2.1 Activity and Distribution Between Low- and High-Density Fractions
As expected, exposing cells to 5 mM MCD for 15–60 min resulted in a gradual decrease of cellular cholesterol level, with a significant decrease observed already after 15 min (Fig. 4A). It is important to point out, however, that cholesterol depletion was not limited to the raft membrane fractions but also was observed in the non-raft fractions (both fractions were depleted 
2-fold after 60 min of incubation). In low-density fractions (fraction 4), cholesterol decreased gradually after 15, 30, and 60 min, with no further decrease observed after 120 min (Fig. 4B). In high-density fractions (fraction 8), although the initial decrease in the level of cholesterol was significant, no further significant changes were observed after the longer MCD exposures (Fig. 4C). It is possible, however, that since the level of cholesterol in fraction 8 was relatively low, gradual changes might not be detectable.
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CD exposure and a further increase observed after 30 and 60 min of MCD exposure (Fig. 5, A and B). The currents and the level of cholesterol were measured in the same cell populations. It is also important to note that, consistent with our earlier study (39), exposing the cells to MCD did not have any effect on membrane capacitance (17 ± 0.9 pF and 18 ± 0.8 pF for control cells and cells exposed to 60 min of MCD, respectively). Reversal potential of the two populations also were very similar (–71.90 ± 0.69 mV and –71.98 ± 1.5 mV for control cells and cells exposed to 60 min of MCD, respectively), indicating that there was no significant contribution of nonspecific membrane conductance (leak). These reversal potentials are slightly more depolarized than the theoretical reversal potential of K+ ions under these recording conditions (–80 mV), which may arise because of small contributions of nonselective cation and/or Cl– conductance that are typically present in most mammalian cells. The currents were recorded immediately after the MCD treatment and within the time window of 3 h after the end of the treatment. Both the levels of cholesterol and the current amplitudes remained unchanged in this window. We also have shown that cholesterol depletion had a similar facilitatory effect on Kir2.3 current (Fig. 5, C and D). Kir2.1 and Kir2.3 currents are recorded under exactly the same experimental conditions and using the same voltage protocols (a linear voltage ramp from –100 to +40 mV). As we showed earlier (38), however, Kir2.3 currents are smaller than Kir2.1 currents, and the sensitivity of Kir2.3 channels to cholesterol depletion appears to be lower than that of Kir2.1 channels.
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CD-Induced Cholesterol Enrichment Occurs Only in Cholesterol-Rich Membrane Fractions: Correlation With Kir2.1 Activity and Targeting to Lipid Rafts
In contrast to cholesterol depletion, exposing the cells to MCD saturated with cholesterol for 15–60 min resulted in an increase in membrane cholesterol exclusively in the raft membrane fractions, whereas the level of cholesterol in the high-density fractions was not affected (Fig. 7). These observations provide the basis to discriminate between the effects of cholesterol residing in the two types of membrane domains. Indeed, the time course of cholesterol increase in rafts correlated closely with the time course of Kir2.1 suppression (Fig. 8, A and B). Similarly, cholesterol enrichment also resulted in the suppression of Kir2.3 currents (Fig. 8, C and D). The sensitivity of the Kir2.3 to cholesterol enrichment also appeared to be lower than that of Kir2.1 channels. Together, these observations suggest that an increase in cholesterol content of the raft domains is sufficient to induce suppression of Kir2.1 and Kir2.3 currents.
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20% in fractions 4 and 5) and a strong shift in the second peak of the distribution from fraction 8 to fraction 7. Although an increase in partitioning of the channels to cholesterol-rich fractions suggests that more channels are included in the raft fractions, the significance of the shift of the second peak is not clear, and further studies are needed to address this issue. Changes in the distribution of Kir2.3 seem to be more straightforward: the channels clearly shifted from the high-density to low-density membrane fractions, although Kir2.3 was less sensitive to changes in membrane cholesterol than Kir2.1 (Fig. 9, C and D). Together, our observations suggest that translocation of the channels between different membrane domains may be one of the important mechanisms regulating Kir 2 function.
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CD and MCD-cholesterol result in opposite effects on both Kir2.1/Kir2.3 activities and the distribution of channels between the different membrane domains, we can exclude the position that the observed effects are due to nonspecific effects of MCD. These observations are consistent with our earlier studies showing that altering MCD/cholesterol ratios, although maintaining a constant level of MCD, has a significant effect on the Kir2.1/Kir2.3 currents (38) and that suppression of endothelial Kir by cholesterol enrichment is reversed by removing the surplus of cellular cholesterol (6). |
DISCUSSION |
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CD results in redistribution of several types of ion channels, including Ca2+-sensitive K+ channels (51), voltage-gated K+ channels (24, 25, 30), and transient receptor potential (TRP) channels (3, 20), from membrane fractions defined as rafts to fractions defined as non-rafts. It has not been established, however, whether it is cholesterol depletion of the raft domains or a global depletion of cellular cholesterol that is responsible for these effects. In addition, although cholesterol enrichment has been shown to affect multiple types of ion channels (14, 21, 38, 40, 48), it has not been established how an increase in cellular cholesterol affects targeting of ion channels to the raft domains. The main findings of this study are 1) both Kir2.1 and Kir2.3 have double-peak distributions between low- and high-density membrane fractions, suggesting that the channels are targeted to both raft and non-raft domains; 2) although MCD depletes cholesterol from both raft and non-raft fractions, cholesterol enrichment results in cholesterol increase exclusively in the raft fractions; 3) the time courses of both depletion-induced Kir2.1 enhancement and enrichment-induced Kir2.1 suppression correlate with the changes in the level of raft cholesterol; and finally, 4) not only does cholesterol depletion shift the distribution of the channels from low- to high-density membrane fractions but also cholesterol enrichment has the opposite effect. Thus this study provides further support for the hypothesis that partitioning to lipid rafts plays an important role in the sensitivity of Kir2.1 channels to cholesterol. Furthermore, our observations provide the first evidence that cholesterol enrichment of the raft fractions is sufficient to suppress Kir2.1 current and to shift Kir2.1 distribution from non-raft to raft domains.
Membrane rafts are generally defined as cholesterol-rich membrane domains (5, 33, 34, 43), but there is no general consensus on the cholesterol levels in raft and non-raft fractions. Specifically, lipid rafts have been shown to contain 30% of total cholesterol in Madin-Darby canine kidney cells (2), 30% in Jurkat T cells (41), 30–40% in macrophages (9), 30–50% in THP-1 monocytes (10), and 4% in human fibroblasts (44). These discrepancies may reflect the differences in raft composition or in relative amount/size of rafts in different cell types. In part, however, the variability also may be attributed to the different protocols used in raft isolation. In this study, we have shown that low-density fractions of CHO cells isolated using a detergent-free protocol contained 80% of the membrane cholesterol and had a much higher cholesterol/protein ratio than the rest of the membrane. An amount of cholesterol in Triton-insoluble membrane fractions isolated from the same cells was significantly lower (25%). A comparison between the two methods in the same cells clearly shows that the amount of cholesterol recovered from the fractions defined as lipid rafts depended strongly on the method of the isolation. Indeed, similar observations were reported earlier by Gaus et al. (10), who showed that although lipid rafts isolated from macrophages using the detergent-free method or a low concentration of Triton (0.2%) contained 40–45% of cholesterol, rafts isolated using 1% Triton contained only 6% of total membrane cholesterol. The question is, which of the two approaches more accurately reflects the composition of rafts in living cells. In general, although detergent-free sonication also may introduce some artifacts, it is considered to be less prone to artifacts than Triton extraction, and the most recent definition of membrane rafts, therefore, does not include detergent resistance (34). Interestingly, our observation that 80% of cholesterol was found in the low-density membrane fractions is consistent with a recent study of Swamy et al. (46), who estimated the portion of the plasma membrane that exists in a liquid-ordered state in living cells by using spin-label electron spin resonance spectroscopy. Swamy et al. (46) found that most of the plasma membrane existed in this phase and that it contained most of the membrane cholesterol. The similarity between the two observations suggests that cholesterol-rich membrane fractions isolated using a detergent-free method indeed reflect the partitioning of cholesterol into different membrane domains in cellular membranes.
We also have shown that, as expected, cholesterol-rich membrane fractions were enriched with caveolin and flotilin, two proteins often associated with membrane rafts, but neither of these proteins is found exclusively in cholesterol-rich fractions. This is not surprising, because no one protein has ever been shown to be a perfect marker and could be expected to have an absolute specificity for raft subdomains [for example, caveolin-1 has been shown to partition to both high- and low-density membrane fractions (22, 49)]. Our study reveals that Kir 2 channels show a clear biphasic distribution between the raft and the non-raft membrane fractions. This distribution of the channels provides a possible mechanistic basis for our earlier hypothesis that the channels may exist in two different modes: the "silent mode" and the "active mode" (38, 39). The following observations led us to hypothesize that the Kir2 channels may exist on the membrane in two modes. First, neither cholesterol enrichment nor cholesterol depletion affects the expression of Kir2.1 channels (38). Second, these treatments have no effect on membrane capacitance, indicating that changes in membrane cholesterol do not induce major changes in the membrane area. This is in contrast to the regulation of Kir2.1 by tyrosine phosphorylation that is accompanied by a significant decrease in membrane capacitance (47). Finally, we have also demonstrated directly that cholesterol treatments do not affect the surface expression of Kir2.1 channels as assessed by visualization of HA-Kir2.1 constructs in nonpermeabilized cells (38). We suggested, therefore, that it is the lateral separation of membrane lipids that is responsible for the regulation of Kir2 channels by cholesterol. This study provides the first evidence to support this hypothesis. Specifically, we suggest that when Kir2 channels partition into the raft domains, they exist in a silent mode, whereas when they partition into non-raft domains, they exist in an active mode. It is also important to note that biphasic distribution between low- and high-density membrane fractions is not unique for Kir2. Specifically, several types of ion channels, such as cardiac Na+ channels (54) and voltage-gated K+ channels (Kv2.1 and Kv1.5) (24, 25), have been shown to distribute between the low- and high-density membrane fractions, and the distributions of the channels appear to be biphasic. Most interestingly, Shlyonsky et al. (42) showed that functional epithelial Na+ channels (ENaC) can be found only in Triton-insoluble fractions, whereas the channels partitioning to Triton-soluble fractions were not functional. In terms of the specific effect, this observation is the opposite of what we propose for the Kir2 channels, but this is not surprising because although cholesterol depletion facilitates Kir channels (38, 39), it was shown to suppress ENaC channels (52). The observation that functional ENaC are found only in Triton-insoluble fractions is important as proof of the principle that association with a specific membrane lipid domain may switch a channel from a functional to a nonfunctional mode.
The ability of MCD to extract cholesterol from both raft and non-raft membrane fractions demonstrated presently, consistent with the earlier studies (9, 10, 19, 31, 41), indicates that sensitivity to cholesterol depletion cannot be taken as decisive evidence for the involvement of lipid rafts in a specific process. However, we have shown here that MCD-cholesterol increases cholesterol specifically in the raft fractions, having no apparent effect on the cholesterol level in non-raft fractions. This finding provides a strong clue to discriminate between the roles of raft and non-raft cholesterol in the regulation of Kir2 channels, showing that an increase in the level of the raft cholesterol is sufficient to suppress the current. Furthermore, this is the first study to demonstrate that cholesterol enrichment also causes redistribution of the channels, with an increased amount of the channels partitioning into the raft fractions. Together, these observations suggest that an increase in the cholesterol level of the raft domains and translocation of the channels to these domains play critical roles in the regulation of Kir2 channels. Clearly, these observations also lead to a series of new questions: Does cholesterol enrichment result in an increase in the number, size, or cholesterol density of the rafts? Are channels actively translocated from non-rafts to rafts, or are they just "trapped" in a growing or newly forming raft? How does being in a raft affect the functional properties of the channels? How does partitioning of the channels into rafts affect their association with chaperone proteins, such as PSD-95, SAP97, or AKAP79? Further studies are needed to address these questions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. Tikku and Y. Epshtein contributed equally to this work. 
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REFERENCES |
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|---|
2. Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68: 533–544, 1992.[CrossRef][Web of Science][Medline]
3. Brownlow SL, Harper AGS, Harper MT, Sage SO. A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets. Cell Calcium 35: 107–113, 2004.[CrossRef][Web of Science][Medline]
4. Campbell KP, Kahl SD. Association of dystrophin and an integral membrane glycoprotein. Nature 338: 259–262, 1989.[CrossRef][Medline]
5. Edidin M. The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 32: 257–283, 2003.[CrossRef][Web of Science][Medline]
6. Fang Y, Mohler ER, III, Hsieh E, Osman H, Hashemi SM, Davies PF, Rothblat GH, Wilensky RL, Levitan I. Hypercholesterolemia suppresses inwardly rectifying K+ channels in aortic endothelium in vitro and in vivo. Circ Res 98: 1064–1071, 2006.
7. Fang Y, Schram G, Romanenko VG, Shi C, Conti L, Vandenberg CA, Davies PF, Nattel S, Levitan I. Functional expression of Kir2.x in human aortic endothelial cells: the dominant role of Kir2.2. Am J Physiol Cell Physiol 289: C1134–C1144, 2005.
8. Forsyth SE, Hoger A, Hoger JH. Molecular cloning and expression of a bovine endothelial inward rectifier potassium channel. FEBS Lett: 277–282, 1997.
9. Gaus K, Kritharides L, Schmitz G, Boettcher A, Drobnik W, Langmann T, Quinn CM, Death A, Dean RT, Jessup W. Apolipoprotein A-1 interaction with plasma membrane lipid rafts controls cholesterol export from macrophages. FASEB J: 574–576, 2004.
10. Gaus K, Rodriguez M, Ruberu KR, Gelissen I, Sloane TM, Kritharides L, Jessup W. Domain-specific lipid distribution in macrophage plasma membranes. J Lipid Res 46: 1526–1538, 2005.
11. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[CrossRef][Web of Science][Medline]
12. Heerklotz H. Triton promotes domain formation in lipid raft mixtures. Biophys J 83: 2693–2701, 2002.[Web of Science][Medline]
13. Ishii K, Yamagishi T, Taira N. Cloning and functional expression of a cardiac inward rectifier K+ channel. FEBS Lett 338: 107–111, 1994.[CrossRef][Web of Science][Medline]
14. Jennings LJ, Xu QW, Firth TA, Nelson MT, Mawe GM. Cholesterol inhibits spontaneous action potentials and calcium currents in guinea pig gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 277: G1017–G1026, 1999.
15. Kamouchi M, Van Den Bremt K, Eggermont J, Droogmans G, Nilius B. Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells. J Physiol 504: 545–556, 1997.
16. Karkanis T, Li S, Pickering JG, Sims SM. Plasticity of KIR channels in human smooth muscle cells from internal thoracic artery. Am J Physiol Heart Circ Physiol 284: H2325–H2334, 2003.
17. Kubo Y, Adelman JP, Clapham DE, Jan LY, Karschin A, Kurachi Y, Lazdunski M, Nichols CG, Seino S, Vandenberg CA. International Union of Pharmacology. LIV. Nomenclature and Molecular Relationships of Inwardly Rectifying Potassium Channels. Pharmacol Rev 57: 509–526, 2005.
18. Kubo Y, Baldwin TJ, Jan YN, Jan LY. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127–132, 1993.[CrossRef][Medline]
19. Larbi A, Douziech N, Khalil A, Dupuis G, Gherairi S, Guerard KP, Fulop T Jr. Effects of methyl--cyclodextrin on T lymphocytes lipid rafts with aging. Exp Gerontol 39: 551–558, 2004.[CrossRef][Web of Science][Medline]
20. Lockwich TP, Liu X, Singh BB, Jadlowiec J, Weiland S, Ambudkar IS. Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains. J Biol Chem 275: 11934–11942, 2000.
21. Lundbaek JA, Birn P, Hansen AJ, Sogaard R, Nielsen C, Girshman J, Bruno MJ, Tape SE, Egebjerg J, Greathouse DV, Mattice GL, Koeppe RE II, Andersen OS. Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of micelle-forming amphiphiles and cholesterol. J Gen Physiol 123: 599–621, 2004.
22. Macdonald JL, Pike LJ. A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res 46: 1061–1067, 2005.
23. Maguy A, Hebert TE, Nattel S. Involvement of lipid rafts and caveolae in cardiac ion channel function. Cardiovasc Res 69: 798–807, 2006.
24. Martens JR, Navarro-Polanco R, Coppock EA, Nishiyama A, Parshley L, Grobaski TD, Tamkun MM. Differential targeting of Shaker-like potassium channels to lipid rafts. J Biol Chem 275: 7443–7446, 2000.
25. Martens JR, Sakamoto N, Sullivan SA, Grobaski TD, Tamkun MM. Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae. J Biol Chem 276: 8409–8414, 2001.
26. Melnyk P, Zhang L, Shrier A, Nattel S. Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle. Am J Physiol Heart Circ Physiol 283: H1123–H1133, 2002.
27. Munro S. Lipid rafts: elusive or illusive? Cell 115: 377–388, 2003.[CrossRef][Web of Science][Medline]
28. Nichols C, Lopatin A. Inward rectifier potassium channels. Annu Rev Physiol 59: 171–191, 1997.[CrossRef][Web of Science][Medline]
29. O'Connell KMS, Martens JR, Tamkun MM. Localization of ion channels to lipid raft domains within the cardiovascular system. Trends Cardiovasc Med 14: 37–42, 2004.[CrossRef][Web of Science][Medline]
30. O'Connell KMS, Tamkun MM. Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains. J Cell Sci 118: 2155–2166, 2005.
31. Ottico E, Prinetti A, Prioni S, Giannotta C, Basso L, Chigorno V, Sonnino S. Dynamics of membrane lipid domains in neuronal cells differentiated in culture. J Lipid Res 44: 2142–2151, 2003.
32. Perier F, Radeke CM, Vandenberg CA. Primary structure and characterization of a small-conductance inwardly rectifying potassium channel from human hippocampus. Proc Natl Acad Sci USA 91: 6240–6244, 1994.
33. Pike LJ. Lipid rafts: bringing order to chaos. J Lipid Res 44: 655–667, 2003.
34. Pike LJ. Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J Lipid Res 47: 1597–1598, 2006.
35. Plaster NM, Tawil R, Tristani-Firouzi M, Canun S, Bendahhou S, Tsunoda A, Donaldson MR, Iannaccone ST, Brunt E, Barohn R, Clark J, Deymeer F, George ALJ, Fish FA, Hahn A, Nitu A, Ozdemir C, Serdaroglu P, Subramony SH, Wolfe G, Fu YH, Ptacek LJ. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell 105: 511–519, 2001.[CrossRef][Web of Science][Medline]
36. Raab-Graham KF, Radeke CM, Vandenberg CA. Molecular cloning and expression of a human heart inward rectifier potassium channel. Neuroreport 5: 2501–2505, 1994.[Web of Science][Medline]
37. Reimann F, Ashcroft F. Inwardly rectifying potassium channels. Curr Opin Cell Biol 11: 503–508, 1999.[CrossRef][Web of Science][Medline]
38. Romanenko VG, Fang Y, Byfield F, Travis AJ, Vandenberg CA, Rothblat GH, Levitan I. Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels. Biophys J 87: 3850–3861, 2004.[CrossRef][Web of Science][Medline]
39. Romanenko VG, Rothblat GH, Levitan I. Modulation of endothelial inward rectifier K+ current by optical isomers of cholesterol. Biophys J 83: 3211–3222, 2002.[Web of Science][Medline]
40. Romanenko VG, Rothblat GH, Levitan I. Sensitivity of volume-regulated anion current to cholesterol structural analogues. J Gen Physiol 123: 77–88, 2004.[CrossRef][Web of Science][Medline]
41. Rouquette-Jazdanian AK, Pelassy C, Breittmayer JP, Aussel C. Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor signaling. Cell Signal 18: 105–122, 2006.[CrossRef][Web of Science][Medline]
42. Shlyonsky VG, Mies F, Sariban-Sohraby S. Epithelial sodium channel activity in detergent-resistant membrane microdomains. Am J Physiol Renal Physiol 284: F182–F188, 2003.
43. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1: 31–39, 2000.[CrossRef][Web of Science][Medline]
44. Smart EJ, Ying Y, Mineo C, Anderson RGW. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci USA 92: 10104–10108, 1995.
45. Song KS, Tang Z, Li S, Lisanti MP. Mutational analysis of the properties of caveolin-1. A novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions. J Biol Chem 272: 4398–4403, 1997.
46. Swamy MJ, Ciani L, Ge M, Smith AK, Holowka D, Baird B, Freed JH. Coexisting domains in the plasma membranes of live cells characterized by spin-label ESR spectroscopy. Biophys J 90: 4452–4465, 2006.[CrossRef][Web of Science][Medline]
47. Tong Y, Brandt GS, Li M, Shapovalov G, Slimko E, Karschin A, Dougherty DA, Lester HA. Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel. J Gen Physiol 117: 103–118, 2001.
48. Toselli M, Biella G, Taglietti V, Cazzaniga E, Parenti M. Caveolin-1 expression and membrane cholesterol content modulate N-type calcium channel activity in NG108–15 cells. Biophys J 89: 2443–2457, 2005.[CrossRef][Web of Science][Medline]
49. Travis AJ, Merdiushev T, Vargas LA, Jones BH, Purdon MA, Nipper RW, Galatioto J, Moss SB, Hunnicutt GR, Kopf GS. Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and guinea pig spermatozoa. Dev Biol 240: 599–610, 2001.[CrossRef][Web of Science][Medline]
50. Tristani-Firouzi M, Jensen JL, Donaldson MR, Sansone V, Meola G, Hahn A, Bendahhou S, Kwiecinski H, Fidzianska A, Plaster N, Fu YH, Ptacek LJ, Tawil R. Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome). J Clin Invest 110: 381–388, 2002.[CrossRef][Web of Science][Medline]
51. Wang XL, Ye D, Peterson TE, Cao S, Shah VH, Katusic ZS, Sieck GC, Lee HC. Caveolae targeting and regulation of large conductance Ca2+-activated K+ channels in vascular endothelial cells. J Biol Chem 280: 11656–11664, 2005.
52. West A, Blazer-Yost B. Modulation of basal and peptide hormone-stimulated Na transport by membrane cholesterol content in the A6 epithelial cell line. Cell Physiol Biochem 16: 263–270, 2005.[CrossRef][Web of Science][Medline]
53. Wible BA, De Biasi M, Majumder K, Taglialatela M, Brown AM. Cloning and functional expression of an inwardly rectifying K+ channel from human atrium. Circ Res 76: 343–350, 1995.
54. Yarbrough TL, Lu T, Lee HC, Shibata EF. Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude. Circ Res 90: 443–449, 2002.
55. Zaritsky JJ, Eckman DM, Wellman GC, Nelson MT, Schwarz TL. Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential role of the inwardly rectifying K+ current in K+-mediated vasodilation. Circ Res 87: 160–166, 2000.
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