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Cellular Biology |
From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.
Correspondence to C. Dart, Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK. E-mail cd12{at}le.ac.uk
| Abstract |
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Key Words: KATP channel adenylyl cyclase caveolae compartmentation protein kinase A
| Introduction |
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| Materials and Methods |
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300 g) (Charles River Laboratories, Inc, Margate, UK) killed by stunning and rapid cervical dislocation. The care and euthanasia of animals conformed to the requirements of the UK Animals (Scientific Procedures) Act 1986.
Antibodies, Polyacrylamide Gel Electrophoresis, and Immunoblotting
The following primary antibodies were used: anti-adaptin, anti-caveolin 1, anti-caveolin 2, anti-caveolin 3, and anti-protein kinase RIIß (BD Transduction Laboratories); anti-adenylyl cyclase 3, anti-adenylyl cyclase 5/6, anti-Kir6.1 (R-14; sc-11224), and associated blocking peptide (sc-11224P) and anti-protein kinase RII
(Santa Cruz Biotechnology); anti-smooth muscle
-actin (Sigma-Aldrich). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Jackson Immunochemical Laboratories. HRP-conjugated anti-goat secondary was from Sigma-Aldrich. Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide-Tris gels and transferred electrophoretically onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech). Immunoblotting was performed as described previously.17
Fractionation of Caveolin-Enriched Membrane and Assay for Cholesterol
Rat aortic smooth muscle cells were isolated enzymatically using methodology described previously.17 Cells were cultured in high-glucose Dulbecco Modified Eagle Media (D-MEM) supplemented with 15% fetal calf serum, penicillin (50 U/mL), streptomycin (50 µg/mL), and fungizone (2.5 µg/mL). All media and reagents were from Life Technologies, Inc. Cells were maintained at 37°C in a humidified atmosphere (10% CO2) and harvested between passages 12 to 15. Smooth muscle cell identity was confirmed by immunocytochemical staining with a smooth musclespecific
-actin antibody.
Buoyant caveolae-enriched membrane fractions were isolated under detergent-free conditions from rat aortic smooth muscle cell homogenates by ultracentrifugation on discontinuous sucrose gradients using a method adapted from18 as described by.11 Briefly, four large (175 cm2) flasks of confluent aortic smooth muscle cells were washed twice in ice-cold PBS and scraped into 2 mL of sodium carbonate (500 mmol/L; pH 11). Cells were disrupted by ten strokes in a hand-operated homogenizer and sonication (3x20-second bursts). The homogenate was made up to 45% sucrose by addition of an equal volume of 90% sucrose in MBS (25 mmol/L MES and 150 mmol/L NaCl, pH 6.5) and loaded in a polyethylene-terephthalate thin-walled ultracentrifuge tube. The sample was overlaid with 4 mL of 35% sucrose prepared in MBS with 250 mmol/L sodium carbonate then 4 mL of 5% sucrose again prepared in MBS with 250 mmol/L sodium carbonate. Buoyant and nonbuoyant membrane fractions were isolated by centrifugation at 39 000 rpm for 18 hours at 4°C in a Sorvall ultracentrifuge equipped with TH-641 rotor. To evaluate the fractionation procedure, ten 1-mL fractions were collected from the top to the bottom of the gradient. Equal volumes of the fractions were separated by SDS-PAGE and analyzed by immunoblotting. Cholesterol levels in each of the fractions were determined by analysis with the Amplex Red cholesterol assay kit (Molecular Probes) according to the manufacturers protocol. In subsequent experiments, the top 2 mL of the sucrose gradient was discarded, and a light-scattering band representing the cholesterol-enriched, caveolin-containing membranes was collected from the 5% to 35% sucrose interface. The bottom 4 mL of 45% sucrose were collected as nonbuoyant, noncaveolar membranes.
Cholesterol Depletion and Filipin Staining
Cholesterol was depleted from smooth muscle membranes by incubation of cell suspensions or strips of rat thoracic aorta with 2% methyl-ß-cyclodextrin (Sigma-Aldrich) for 1 to 2 hours.19,20 ß-Cyclodextrins are noninvasive, water-soluble cyclic heptasaccharides containing a hydrophobic core capable of solubilizing cholesterol and extracting it from membranes. After treatment with methyl-ß-cyclodextrin, the degree of cholesterol depletion was assessed by staining a sample of smooth muscle cells with the cholesterol-binding agent filipin according to methodology described previously.21
Coimmunoprecipitation
Control or cholesterol-depleted rat thoracic aorta was homogenized in ice-cold lysis buffer (20 mmol/L Tris-HCl; 250 mmol/L NaCl; 3 mmol/L EDTA; 3 mmol/L EGTA; pH 7.6) containing 1% Triton X100 and protease inhibitors (1:100 dilution, Sigma Protease Inhibitor Cocktail containing AEBSF, aprotinin, bestatin, leupeptin, pepstatin A). Insoluble material was pelleted by centrifugation and the lysate precleared by incubation with protein-A agarose (Santa Cruz Biotechnology; 1 hour, 4°C). 500 µL of cleared lysate was incubated with 5 µg of anti-Kir6.1, 5 µg of anti-adaptin, 5 µg of anti-adenylyl cyclase 5/6, or 5 µg of appropriate nonimmune control serum overnight at 4°C. Antigen-antibody complexes were captured with protein-A agarose (4°C, 2 hours). Agarose beads were washed extensively before removal of bound proteins by boiling in SDS sample buffer. Samples were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and analyzed by immunoblotting.
Electrophysiology
Single smooth muscle cells were isolated enzymatically from small branches of the rat mesenteric artery, as described previously.22 Cells were stored at 4°C, and used on the day of preparation. Whole-cell K+ currents were recorded from single smooth muscle cells using an Axopatch 200B amplifier (Axon Instruments). Recorded membrane currents were filtered at 5 kHz, digitized using a Digidata 1320A interface (Axon Instruments), and analyzed using pCLAMP software. Patch pipettes were pulled from borosilicate glass (outer diameter 1.5 mm, inner diameter 0.86 mm; Clarke Electromedical) and fire polished to give a final resistance of
5 M
when filled. The pipette-filling solution contained (in mmol/L) 110 KCl, 30 KOH, 10 HEPES, 10 EGTA, 1 MgCl2, 1 CaCl2, 1 Na2ATP, 0.1 ADP, 0.5 GTP; adjusted to pH 7.2. The 6 mmol/L K+ extracellular solution contained (in mmol/L) 134 NaCl, 6 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 10 glucose; adjusted to pH 7.4. To separate KATP currents, we recorded at 60 mV to minimize activation of voltage-dependent K+ channels, and raised extracellular [K+] to 140 mmol/L to give a substantial inward driving force for K+. In addition, 140 mmol/L K+ extracellular solution contained (in mmol/L) 140 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 10 glucose; pH 7.4. Pinacidil, glibenclamide, GDPßS, propranolol, 8-SPT, and CGRP 8-37 were from Sigma-Aldrich. 2',5'-Dideoxyadenosine and Rp-cAMPS (the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt) were from Calbiochem. Pinacidil and glibenclamide were dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was less than 0.2%. Experiments were conducted in a temperature-controlled bath at 25°C. Results are expressed as mean±SEM. Intergroup differences were analyzed using ANOVA followed by the Student-Newman-Keuls test for multiple comparisons or Student t test for simple comparisons.
| Results |
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The unusual lipid composition of caveolae (enriched with cholesterol and sphingolipids) gives them distinct properties as compared with the bulk of the plasma membranenamely a highly reduced "buoyant" density. We isolated buoyant membrane fractions under detergent-free conditions from rat aortic smooth muscle cell homogenates by ultracentrifugation on discontinuous sucrose gradients.11,18 To assess the purity of caveolar and noncaveolar fractions, we used Western blot analysis to determine the distribution of specific marker proteins, and an assay to determine the level of cholesterol within each fraction. As a marker for the buoyant caveolar fraction, we used the protein caveolin. Caveolins bind cholesterol and represent the major structural components of caveolae, coating the whole of the cytoplasmic surface of these organelles. They comprise a family of three distinct 21- to 24-kDa isoforms; caveolin-1 and -2 are widely expressed and most likely form a heterooligomeric complex, whereas caveolin-3 is a muscle-specific isoform.14,16 Figure 2A shows Western blot analysis of ten 1-mL fractions collected from top to bottom of the sucrose density gradient. Caveolin-1, was found predominantly in fraction 4 of the gradient, with small residual amounts either side of this layer in fractions 3 and 5. In contrast, ß-adaptin, a marker protein for clathrin-coated pits, was largely excluded from the caveolar fractions and localized to the bottom layers of the gradient. Measurement of cholesterol levels within each fraction showed cholesterol to be enriched in fractions 3 and 4 (Figure 2B), consistent with the idea that fraction 4 represents the caveolae-containing layer of the density gradient. Having established that cholesterol-enriched caveolar membranes can be separated from the noncaveolar membranes by this fractionation procedure, we set out to determine the distribution of various proteins between caveolar and noncaveolar fractions. Consistent with previous studies, we found caveolin-1, -2, and -3 expressed in rat aortic smooth muscle cell lysates and also in the caveolar membrane fractions11,26 (Figure 3A). In agreement with the work of Ostrom et al,11 adenylyl cyclase isoforms 3 and 5/6 also localized primarily to the caveolar membrane fractions (Figure 3B). Other proteins such as the regulatory subunit of PKA were more evenly distributed between the caveolar and noncaveolar membrane regions.
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KATP channels form as 4+4 octomers of Kir6 pore-forming subunits and sulfonylurea receptor (SUR) proteins.27 The dominant channel in most vascular smooth muscle most likely comprises Kir6.1/SUR2B subunits.28 To determine the distribution of KATP channels between the isolated membrane fractions, we used an antibody directed against the Kir6.1 subunit. We detected the presence of Kir6.1 predominantly in the buoyant, caveolin-enriched fraction of the membrane (Figure 4A, top). Importantly, preincubation of the Kir6.1 antibody with a blocking peptide representing the unique antigenic carboxyl-terminal sequence of Kir6.1 specifically reduced the ability of the antibody to detect this protein band (Figure 4A, bottom).
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To verify our membrane fractionation results by an independent method, we undertook coimmunoprecipitation experiments. Aside from being the major structural component of caveolae, caveolins also act as scaffold proteins that interact with many caveolae-localized signaling molecules.29 We therefore hypothesized that if Kir6.1 was indeed resident within caveolae it might be possible to coimmunoprecipitate Kir6.1 with caveolin. Figure 4B shows that antibodies specific to Kir6.1 were able to isolate all three isoforms of caveolin from rat aortic homogenates, suggesting that Kir6.1 and caveolin exist together in a complex in intact cells. This complex may arise through direct protein-protein interaction between Kir6.1 and caveolin or by virtue of the fact that these proteins localize to a membrane microdomain that remains intact throughout the immunoprecipitation process. If this latter case is true, the interaction between Kir6.1 and caveolin should be abolished by the disruption of the caveolar membrane compartment. The integrity of lipid microdomains such as caveolae is heavily dependent on cholesterol. Cholesterol depletion profoundly alters caveolar structure leading to a flattening, or total disappearance, of these membrane invaginations30,31 and dissociation of sequestered proteins from these domains.15,32 We pretreated strips of rat aorta with the cholesterol-depleting agent, methyl-ß-cyclodextrin19,20 for 1 to 2 hours before performing the coimmunoprecipition assay and found that cholesterol depletion had no effect on the ability of the antibody against Kir6.1 to isolate caveolin (Figure 4B, bottom), suggesting that association between Kir6.1 and caveolin may be via specific protein-protein interactions. This ability to isolate caveolin from smooth muscle homogenates was specific to the Kir6.1 antibody because antibodies directed against the clathrin-coated pit protein ß-adaptin failed to coprecipitate any caveolin isoforms (Figure 4C).
Biochemical data therefore suggest compartmentation of both KATP channels and adenylyl cyclase in smooth muscle caveolae. To determine whether this colocalization has functional significance, we investigated the effect of disrupting caveolae on the PKA-dependent tonic activation of KATP channels. We pretreated suspensions of isolated smooth muscle cells with methyl-ß-cyclodextrin for 1 to 2 hours before recording whole-cell currents induced by the KATP channel opener, pinacidil (10 µmol/L). In these experiments, the application of 100 µmol/L of the PKA inhibitor Rp-cAMPS to control cells caused a 41.4±5.8% (n=7) inhibition of the whole cell pinacidil-induced current (Figure 5A). This PKA-sensitive component of the whole cell KATP current was significantly reduced in cholesterol-depleted cells, where application of 100µmol/L Rp-cAMPS inhibited only 11.7±5.0% of the evoked current (Figure 5B, n=7, P<0.01). This suggests that in cholesterol-depleted cells, KATP channels are no longer subject to a high level of tonic PKA-dependent activation. Depletion of cholesterol with methyl-ß-cyclodextrin has previously been shown to have no effect on forskolin-stimulated adenylyl cyclase activity.33 Similarly, the ability of the KATP channel to respond to the opener pinacidil was essentially the same in control and depleted cells, suggesting that the channel itself was largely unaffected by reduced cholesterol levels. Although we did not observe a significant reduction in current density in cholesterol-depleted cells as might be expected if a sustained background drive on the channel has been reduced (Figure 5D), such a change would be hard to detect because of the high cell-to-cell variability of whole cell current amplitudes induced by pinacidil. Near complete inhibition of tonic PKA drive by the potent blocker of adenylyl cyclase, 2',5'-dideoxyadenosine, did produce a significant reduction in evoked KATP current (Figure 1F). We suggest that to be susceptible to PKA-dependent regulation, KATP channels need to be localized in the same lipid compartment as adenylyl cyclase, and that disruption of the caveolae by cholesterol depletion reduces the PKA-sensitive component of the KATP current by functionally uncoupling adenylyl cyclase and the channel.
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| Discussion |
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In smooth muscle cells, adenylyl cyclase resides predominantly in caveolae, small vesicular invaginations of the plasma membrane that associate with the cholesterol-binding protein caveolin.11,12 These specialized lipid microdomains have previously been implicated in generating subcellular signaling compartments by aggregating interacting signaling molecules.1316 We find that, like adenylyl cyclase, KATP channels localize to the same cholesterol-enriched smooth muscle membrane fractions as the caveolae marker caveolin. Additionally, an antibody specific to the pore-forming subunit of KATP channels coimmunoprecipitates caveolin from arterial homogenates, suggesting that KATP channels and caveolin exist together in a complex within cells. This association could be via specific protein-protein interactions between Kir6.1 and caveolin, a known scaffold protein that interacts with many caveolae-localized signaling molecules,29 or by virtue of the fact that both proteins reside in a lipid compartment that remains intact throughout the immunoprecipitation process. The voltage-gated potassium channel, Kv1.5, for example, targets to caveolae and coimmunoprecipitates with caveolin-139 through Kv1.5-mediated immunoisolation of a protein-lipid complex harboring caveolin. Our results show that exposure to the cholesterol-depleting agent methyl-ß-cyclodextrin has no effect on the ability of antibodies directed against Kir6.1 to coprecipitate caveolin, suggesting a tight association between the proteins is maintained following disruption of their native lipid environment. This points to a direct protein-protein interaction between Kir6.1 and caveolin, an idea that is supported by the finding that antibodies against another caveolae-resident protein, adenylyl cyclase, failed to coprecipitate caveolin (data not shown).
The integrity of the membrane compartments generated by caveolae seems important in maintaining normal KATP channel regulation. This is demonstrated by the finding that disruption of caveolae by cholesterol depletion significantly reduces the PKA-sensitive component of KATP channel current, indicating that tonic PKA-dependent channel activation may rely on the spatial confinement of adenylyl cyclase and KATP channels. This would be consistent with previous findings in cardiac muscle that show that the distance cAMP can diffuse from its site of production (ie, adenylyl cyclase) is severely restricted by the action of phosphodiesterases.23 The major target for cAMP, PKA, is generally anchored in close proximity to its intended substrate through the action of A-kinase anchoring proteins or AKAPs,4042 and we have previously demonstrated the involvement of an unidentified AKAP in both steady state and receptor-driven activation of KATP channels.24 Recent evidence suggests that PKA phosphorylates sites on the channel subunits themselves to cause activation.43 It seems likely that efficient PKA-dependent channel regulation will depend on KATP channels and their associated kinases being in the vicinity of cAMP production and therefore adenylyl cyclase.
The compartmentation of adenylyl cyclase and KATP channels presumably represents just one element of the signaling machinery surrounding these channels. Opening of arterial KATP channels causes membrane hyperpolarization, a decrease in Ca2+ influx through voltage-dependent L-type Ca2+ channels and vasorelaxation. Thus, vasodilators open KATP channels, whereas vasoconstrictors close them, and such modulation probably represents a major component of their physiological regulation. Many vasodilators elevate KATP channel activity by acting at Gs-coupled receptors to stimulate adenylyl cyclase and activate PKA.3 Additionally, vasoconstrictors such as angiotensin II that activate Gq/11-coupled receptors to inhibit KATP channels via stimulation of protein kinase C may also suppress KATP channel activity by Gi/o-mediated inhibition of adenylyl cyclase and a reduction in steady-state PKA-dependent channel phosphorylation.22 Adenylyl cyclase is therefore a pivotal enzyme in both tonic and receptor-driven regulation of KATP channels, and its localization to caveolae, which in other tissues have been shown to be highly enriched with both G proteincoupled receptors and G proteins,14,16 makes it likely that larger, more elaborate signaling complexes exist within these domains. Caveolae have already been implicated as integration sites for smooth muscle Ca2+ signaling because of their ability to aggregate proteins involved in Ca2+ regulation and excitation-contraction coupling.44,45 The subcellular distribution of the major receptors that couple to arterial KATP channels is largely unknown, but activated receptors for angiotensin II have been shown to congregate in caveolae of rat aortic smooth muscle.26,31 Finally, it is worth noting that phosphatidylinositol-4,5-bisphosphate (PIP2), an important determinant of KATP channel activity and otherwise minor phospholipid component of the bilayer, is greatly concentrated in caveolae.46 Thus, by sequestering both proteins and lipids necessary for normal channel function, caveolae may be used by smooth muscle as a means of compartmentalizing and organizing the complex regulatory pathways that modulate KATP channel activity and so vascular tone.
| Acknowledgments |
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| Footnotes |
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| References |
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