Published online before print July 28, 2008, doi: 10.1161/HYPERTENSIONAHA.108.110817
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(Hypertension. 2008;52:499.)
© 2008 American Heart Association, Inc.
Original Articles |
Protein Kinase C-
Induces Caveolin-Dependent Internalization of Vascular Adenosine 5'-Triphosphate–Sensitive K+ Channels
From the Division of Pharmacology, College of Pharmacy (J.J., V.G., T.S.E., K.H.), and Davis Heart and Lung Research Institute (T.S.E.), Ohio State University, Columbus; and the Department of Pharmacology (B.Y.), Harbin Medical University, Harbin, Heilongjiang, China.
Correspondence to Keli Hu, 530 Parks Hall, Division of Pharmacology, College of Pharmacy, 500 W 12th Ave, Ohio State University, Columbus, OH 43210. E-mail hu.175{at}osu.edu
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Abstract |
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Vascular ATP-sensitive K+ (KATP) channels are critical regulators of arterial tone and, thus, blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is known that protein kinase C (PKC) activation inhibits KATP channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Here we report that caveolin-dependent internalization is involved in PKC-
–mediated inhibition of vascular KATP channels (Kir6.1 and SUR2B) by phorbol 12-myristate 13-acetate or angiotensin II in human embryonic kidney 293 cells and human dermal vascular smooth muscle cells. We showed that Kir6.1 substantially overlapped with caveolin-1 at the cell surface. Cholesterol depletion with methyl-β-cyclodextrin significantly reduced, whereas overexpression of caveolin-1 largely enhanced, PKC-induced inhibition of Kir6.1/SUR2B currents. Importantly, we demonstrated that activation of PKC- caused internalization of KATP channels, the effect that was blocked by depletion of cholesterol with methyl-β-cyclodextrin, expression of dominant-negative dynamin mutant K44E, or knockdown of caveolin-1 with small interfering RNA. Moreover, patch-clamp studies revealed that PKC-–mediated inhibition of the KATP current induced by PMA or angiotensin II was reduced by a dynamin mutant, as well as small interfering RNA targeting caveolin-1. The reduction in the number of plasma membrane KATP channels by PKC activation was further confirmed by cell surface biotinylation. These studies identify a novel mechanism by which the levels of vascular KATP channels could be rapidly downregulated by internalization. This finding provides a novel mechanistic insight into how KATP channels are regulated in vascular smooth muscle cells.
Key Words: KATP • ion channel • caveolin-1 • angiotensin II • dynamin
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Introduction |
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Vascular ATP-sensitive K+ (KATP) channels are highly abundant plasma membrane proteins responsible for linking the cellular energy levels to cellular excitability.1–4 In the vascular smooth muscles, they play an important role in the regulation of vascular tone and, thus, blood flow in response to local metabolic needs and vasoactive transmitters.4–9 They are targets of a wide variety of vasodilators and constrictors and are important drug targets for the treatment of hypertensive emergency and angina.10 As a major vascular isoform, Kir6.1 knockout mice exhibited a high rate of sudden death associated with coronary vasospasm and elevated resting blood pressure11,12 and were found to have no detectable KATP currents and lack all vasodilatory responses to KATP channel agonists.
It is known that vascular KATP channels, which are composed of Kir6.1 and SUR2B, are inhibited by protein kinase C (PKC) in the heterologous expression system.13 Several vasoconstrictors have also been shown to inhibit KATP channels via activation of PKC.14,15 Recent studies have revealed that Kir6.1 is mainly localized in the caveolae of aortic smooth muscle cells.16 Angiotensin II, a potent physiological vasoconstrictor, was reported to recruit a novel PKC isoform, PKC-, to arterial smooth muscle caveolae, indicating a potential role for caveolae in the regulation of KATP channel function.17 However, the molecular mechanism by which PKC inhibits vascular KATP channels remains unknown.
Caveolae, subsarcolemmal membrane compartments, have been implicated in signal transduction and vesicular trafficking.18–23 Caveolae are capable of removing proteins from the plasma membrane by sequestration and endocytotic mechanism. In the present study, we investigated the possible involvement of caveolae-dependent internalization in PKC-mediated inhibition of KATP channels. Given that caveolin-1 is a major structural component and key regulator of caveolae in vascular smooth muscles, we studied the role of caveolin-1 in PKC-induced internalization of KATP channels in human embryonic kidney (HEK) 293 cells and human dermal vascular smooth muscle cells (VSMCs). We provided compelling evidence that activation of PKC- by either phorbol 12-myristate 13-acetate (PMA) or angiotensin II facilitates internalization of vascular KATP channels in a caveolin-1–dependent and dynamin-mediated manner.
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Materials and Methods |
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cDNA Constructs
cDNAs encoding Kir6.1 (rat) and SUR2B (rat) were expressed in pcDNA3 and pCMV, respectively. Green fluorescent protein (GFP)–tagged caveolin-1 (caveolin-1-GFP) was a kind gift from Dr Mark A. McNiven (Mayo Clinic, Rochester, Minn). Please see the data supplement available online at http://hyper.ahajournals.org.
Drugs and Chemicals
Please see the data supplement for more information.
Cell Culture and Transfection
HEK293 cells were maintained in DMEM/F12 supplemented with 10% FBS, 2 mmol/L of glutamine, and penicillin-streptomycin.15 Primary dermal arteriolar VSMCs were cultured from punch biopsies as reported previously.24,25 These cells were maintained in the same medium as that for HEK293 cells and cultured in serum-free medium for 12 to 24 hours before experiments. Cells were transfected with cDNA using FuGENE6.
Small Interfering RNA
Predesigned caveolin-1-specific small interfering RNA (siRNA; human) was purchased from Ambion. siRNA oligonucleotides (50 nM) were transfected into human VSMCs with X-Tremegene siRNA transfection reagent (Ambion). Western blotting was carried out to examine the knockdown of caveolin-1 48 to 72 hours after transfection. A control scrambled siRNA was used to monitor nonspecific effects. Experiments were performed 48 to 72 hours after transfection.
Western Blotting
Immunoblot analysis was carried out as described previously26 (please see the data supplement).
Cell Surface Biotinylation
The levels of plasma membrane KATP channels in HEK293 cells were determined using cell surface biotinylation techniques. In brief, HEK293 cells transiently transfected with Kir6.1 and SUR2B were first incubated with PMA or PMA+PKC V1-2 at 37°C for 15 minutes and then treated with sulfo-NHS-SS-biotin (1 mg/mL, Pierce) to label proteins on the cell surface. The biotinylated proteins were then isolated by NeutrAvidin-agarose beads. The level of surface Kir6.1 was analyzed by SDS-PAGE and anti-Kir6.1 antibody (please see the data supplement).
Immunofluorescence Microscopy
For colocalization experiments,26 the cells were fixed and permeabilized before they were labeled with primary antibody. For cell surface staining of extracellular hemagglutinin (HA)-tagged Kir6.1 (internalization assay),27 live cells were labeled with primary antibody (anti-HA, 2 hours at 4°C) before the treatment with PMA or other agents at 37°C for 15 minutes (please see the data supplement).
Electrophysiology
Whole-cell KATP currents were recorded by patch clamp technique as described preciously.28 For the time course of KATP currents in HEK293 cells, whole-cell currents were elicited by 25-ms voltage steps to –60 mV from a holding potential of 0 mV. For recording in human VSMCs, the cell membranes were held at –60 mV and 300-ms voltage steps to potentials between –100 and +40 mV in 20-mV increments. The amplitude of the KATP current was measured as a glibenclamide-sensitive current (please see the data supplement).
Statistics
Group data were presented as means±SEs. The paired t test was used to compare within the same group. Unpaired t test was used to compare between groups. Multiple group means were compared by ANOVA followed by least-squares difference posthoc test. Differences with a 2-tailed P<0.05 were considered statistically significant.
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Results |
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Effect of Dominant-Negative Dynamin Mutant in PKC-
–Mediated Inhibition of Kir6.1/SUR2B Currents by PMAWhole-cell current recordings in HEK293 cells transfected with Kir6.1 and SUR2B were made in a symmetrical K+ concentration (extracellular and intracellular equal to 140 mmol/L), and pipette solutions were supplemented with 1 mmol/L of ATP and 500 µmol/L of ADP. The time course of whole-cell currents was elicited by 25-ms voltage steps to –60 mV from a holding potential of 0 mV. Because KATP channels are usually inactive, we used a KATP channel opener, pinacidil, to activate KATP channels. Pinacidil was applied
5 minutes after the membrane rupture to ensure complete dialysis of the pipette solution. As shown in Figure S1, pinacidil at 100 µmol/L activated a large inward current. Application of PMA (1 µmol/L) significantly decreased the pinacidil-induced current by approximately half (52.0±9.1%; n=5; P<0.01), whereas its inactive congener 4
-PMA (1 µmol/L) had no effect (93.0±4.2%; n=4; P value not significant). When the PKC- inhibitor peptide PKC- V1-2 (200 µmol/L) was included in the pipette solution, PMA no longer inhibited pinacidil-induced KATP currents (92.0±3.1%; n=4; P value not significant). All of the pinacidil-induced currents were subsequently inhibited by a selective KATP channel blocker glibenclamide (10 µmol/L). These results are consistent with the previous report that activation of PKC- inhibits recombinant vascular KATP channels in HEK293 cells transfected with Kir6.1 and SUR2B.7,13,14 To determine the role of dynamin in PKC-mediated inhibition of recombinant KATP channels, we used a dynamin mutant K44E that lacks GTPase activity and blocks dynamin-dependent internalization in a dominant-negative fashion.29 PMA inhibited pinacidil-induced currents in cells cotransfected with wild-type dynamin but not in cells cotransfected with dynamin mutant K44E (Figure 1A and 1B). The averaged data show that the percentage of inhibition of KATP currents by PMA was significantly higher in cells transfected with wild-type dynamin compared with that in cells transfected with dynamin-mutant K44E (Figure 1C; 49.9±4.8% versus 26.5±2.9%; n=5; P<0.01). Western blot analysis demonstrated a substantially higher level of expression for dynamin in cells transfected with wild-type dynamin or dynamin K44E than that in nontransfected cells (Figure 1D).
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Effect of Methyl-β-Cyclodextrin in PMA-Induced Inhibition of Kir6.1/SUR2B Currents
Identification of KATP channels and PKC- in caveolae-enriched fractions isolated from arterial smooth muscles cells led us to examine whether caveolae were involved in PKC-induced inhibition of KATP channels expressed in HEK293 cells.16,17 We evaluated the effect of methyl-β-cyclodextrin (MβCD) on PMA-induced inhibition of KATP currents in HEK293 cells transfected with Kir6.1/SUR2B. MβCD is a drug that disrupts caveolae and lipid rafts by depleting the cell membrane of cholesterol. Cells were incubated with MβCD (10 mmol/L) for 30 minutes before the application of pinacidil.30 Incubation of cells with MβCD saturated with cholesterol at an MβCD:cholesterol molar ratio of 10:1 was used as a control.31 MβCD-treated cells showed significantly less inhibition of KATP currents by PMA compared with the control cells but exhibited no effect on pinacidil-induced KATP currents (51.9±10.2% versus 17.2±5.6%; n=5; P<0.05; Figure 2A, 2B, and 2D). Cholesterol-saturated MβCD did not have any significant effect on PMA-induced inhibition of KATP currents (41.9±5.0% versus control; P value not significant; Figure 2C and 2D). Quantification of the cellular cholesterol level indicated a 65% reduction in the MβCD-treated group when normalized with the control group (64.8±2.9%).
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Effect of Caveolin-1 in PMA-Induced Inhibition of Kir6.1/SUR2B Currents
To assess the functional role of caveolin-1 in PKC-induced inhibition of Kir6.1/SUR2B currents, we created a gain-of-function situation by expressing caveolin-1-GFP in HEK293 cells where the endogenous level of caveolin-1 is very low.32,33 Caveolin-1-GFP has been shown to preserve the subcellular distribution and function of endogenous caveolin-1.34 We recorded whole-cell KATP currents in HEK293 cells transfected with either Kir6.1/SUR2B/GFP or Kir6.1/SUR2B/caveolin-1-GFP. Overexpression of caveolin-1-GFP significantly increased the inhibitory effect of PMA on the KATP current compared with the control group (68.3±8.5% versus 36.8±4.3%; n=5; P<0.01; Figure 3A and 3B). Western blot data indicated that the same level of endogenous caveolin-1 at a molecular mass of 
21 kDa was detected in both control and transfected cells, whereas caveolin-1-GFP–transfected cells showed significant expression of caveolin-1-GFP at 50 kDa. Both transfected and nontransfected cells showed a band at a similar molecular mass close to 50 kDa, indicating a dimer of caveolin-1 (Figure 3C). Immunofluorescence further demonstrated that cell surface punctuate areas of Kir6.1 and caveolin-1 staining were clearly observed in these cells. Merged images showed substantial colocalization of Kir6.1 with caveolin-1 along the plasma membrane but not intracellular compartments (Figure S2).
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Internalization of Kir6.1/SUR2B Channels by Activation of PKC- in HEK293 Cells
To examine whether PKC activation induces the internalization of KATP channels, we evaluated the effect of PKC on subcellular distribution of KATP channels in HEK293 cells transiently transfected with Kir6.1/SUR2B. Cell surface immunostaining was performed at 4°C with anti-HA antibody, which labeled only cell surface Kir6.1 channels with extracellular HA epitope and then were treated with various agents before fixation, permeabilization, and labeling with fluorescence-conjugated secondary antibodies. In the absence of PMA, cells showed significant plasmalemmal staining, indicating that most of the KATP channels are on the cell surface (Figure 4A). Treatment with PMA (1 µmol/L) for 15 minutes at 37°C caused significant internalization of KATP channels, which was evident by the redistribution of the staining from the cell surface to an intracellular location. The PMA-induced internalization of KATP channels, as indicated by intracellular punctuate staining, was significantly reduced by not only the treatment with the PKC- inhibitor peptide PKC- V1-2 (200 µmol/L; Figure 4A, bottom left) but also MβCD (10 mmol/L; Figure 4A, bottom right). Either PKC- V1-2 or MβCD treatment alone did not cause any significant changes in the Kir6.1 staining pattern (data not shown). For quantitative analysis, transfected cells were scored positive (5 punctae) or negative (<5 punctae) for internalization of HA-tagged KATP channels. Among a total of 254 cells analyzed from 3 independent experiments, 33% of cells without PMA treatment and 91% of cells treated with PMA showed internalization (32.7±4.3% versus 90.6±9.0%). Both the PKC- inhibitor peptide and MβCD significantly reduced the number of cells showing internalization (35.8±3.8% versus 38.8±5.8%).
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Role of Dynamin in PKC-Mediated Internalization of KATP Channels in HEK293 Cells
Dynamin is known to mediate caveolae-dependent internalization.22,35 To define the functional role of dynamin in PKC-induced internalization of KATP channels, we examined the effect of the dynamin mutant (K44E) in HEK293 cells transfected with Kir6.1/SUR2B and dynamin K44E. Wild-type dynamin was used as a control. Whereas PMA facilitated internalization of Kir6.1/SUR2B channels in cells cotransfected with wild-type dynamin, as indicated by increased intracellular punctuate staining (percentage of cells showing internalization: 91.1±8.7% versus 29.4±3.2%, PMA versus no PMA), this effect was largely abolished in cells transfected with dynamin K44E (Figure 4B; 41.7±5.7%). More than 75 cells were analyzed in each group from 3 independent experiments.
Effect of PMA on Cell Surface Levels of KATP Channels in HEK293 Cells
The increase in PKC-–induced internalization suggested that PMA treatment will decrease the level of KATP channels on the cell surface. To test this hypothesis, the level of KATP channels in the plasma membrane was analyzed after PMA treatment using cell surface biotinylation (Figure 4C). As expected, there was a significant decrease in the level of cell surface Kir6.1 protein after PMA treatment, and this effect was prevented by PKC V1-2. This result is consistent with the observation that PKC activation inhibits whole-cell KATP currents in HEK293 cells, possibly by reducing number of functional KATP channels on the cell surface via internalization.
Effect of Dominant-Negative Dynamin in PKC-Mediated Inhibition of KATP Currents by Angiotensin II in Human VSMCs
To further demonstrate that dynamin-dependent internalization is involved in PKC-mediated inhibition of endogenous KATP channels, we studied the effect of angiotensin II on whole-cell KATP currents in human dermal VSMCs. Pinacidil (10 µmol/L) caused a small but significant increase in outward currents at 20 mV from a holding potential of –60 mV (Figure 5). The application of angiotensin II (100 nmol/L) partially inhibited pinacidil-induced currents. The remaining pinacidil-induced currents were subsequently reduced by glibenclamide (10 µmol/L; Figure 5A). In the absence of angiotensin II, the pinacidil-activated current was completely inhibited by glibenclamide, indicating activation of glibenclamide-sensitive currents (10 µmol/L; Figure 5B, inset). Angiotensin II–induced inhibition of currents was largely blocked by PKC- V1-2 (100 µmol/L; 61.9±4.0% versus 19.3±4.5%; n=3; P<0.01), suggesting that angiotensin II inhibits KATP currents via activation of PKC- in the human VSMCs (Figure 5A and 5B). When these cells were transfected with dynamin K44E, the inhibitory effect of angiotensin II on KATP channels was significantly reduced (61.3±3.7% versus 34.3±7.0%; n=6; P<0.05; Figure 5C and 5D). Western blot analysis with antidynamin antibody showed significant expression of dominant-negative dynamin in cells transfected with dynamin K44E (950.8±18.8% versus nontransfected cells; n=3; Figure 5D, inset).
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Effect of siRNA Targeting Caveolin-1 on PKC-Mediated Inhibition of KATP Channels by Angiotensin II in Human VSMCs
To define the role of caveoin-1 in PKC-mediated inhibition of endogenous KATP channels, we studied the effect of suppressing caveolin-1 on angiotensin II–induced inhibition of KATP channels in human VSMCs. VSMCs were transiently transfected with siRNA targeting caveolin-1 or control siRNA. The siRNA targeting caveolin-1 significantly suppressed endogenous caveolin-1 protein level by 60% when normalized to the control (43.0±8.1% versus control; n=3; Figure 6A). Angiotensin II (100 nmol/L) inhibited KATP currents in control cells. However, in cells transfected with siRNA targeting caveolin-1, the inhibitory effect of angiotensin II was much smaller (62.4±7.6% versus 31.5±9.8%; n=6; P<0.05; Figure 6B and 6C). Immunofluorescence microscopic analysis demonstrated that the human VSMCs were strongly labeled with both Kir6.1 and caveolin-1. When these images were merged, there was substantial colocalization along the plasma membrane in every cell analyzed (75 cells scored; Figure 6D).
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Caveolin-1–Dependent and Dynamin-Mediated Internalization of KATP Channels by Angiotensin II in Human VSMCs
To determine the physiological relevance of our findings, we examined the effect of angiotensin II, an endogenous PKC activator, on subcellular distribution of KATP channels in the human dermal VSMCs. We transfected the VSMCs with HA-Kir6.1 alone, with the expectation that HA-Kir6.1 would coassemble with native subunits and reach the cell surface, thereby enabling the use of anti-HA antibody to follow internalization. HA-Kir6.1 levels at the cell surface would not exceed the endogenous levels, because HA-Kir6.1 cannot reach the cell surface unless its endoplasmic reticulum retention signals are masked by coassembling with a endogenous SUR subunit.36 As shown in Figure 7A, endogenous SUR2B expression was not altered by overexpression of HA-Kir6.1, whereas human VSMCs expressed Kir6.1 and SUR2B (Figure 7A, left). The density of glibenclamide-sensitive KATP currents induced by pinacidil (data not shown) and the degree of channel inhibition by angiotensin II were not significantly affected by overexpression of HA-Kir6.1 (Figure 7A, right). As predicted, exogenously expressed HA-Kir6.1 showed surface expression (Figure 7B). However, channels bearing HA-Kir6.1 showed significant internalization by angiotensin II (100 nmol/L), as indicated by an increase in intracellular punctate staining. We scored 75 cells in each group and found that 96% of cells treated with angiotensin II showed significant internalization, whereas only 27% of control cells (no angiotensin II) showed positive staining for internalization (26.5±3.2% versus 95.6±10.0%). This effect was blocked by pretreatment with PKC- V1-2 (100 µmol/L) for 15 minutes before the application of angiotensin II. These data eliminate uncertainties associated with the other cell lines, where channels could be overexpressed leading to erroneous results.
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The role of caveolin-1 in this process was assessed in the VSMCs transfected with or without siRNA targeting caveolin-1. Cells transfected with caveolin-1–specific siRNA significantly reduced angiotensin II–induced internalization of KATP channels compared with control cells (Figure 7C; 30.3±5.0% versus 92.3±8.7%; >70 cells scored in each group). Consistent with the observations in HEK293 cells, dominant-negative dynamin mutant K44E largely reduced internalization of KATP channels by angiotensin II in human VSMCs (Figure 7C; 39.0±4.1% versus 94.1±9.2%; >75 cells scored in each group).
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Discussion |
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Our findings provide the first evidence that dynamin and caveolin-1–dependent internalization are involved in PKC-mediated inhibition of vascular KATP channels by PMA and angiotensin II in HEK293 cells and primary human dermal VSMCs. Specifically, we found that PKC-
–induced inhibition of KATP channels was largely reduced by cholesterol depletion, siRNA knockdown of caveolin-1 expression, or dominant-negative suppression of dynamin function. Importantly, we showed that activation of PKC- facilitated internalization of KATP channels, and this effect involved a caveolin-1 and dynamin-dependent mechanism.
A number of vasoconstrictors have been reported to inhibit vascular KATP channels via activation of PKC.15,37 However, the mechanism by which PKC inhibits KATP channels is unknown. Both PKC- and Kir6.1 are localized in the caveolae of arterial smooth muscles,16,17 which are specialized microdomains in the plasma membrane and serve to compartmentalize and integrate numerous signaling events.38 Caveolin-1 is a major integral membrane component of caveolar membranes in vascular smooth muscles. In addition to its structural role, caveolin-1 is believed to play a scaffolding function to organize signaling molecules and participate in signaling events and endocytic trafficking.22,39 Interestingly, both Kir6.1 and PKC- have been reported to coimmunoprecipitate with caveolin-1.17 This caveolin-1 association could serve to cluster PKC and signaling molecules with KATP channels, allowing for specific modulation while preventing crosstalk with other pathways. In the present study, we found that caveolae disruption with MβCD or caveolin-1–specific siRNA prevented not only an inhibitory effect of PKC on KATP currents but also PKC-induced internalization of KATP channels, suggesting the functional importance of this compartmentalization. HEK293 cells have been reported to express a low but physiologically important level of caveolin-1.32,33 These cells are highly enriched in the caveolin-functional homologous flotillins and exhibit caveolae-like structures.40 Thus, the observed effect of PKC on recombinant KATP channels in HEK293 cells could depend on lipid raft microdomains and occurred at caveolin-1 levels below the threshold for caveolae formation. By overexpression of caveolin-1 in these cells, we found that caveolin-1 significantly enhanced PKC-induced inhibition of KATP channels in HEK293 cells, implying that caveolin-1 is essential for PKC-induced inhibition of KATP channels.
Although it is currently unclear whether different dynamin isoforms perform redundant functions or participated in the distinct endocytic process, dynamin is a family of signal-transducing GTPases that has been widely reported to be involved in both clathrin and caveolin-mediated internalization.22,35,41 They are known to localize to and mediate the scission of caveolae from the plasma membrane.42 In the present study, expression of the dominant-negative dynamin mutant largely blocked PKC-mediated inhibition of KATP currents in both HEK293 cells and human VSMCs, indicating that dynamin-dependent internalization is likely to contribute to the inhibitory effect of PKC on KATP channels. Consistent with this finding, immunofluorescence microscopy and biotinylation experiments revealed that Kir6.1 was translocated from a preferential plasma membrane to cytosolic localization on PKC stimulation with PMA or angiotensin II, and the amount of Kir6.1 on the plasma membrane was significantly reduced. This effect was prevented by the expression of a dominant-negative dynamin mutant.
Although we did not find any significant changes in the biophysical properties of KATP channels (eg, voltage dependence) by PKC except a reduction in KATP current amplitude, our findings do not rule out the possibility that a direct functional modulation of KATP channel by PKC exists. A previous report has shown that PKC inhibits the KATP channel open probability possibly by increasing interburst intervals in recombinant KATP channels composed of Kir6.1 and SUR2B, as well as KATP channels in the rat portal vein,13,43 indicating that a direct functional modulation of KATP channels by PKC may contribute, in part, to the inhibitory effect of PKC on KATP channels. Our data show that PKC-induced inhibition of KATP channels was not completely prevented by pretreatment with MβCD or expression of a dominant-negative dynamin mutant. One possible explanation could be incomplete disruption of lipid rafts or endogenous dynamin function. Alternatively, PKC may directly modulate KATP channel kinetics. Nevertheless, our findings uncover a novel mechanism in regulating KATP channel function in arterial smooth muscle-caveolin–dependent internalization.
In the present study, we evaluated the effect of PKC on recombinant Kir6.1/SUR2B in HEK293 cells. Although the molecular identity of smooth muscle KATP channels is not established with certainty, increasing evidence supports the composition of Kir6.1 and SUR2B in native vascular smooth muscles. Not only do channels reconstituted from Kir6.1 and SUR2B have properties similar to KATP channels in native VSMCs,44 but also Kir6.1 knockout mice show a lack of vasodilatation in response to pinacidil.11 More importantly, the modulation of recombinant Kir6.1/SUR2B but not Kir6.2/SUR2B by PKC mimics that of native vascular KATP channels.13 Our data from both HEK293 cells and human dermal VSMCs further support this notion.
There is ample evidence that KATP channels contribute to the resting blood flow in the number of VSMCs.4 Thus, it is expected that KATP channel inhibition contributes to the effects of vasoconstrictors in reducing blood flow.15,45 Our observations reveal an important novel mechanism allowing KATP channels to be downregulated by PKC-mediated and caveolin-dependent internalization. This mechanism may play a critical role in regulating KATP channels and, thus, vascular function.
Perspectives
To our knowledge, our study is the first to show that vascular KATP channels can be controlled via traffic regulation. This regulatory mechanism may play an important role in controlling KATP channel function and, thus, vascular tone in response to local metabolic needs. The molecular basis by which cell surface KATP channels in vascular smooth muscles are targeted to the endocytic pathway after PKC stimulation remains to be elucidated. It would be interesting to determine whether PKC-mediated phosphorylation is critical for caveolin-dependent internalization of KATP channels and whether there is an endocytic motif in Kir6.1/SUR2B that is required for channel regulation. Nevertheless, our current finding suggests that interference with vascular KATP channel trafficking may represent an important approach for controlling blood flow.
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Acknowledgments |
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Sources of Funding
This study was supported in part by the American Heart Association (K.H.) and National Institutes of Health/National Heart, Lung, and Blood Institute (T.S.E.).
Disclosures
None.
Received January 25, 2008; first decision February 18, 2008; accepted July 8, 2008.
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References |
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1. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983; 305: 147–148.[CrossRef][Medline] [Order article via Infotrieve]
2. Nichols CG, Ripoll C, Lederer WJ. ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction. Circ Res. 1991; 68: 280–287.
3. Aguilar-Bryan L, Bryan J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev. 1999; 20: 101–135.
4. Quayle JM, Nelson MT, Standen NB. ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev. 1997; 77: 1165–1232.
5. Clapp LH, Tinker A. Potassium channels in the vasculature. Curr Opin Nephrol Hypertens. 1998; 7: 91–98.[Medline] [Order article via Infotrieve]
6. Suzuki M, Li RA, Miki T, Uemura H, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Ogura T, Seino S, Marban E, Nakaya H. Functional roles of cardiac and vascular ATP-sensitive potassium channels clarified by Kir6.2-knockout mice. Circ Res. 2001; 88: 570–577.
7. Quinn KV, Cui Y, Giblin JP, Clapp LH, Tinker A. Do anionic phospholipids serve as cofactors or second messengers for the regulation of activity of cloned ATP-sensitive K+ channels? Circ Res. 2003; 93: 646–655.
8. Tang G, Wu L, Wang R. The effect of hydroxylamine on KATP channels in vascular smooth muscle and underlying mechanisms. Mol Pharmacol. 2005; 67: 1723–1731.
9. Jackson WF. Ion channels and vascular tone. Hypertension. 2000; 35: 173–178.
10. Jahangir A, Terzic A, Shen WK. Potassium channel openers: therapeutic potential in cardiology and medicine. Expert Opin Pharmacother. 2001; 2: 1995–2010.[CrossRef][Medline] [Order article via Infotrieve]
11. Miki T, Suzuki M, Shibasaki T, Uemura H, Sato T, Yamaguchi K, Koseki H, Iwanaga T, Nakaya H, Seino S. Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1. Nat Med. 2002; 8: 466–472.[CrossRef][Medline] [Order article via Infotrieve]
12. Chutkow WA, Pu J, Wheeler MT, Wada T, Makielski JC, Burant CF, McNally EM. Episodic coronary artery vasospasm and hypertension develop in the absence of Sur2 K(ATP) channels. J Clin Invest. 2002; 110: 203–208.[CrossRef][Medline] [Order article via Infotrieve]
13. Thorneloe KS, Maruyama Y, Malcolm AT, Light PE, Walsh MP, Cole WC. Protein kinase C modulation of recombinant ATP-sensitive K(+) channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B. J Physiol. 2002; 541: 65–80.
14. Hayabuchi Y, Davies NW, Standen NB. Angiotensin II inhibits rat arterial KATP channels by inhibiting steady-state protein kinase A activity and activating protein kinase Ce. J Physiol. 2001; 530: 193–205.
15. Shi W, Cui N, Shi Y, Zhang X, Yang Y, Jiang C. Arginine vasopressin inhibits Kir6.1/SUR2B channel and constricts the mesenteric artery via V1a receptor and protein kinase C. Am J Physiol Regul Integr Comp Physiol. 2007; 293: R191–R199.
16. Sampson LJ, Hayabuchi Y, Standen NB, Dart C. Caveolae localize protein kinase A signaling to arterial ATP-sensitive potassium channels. Circ Res. 2004; 95: 1012–1018.
17. Sampson LJ, Davies LM, Barrett-Jolley R, Standen NB, Dart C. Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels. Cardiovasc Res. 2007; 76: 61–70.
18. Okamoto T, Schlegel A, Scherer PE, Lisanti MP. Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J Biol Chem. 1998; 273: 5419–5422.
19. Cheng ZJ, Singh RD, Marks DL, Pagano RE. Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids. Mol Membr Biol. 2006; 23: 101–110.[CrossRef][Medline] [Order article via Infotrieve]
20. O'Connell KM, Martens JR, Tamkun MM. Localization of ion channels to lipid Raft domains within the cardiovascular system. Trends Cardiovasc Med. 2004; 14: 37–42.[CrossRef][Medline] [Order article via Infotrieve]
21. Maguy A, Hebert TE, Nattel S. Involvement of lipid rafts and caveolae in cardiac ion channel function. Cardiovasc Res. 2006; 69: 798–807.
22. Lajoie P, Nabi IR. Regulation of raft-dependent endocytosis. J Cell Mol Med. 2007; 11: 644–653.[CrossRef][Medline] [Order article via Infotrieve]
23. Anderson RG, Kamen BA, Rothberg KG, Lacey SW. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992; 255: 410–411.
24. Chotani MA, Mitra S, Su BY, Flavahan S, Eid AH, Clark KR, Montague CR, Paris H, Handy DE, Flavahan NA. Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells. Am J Physiol Heart Circ Physiol. 2004; 286: H59–H67.
25. Martin MM, Buckenberger JA, Jiang J, Malana GE, Nuovo GJ, Chotani M, Feldman DS, Schmittgen TD, Elton TS. The human angiotensin II type 1 receptor +1166 A/C polymorphism attenuates microrna-155 binding. J Biol Chem. 2007; 282: 24262–24269.
26. Garg V, Hu K. Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane. Am J Physiol Heart Circ Physiol. 2007; 293: H322–H332.
27. Hu K, Huang CS, Jan YN, Jan LY. ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C. Neuron. 2003; 38: 417–432.[CrossRef][Medline] [Order article via Infotrieve]
28. Hu K, Duan D, Li GR, Nattel S. Protein kinase C activates ATP-sensitive K+ current in human and rabbit ventricular myocytes. Circ Res. 1996; 78: 492–498.
29. Herskovits JS, Burgess CC, Obar RA, Vallee RB. Effects of mutant rat dynamin on endocytosis. J Cell Biol. 1993; 122: 565–578.
30. Balijepalli RC, Foell JD, Hall DD, Hell JW, Kamp TJ. Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation. Proc Natl Acad Sci U S A. 2006; 103: 7500–7505.
31. Christian AE, Haynes MP, Phillips MC, Rothblat GH. Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res. 1997; 38: 2264–2272.[Abstract]
32. Wei Y, Yang X, Liu Q, Wilkins JA, Chapman HA. A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling. J Cell Biol. 1999; 144: 1285–1294.
33. Burgueno J, Canela EI, Mallol J, Lluis C, Franco R, Ciruela F. Mutual regulation between metabotropic glutamate type 1alpha receptor and caveolin proteins: from traffick to constitutive activity. Exp Cell Res. 2004; 300: 23–34.[CrossRef][Medline] [Order article via Infotrieve]
34. Mundy DI, Machleidt T, Ying YS, Anderson RG, Bloom GS. Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton. J Cell Sci. 2002; 115: 4327–4339.
35. Oh P, McIntosh DP, Schnitzer JE. Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium. J Cell Biol. 1998; 141: 101–114.
36. Zerangue N, Schwappach B, Jan YN, Jan LY. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron. 1999; 22: 537–548.[CrossRef][Medline] [Order article via Infotrieve]
37. Bonev AD, Nelson MT. Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C. J Gen Physiol. 1996; 108: 315–323.
38. Shaul PW, Anderson RG. Role of plasmalemmal caveolae in signal transduction. Am J Physiol. 1998; 275: L843–L851.[Medline] [Order article via Infotrieve]
39. Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev. 2002; 54: 431–467.
40. Rybin VO, Xu X, Lisanti MP, Steinberg SF. Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem. 2000; 275: 41447–41457.
41. Damke H, Baba T, Warnock DE, Schmid SL. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol. 1994; 127: 915–934.
42. Henley JR, Krueger EW, Oswald BJ, McNiven MA. Dynamin-mediated internalization of caveolae. J Cell Biol. 1998; 141: 85–99.
43. Cole WC, Malcolm T, Walsh MP, Light PE. Inhibition by protein kinase C of the K(NDP) subtype of vascular smooth muscle ATP-sensitive potassium channel. Circ Res. 2000; 87: 112–117.
44. Cui Y, Tran S, Tinker A, Clapp LH. The molecular composition of K(ATP) channels in human pulmonary artery smooth muscle cells and their modulation by growth. Am J Respir Cell Mol Biol. 2002; 26: 135–143.
45. Tateishi J, Faber JE. ATP-sensitive K+ channels mediate alpha 2D-adrenergic receptor contraction of arteriolar smooth muscle and reversal of contraction by hypoxia. Circ Res. 1995; 76: 53–63.
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