1Department of Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa; and 2Department of Pharmacology, University of Michigan, Ann Arbor, Michigan
Submitted 15 August 2004 ; accepted in final form 8 February 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
potassium channel; membrane microdomain
Myometrial smooth muscle contains an extensive number of plasma membrane invaginations termed caveolae, which can increase the membrane surface area by 70% (7). These morphologically identifiable invaginations are rich in cholesterol and sphingolipids, and they contain the marker protein caveolin. These structures act as a scaffold to congregate multiple proteins for Ca2+ handling in smooth muscle cells, including Ca2+ channels, Ca2+-binding proteins, and Ca2+ pumps (10). In addition, recent investigations have revealed that caveolae can be converging centers to regulate ion channel activity (28). Maxi-K channels associate in lipid rafts in both heterologous expression systems and ureter smooth muscle (2, 5) and may modulate phasic contractions (2). It is interesting to note that signaling molecules known to modulate maxi-K channel activity, including protein kinase C (46), are also associated with raft caveolar domains (31). An assessment of whether myometrial maxi-K channels are directly regulated by cell signaling molecules or indirectly modulated by other proteins assembled in macromolecular complexes containing the channel protein would provide important information regarding the complex regulation of this channel protein.
Studies have demonstrated that the actin cytoskeleton plays a role in anchoring caveolae to the plasma membrane (30, 41). Actin filament disruption by latrunculin A causes redistribution of caveolin-1 and ultimately internalization (30). Depolymerization of the actin cytoskeleton in neuronal cells resulted in maxi-K channel inhibition (20), while channel activity increased in cultured smooth muscle cells (13). The reasons for these contrasting results have not been investigated; however, one potential mechanism is that actin-channel interactions are indirect and complex with other regulatory proteins. Therefore, the actin cytoskeleton may play a role in controlling the proximity of membrane proteins with their signaling molecules by controlling caveolar organization. Channel-cytoskeleton microdomain complexes have been hypothesized for at least two families of K+ channels. The inwardly rectifying Kir2.1 channel interacts with filamin, an actin-binding protein that associates with the caveolin-associated protein caveolin-1 (39). Voltage-gated family member Kv4.2 also has been shown to interact with filamin. For both channels, filamin regulated surface expression and distribution in either vascular smooth muscle (Kir2.1) (37) or neurons (Kv4.2) (35), and it altered current expression in their respective cell types.
To understand the complex regulation of maxi-K channels in MSMCs, we tested whether this channel is compartmentalized in caveolar microdomains. Sequence analysis identified a consensus caveolin-binding motif on the COOH-terminal portion of the maxi-K channel, and coimmunoprecipitation experiments demonstrated that this channel associates with caveolin in human MSMCs (hMSMCs). On the basis of the evidence that actin plays a role in anchoring caveolae in the plasma membrane, we further hypothesized that the maxi-K channel, caveolin, and actin form a macromolecular complex within caveolae. Both immunofluorescence and immunoelectron microscopy demonstrate the proximity of actin and the maxi-K channel within cell surface caveolae. In addition, disruption of the actin cytoskeleton and perturbation of caveolar organization dramatically increase maxi-K channel function. In summary, our data demonstrate that the MSMC maxi-K channels are localized to caveolae on the cell surface as part of a macromolecular complex of proteins that includes actin and caveolin. We report the first data demonstrating that a protein complex containing the maxi-K channel in myometrial smooth muscle may be one mechanism by which to regulate current expression in uterine smooth muscle.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell isolation and culturing. Freshly isolated human myometrial tissue was disaggregated with 1 mg/ml collagenase (type 1; Worthington Biochemical, Lakewood, NJ) in Hanks' balanced salt solution at 37°C with mild agitation for 1 h. Partially digested explants were plated in DMEM-Ham's F-12 medium supplemented with 5% FBS, 4 ng/ml basic fibroblast growth factor (Sigma, St. Louis, MO), 1 ng/ml epithelial growth factor (Sigma), 50 µg/ml gentamicin (GIBCO-BRL, Grand Island, NY), and 5 µg/ml Fungizone (GIBCO-BRL). This smooth muscle cell-defined medium enhances smooth muscle cell growth (14) and inhibits the growth of other cell types present in myometrial tissue samples (e.g., fibroblasts, endothelial cells). Cells were grown in a humidified incubator with 5% CO2 at 37°C for 2 days until smooth muscle cell outgrowth was observed. At that point, the explants were removed, and adherent cells were trypsinized, plated on fresh dishes, and grown for an additional 2 days in the medium described above (with Fungizone removed) until they reached 7080% confluence. Subsequently, cells were differentiated in DMEM-Ham's F-12 medium supplemented with low (0.5%)-concentration FBS before experiments to induce the contractile phenotype similar to that observed in vivo. Cells were stimulated with 30 mM KCl to ensure that cells maintained a contractile phenotype after each passage. Cells were used at low passage number (up to passage 5) because cell phenotype changed irreversibly to a proliferative one with further passages.
Isolation of detergent-resistant membrane rafts. Human uterine tissue (0.75 g) was homogenized in 6 ml of MES-buffered saline (24 mM MES, pH 6.5, and 0.15 NaCl) plus 1% Triton X-100, spun down at 3,000 g for 5 min at 4°C, and 4 ml of the supernatant were made to compose 40% sucrose. This solution was placed in a 12.5-ml Beckman centrifuge tube (Beckman Coulter, Fullerton, CA) with a 530% sucrose gradient layered on top and then spun at 39,000 rpm for 24 h at 4°C in a Beckman SW-41 rotor. After being spun, 600-µl fractions of the solution were collected. For all lipid raft isolations, detergent solubilization was assessed using fractionation of the transferrin receptor. Insufficient detergent solubilization led to the transferrin receptor floating to a low buoyant density. Increasing the detergent concentration led to full solubilization of the transferrin receptor, while true raft-associated proteins (i.e., caveolin) floated to raft fractions. Extraction in 1% Triton X-100 led to nearly complete solubilization of the transferrin receptor as determined by pelleting insoluble proteins and/or complexes with a high-speed spin in extraction buffer. Therefore, we successfully isolated lipid rafts under these conditions, according to the operational definition of lipid rafts (4, 19, 38).
Preparation of uterine cytosol. NP and LP uterine tissue (0.751 g) were homogenized in 1.5 ml of membrane prep solution (0.25 M sucrose, 0.05 M MOPS, 2 mM EDTA, and 2 mM EGTA, pH 7.4) plus a Complete protease inhibitor tablet (Roche, Indianapolis, IN). This mixture was spun at 10,000 g for 5 min at 4°C, and the supernatant was spun at 14,000 g for 15 min and 100,000 g for 1 h at 4°C. The membrane pellet was solubilized with membrane prep solution with 1% Triton X-100 and was cleared at 14,000 g for 15 min at 4°C. Protein concentration was determined using a bicinchoninic acid protein assay (Sigma).
Immunoprecipitation. Two milligrams of antibody (maxi-K or pan-caveolin; Transduction Laboratories, Lexington, KY) were bound to 60 µl of protein G-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature (RT) with rocking. Ten volumes of 0.2 M sodium borate was added, mixed, and centrifuged at 3,500 g for 5 min at 4°C. Beads were resuspended in 10 volumes of 0.2 M sodium borate, and then dimethyl pimelimdate·2HCl was added to a concentration of 20 mM and mixed for 30 min at RT. Beads were spun as described above, washed once with 1 ml of 0.1 M ethanolamine, resuspended in 400 µl of 0.1 M ethanolamine, and incubated for 2 h at RT with rocking. Beads were spun as described above and resuspended in 450 µl of PBS, to which 25 µl of each lipid raft fraction containing the opaque band were added and incubated overnight at 4°C with rocking. Beads were washed twice with 1 ml of buffer A (150 mM NaCl, 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, and 0.5% Triton X-100) and resuspended in 20 µl of 2x SDS. Control immunoprecipitation was performed in the absence of primary antibody.
Actin spin-down assay. Assays were performed as described previously (15). NP and LP human uterine cytosol solutions (1.5 mg) were incubated with 20 nM phalloidin, 2 mM ATP, and 100 µM nocodozole for 30 min at RT. This mixture was spun through a 2 M sucrose cushion at 48,000 rpm for 1 h at 4°C. The pellet was saved and examined using Western blot analysis.
Immunoblotting.
Immunoprecipitates, lipid raft fractions, and actin spin-down products were fractionated using SDS-PAGE and stained with Coomassie blue for 1 h or immunoblotted as described previously (3). Primary antibodies used were against the maxi-K channel (1:250 dilution; Transduction Laboratories); caveolin-1, caveolin-2, caveolin-3, and pan-caveolin (1:2,500 dilution; Transduction Laboratories); transferrin receptor (1:1,000 dilution; Zymed Laboratories, South San Francisco, CA); -tubulin (clone E7; University of Iowa Hybridoma Facility, Iowa City, IA); and
-actin (Sigma) and
-actin (1:1,000 dilution; Chemicon International, Temecula, CA). Secondary antibodies used included goat anti-mouse (1:3,000 dilution), goat anti-rabbit (1:3,000 dilution), and rabbit anti-sheep (1:3,000 dilution; Jackson ImmunoResearch, West Grove, PA). All antibodies were diluted in Tris-buffered saline containing Tween 20 and 3% nonfat dry milk. Blots were detected using enhanced chemiluminescence Western blot detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunoelectron microscopy. Thin slices of NP or LP human uterine tissue sample were fixed in 2% paraformaldehyde and 0.25% glutaraldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, and inactive aldehyde groups were quenched with 0.05 M glycine in PBS for 15 min. Tissue was then permeabilized with 0.01% Triton X-100 in PBS for 15 min at RT, blocked in PBS containing 5% BSA, 0.1% coldwater fish skin gelatin, and 5% FBS. Tissue was washed in PBS and incubated overnight at 4°C with gentle agitation with a mouse monoclonal anti-actin antibody (Sigma) diluted 1:500 in PBS. After being washed with PBS, the tissue was incubated with a goat anti-mouse 1.4-nm gold-conjugated secondary antibody (1:2,500 dilution; Nanoprobes, Yaphank, NY) and incubated for 2 h at RT. After being washed with PBS, the tissue was blocked as described above and incubated with rabbit anti-maxi-K polyclonal antibody (1:250 dilution; Chemicon International) in PBS overnight at 4°C. After being washed in PBS, the tissue was incubated with a goat anti-rabbit 10-nm gold-conjugated secondary antibody (Nanoprobes) diluted 1:2,500 in PBS for 2 h at RT. After being washed with PBS, the tissue was postfixed with 2% paraformaldehyde and 0.25% glutaraldehyde in PBS for 5 min and washed with PBS and distilled water. The tissue slices were then enhanced with gold according to the protocol provided by Nanoprobes.
The tissue slices were prepared for transmission electron microscopy by performing osmication for 90 min in osmium fixation solution containing 0.2 M sodium cacodylate, 1% osmium, and 6% potassium ferrocyanide at a ratio of 2:1:1, and they were washed with 0.1 M sodium cacodylate for 20 min followed by a 20-min wash with distilled water. The tissue was then dehydrated using incubation in 50% ethanol for 15 min at RT, 75% ethanol for 15 min at 4°C, 95% ethanol for 30 min at 20°C, and 100% ethanol for 1 h at 20°C. The tissue was subsequently incubated in two parts ethanol and one part Epon-812 (Electron Microscopy Sciences) for 30 min at RT, one part ethanol and two parts Epon-812 for 2 h at RT, and Epon-812 overnight at RT. The tissue was placed in a Beem capsule in fresh Epon-812 and cured overnight at 60°C. After the blocks had cooled, they were sectioned (40-nm sections) and mounted on no. 300 nickel grids backed with Formvar and carbon (Electron Microscopy Sciences). The grids were subsequently stained using a standard electron microscopy grid-staining technique with 5% uranyl acetate and lead citrate. The samples were viewed using a Hitachi H-7000 electron microscope (Hitachi North America, Pleasanton, CA), and images were captured using Kodak 4489 film.
Immunocytochemistry.
Cultured hMSMCs were fixed in 2% paraformaldehyde and 0.01% Triton X-100 for 30 min at RT, blocked with 10% heat-inactivated FBS and 1% heat-inactivated donkey serum (blocking buffer; 30 min at 37°C). For colocalization studies, hMSMCs were incubated with mouse maxi-K channel antibody (1:250 dilution; Transduction Laboratories), biotin-conjugated donkey anti-mouse secondary antibody (1:1,000 dilution; Jackson ImmunoResearch). Signal was enhanced using mouse anti-biotin antibody (1:500 dilution; Jackson ImmunoResearch), a biotin-conjugated donkey anti-mouse secondary antibody, and a Cy2-conjugated streptavidin (1:1,000 dilution; Jackson ImmunoResearch). Cells were incubated in blocking buffer and then with a pan-caveolin antibody (1:500 dilution) or a caveolin-1 antibody (1:500 dilution) and Cy5-conjugated donkey anti-rabbit secondary antibody (1:1,000 dilution; Jackson ImmunoResearch). Cells were blocked and incubated with either Cy3-conjugated anti--actin (1:500 dilution; Sigma) or
-actin antibodies (1:500 dilution; Chemicon International). Cells treated with
-actin antibody were incubated with a biotin-conjugated donkey anti-sheep secondary antibody and a Cy3-conjugated streptavidin (both 1:1,000 dilution; Jackson ImmunoResearch). Signals were visualized using a confocal scanning microscope (model 510; Zeiss, Oberkochen, Germany), and images were obtained using LSM 5 image browser software (Zeiss, Jena, Germany). For controls, cultured hMSMCs were incubated either with primary antibodies alone for 30 min at 37°C or with secondary antibodies alone for 15 min at 37°C. Leakage of fluorescent signals into the neighboring channels was controlled by turning off all but one laser at a time and recording images in all three channels. Laser power, pinhole size, and detector gain were then adjusted to ensure that images appeared in their respective channels only.
Electrophysiology.
All patch-clamp experiments were performed at RT (22°C). Cultured hMSMCs were placed in a pH 7.4 solution containing (in mM) 145 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES. Borosilicate glass pipettes of 615 M
were filled with a pH 7.2 solution containing (in mM) 145 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES. High (330 G
)-resistance patch seals were used for cell-attached measurements. A membrane potential of +40 mV was applied to membrane patches for up to 2 min using an Axopatch 200B voltage-current amplifier (Axon Instruments, Union City, CA). The elicited currents were recorded using pCLAMP 6.0 software (Axon Instruments). The number of open events was measured using the Fetchan function of pCLAMP 6.0 software. The open-state probability was calculated using the pStat program of pCLAMP 6.0. Statistical significance was calculated using Student's t-test for paired observations (SigmaPlot software; SPSS, Chicago, IL). Differences were considered significant at P < 0.05. Whole cell recording was performed and analyzed as described previously (3). Briefly, current was measured using an Axopatch 200-B amplifier (Axon Instruments). Signals were filtered with a cutoff frequency of 5 kHz. Data acquisition was controlled using commercially available pCLAMP 6.0.3 software (Axon Instruments), and data were digitized using a Digidata 1200 interface (Axon Instruments). Iberiotoxin (100200 nM; Sigma) was used to confirm the presence of maxi-K channel current. Membrane area was estimated on the basis of integrating capacitive currents generated by a 5-mV pulse after cancelation of the patch-pipette capacitance. Currents were measured using a holding potential of 80 mV and prepulsing to 100 mV, and they were elicited at step potentials from 80 to +120 in 20-mV intervals. The bath solution contained (in mM) 135 NaCl, 4.7 KCl, 1 MgCl2, 10 glucose, 2 CaCl2, and 5 HEPES (pH 7.4). The pipette solution contained (in mM) 140 KCl, 0.5 MgCl2, 1 EGTA, 5 ATP, and 5 HEPES (pH 7.2). Mean sustained K+ current amplitudes were calculated using the Clampfit 6.0.4 software program (Axon Instruments) and plotted as pA/pF to normalize for differences in cell size. Results are plotted as means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Maxi-K channels are part of a macromolecular complex containing both caveolin and actin on the cell surface in myometrial cells. Recent evidence has demonstrated that different K+ channel isoforms can localize to distinct raft populations (28). While caveolae share many of the biochemical properties of noncaveolar raft domains, isolation of detergent-resistant membrane fractions does not discriminate between distinct cell surface microdomains. To determine whether the maxi-K channel and caveolin isoforms are localized to the same membrane area, immunocytochemistry was performed on cultured LP human myometrial cells using maxi-K channel (Fig. 2A, green) and caveolin-1 antibodies (Fig. 2A, red). Maxi-K channels are localized primarily on the plasma membrane, whereas caveolin is localized to intracellular and plasma membrane compartments. The proteins colocalize primarily on the MSMC membrane as indicated by the signal overlap in the merged figure (Fig. 2A, yellow) and the corresponding area in the differential interference contrast image (DIC). Control studies using primary or secondary antibodies alone did not produce fluorescent signals.
|
To identify potential proteins associated with the maxi-K channel-caveolin complex during pregnancy, additional immunoprecipitation experiments were performed with LP human myometrial protein using an antibody against the maxi-K channel. This was separated using SDS-PAGE and stained with Coomassie blue (Fig. 3A). A prominent 43-kDa band was immunoprecipitated with the maxi-K channel antibody. This band was identified using matrix-assisted laser desorption time-of-flight mass spectrometry in the LP sample as a mixture of
- and
-actin. To elucidate the nature of the interaction between actin and the maxi-K channel, actin spin-down assays were performed using previously described methods (15). NP and LP human uterine cytosol (1.5 mg) were incubated with 20 nM phalloidin, 2 mM ATP, and 100 µM nocodozole for 30 min at RT and spun through a sucrose cushion. Pellets were then immunoblotted for the maxi-K channel as well as for
- and
-actin. The interaction between the maxi-K channel and actin was more prevalent in NP than in LP myometrium. In addition, although similar amounts of
-actin were spun down at these two phases of pregnancy,
-actin was more predominant at LP stages, similar to what has been reported previously in the literature (7). Positive control experiments using the actin-binding protein filamin indicate that actin is able to pellet associated proteins. Negative controls using
-tubulin (which would be disrupted by nocodazole) were not pelleted after actin polymerization. These experiments demonstrate that the maxi-K channel is associated with actin in both NP and LP human myometrium. Even though the interactions between actin and the maxi-K channel are stronger in NP than in LP myometrium, the availability of fresh human uterine tissue from LP samples was greater; thus characterization of these macromolecular interactions was performed in LP hMSMCs.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of caveolae in converging multiple signaling processes has been described in various tissues (36). To date, little is known about the role of caveolae in the uterus, despite morphological data demonstrating an abundance of caveolae in myometrial smooth muscle (7). Although quantitative studies indicate that the number of caveolae do not differ between NP, term-pregnant (i.e., LP), and laboring human myometrium (8), the levels of caveolin-1 and -2 proteins do change. Each appears to demonstrate low expression during the first part of the pregnancy and then gradually increases in expression until the day of delivery in the pregnant rat (40). Although these results appear contrary, caveolins directly bind signaling molecules, and the dynamic nature of these interactions could elicit changes in myometrial excitability without altering the abundance of caveolae. Both caveolin-1 and caveolin-2 are suppressed by estrogen (40), indicating that hormonal status regulates caveolar proteins and perhaps associated signaling processes during pregnancy.
Our data, which complement the caveolin-binding motif in the channel, suggest a direct association between the maxi-K channel and caveolin-1 and -2 in human myometrium in both NP and LP states. Although caveolin-3 is found in vascular smooth muscle (12), our studies and others have not detected this isoform in great abundance in uterine tissue (18). In addition, we found only a weak association between the maxi-K channel and caveolin-3. While our data suggest a role for a channel-caveolin-actin complex in the regulation of maxi-K current in the uterus, the complexity and physiological role of channel-caveolin interactions require additional study. It is tempting to hypothesize that this interaction is important for channel transport. Caveolae could be involved in the trafficking of the maxi-K channel to the plasma membrane, with caveolin acting as a tether helping to anchor the channel to a caveolar vesicle. It is interesting that, using immunoelectron microscopy, we could detect electron-dense particles corresponding to the maxi-K channel in unfused caveolae beneath the cell surface (Fig. 5B).
Maxi-K channels contain a putative caveolin-binding motif in the COOH terminus (9). Recent studies have demonstrated loss of membrane expression in maxi-K channels lacking this region, perhaps implicating this region in transport (42). Caveolae are also thought to be able to open and close when on the membrane (32), and perhaps localization of the maxi-K channel to cell surface caveolae allows cells to dynamically regulate their electrical excitability by the opening and closing of the caveolar neck. As shown in ureter smooth muscle, disruption of lipid rafts, which contain maxi-K channels, alters phasic contraction (2). However, it is unlikely that all maxi-K channels are localized to caveolar microdomains; there may be a basal amount on the cell surface, with additional channels added as needed to buffer cell excitation. Therefore, caveolae could open and expose more channels to the membrane, buffering cell excitability and thereby keeping the cells relaxed, and may thus explain partially the increase in current density observed after caveolar disruption (Fig. 7A). In contrast, caveolae could close off from the membrane, engulfing channels with them to promote conditions required for uterine contraction.
The maxi-K channel plays a role in relaxing the uterus by keeping it in a quiescent state throughout pregnancy; however, its role at the onset of labor is unknown. Studies have shown a switch in channel phenotype at the onset of labor (23, 24) from a typical voltage and Ca2+-sensitive maxi-K current to one that is constitutively active, lacking sensitivity to these factors. While this may represent a novel channel type, it may also represent differential regulation of the maxi-K channel. There is precedent for this channel to be regulated differently by PKA during pregnancy (34), which is likely due in part to alternative splicing that results in expression of different maxi-K channel isoforms. Although our studies were performed with LP nonlaboring human myometrium and primary cultured myometrial cells from this stage, our results suggest that changes in channel macromolecular complexes, including caveolin, may lead to dynamic regulation of proteins associated with the maxi-K channel. The culturing of myometrial cells is an experimental limitation because phenotypic changes can occur. However, results obtained using these cells are in agreement with data collected using myometrial tissue and suggest that changes in channel-containing macromolecular complexes may regulate channel activity and thereby contribute to changes in the current phenotype observed at the onset of labor.
Our data demonstrate an interaction between the maxi-K channel and actin that may be regulated in part by caveolae (Fig. 7B). Caveolae are thought to be membrane-organizing centers that recruit lipids and proteins for participation in intracellular trafficking and signal transduction (25). As functionally noted, disruption of the actin cytoskeleton could elicit a series of events that lead directly to channel activation and/or release of an inhibitory factor. In either case, disruption of the actin cytoskeleton in cultured LP hMSMCs produces a phenotype similar to that observed during labor. It is important to note that on the basis of our biochemical fraction data (Fig. 1A), not all maxi-K channels are associated with rafts and/or caveolin. The actin pull-down assays performed in the present study cannot discriminate raft- and non-raft-associated channels. Although our immunolocalization data suggest that the three proteins colocalize on the cell surface, it is possible that non-raft- as opposed to raft-associated maxi-K channels associate with actin.
Remodeling of the actin cytoskeleton in the myometrium has not been examined during pregnancy; however, recent reports have indicated that localized mechanical stress can induce actin remodeling and stiffening in smooth muscle cells (11). Whether this occurs in the myometrium during labor and how this may affect electrical activity are interesting points to consider. Actin has been implicated in the spatial organization of caveolae on the plasma membrane (39). This finding, coupled with our data showing the proximity of the maxi-K channel to actin within the caveolae, may indicate that their compartmentation is one mechanism by which to regulate uterine function. While the link between the maxi-K channel, caveolin, and actin is not known, it may involve additional actin-binding proteins (i.e., filamin) or a lipid intermediate. Nevertheless, understanding this interaction will be an important step in identifying other mechanisms that may regulate K+ channel activity.
The increase in the rate of premature births (43) and the need for more effective tocolytic therapy have prompted new studies to understand the proteins involved in this process. With much to elucidate about the regulation of the maxi-K channel during pregnancy and at the onset of labor, these data are the first to show that maxi-K channels target to caveolar membrane microdomains, associate with caveolin, and interact with the actin cytoskeleton in hMSMCs.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Babiychuk EB, Smith RD, Burdyga T, Babiychuk VS, Wray S, and Draeger A. Membrane cholesterol regulates smooth muscle phasic contraction. J Membr Biol 198: 95101, 2004.[ISI][Medline]
3. Benkusky NA, Fergus DJ, Zucchero TM, and England SK. Regulation of the Ca2+-sensitive domains of the maxi-K channel in the mouse myometrium during gestation. J Biol Chem 275: 2771227719, 2000.
4. Brady JD, Rich TC, Le X, Stafford K, Fowler CJ, Lynch L, Karpen JW, Brown RL, and Martens JR. Functional role of lipid raft microdomains in cyclic nucleotide-gated channel activation. Mol Pharmacol 65: 503511, 2004.
5. Bravo-Zehnder M, Orio P, Norambuena A, Wallner M, Meera P, Toro L, Latorre R, and González A. Apical sorting of a voltage- and Ca2+-activated K+ channel -subunit in Madin-Darby canine kidney cells is independent of N-glycosylation. Proc Natl Acad Sci USA 97: 1311413119, 2000.
6. Brown DA and Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68: 533544, 1992.[CrossRef][ISI][Medline]
7. Cavaille F. The contractile proteins of the human myometrium. Acta Physiol Hung 65: 453460, 1985.[ISI][Medline]
8. Ciray HN, Guner H, Hakansson H, Tekelioglu M, Roomans GM, and Ulmsten U. Morphometric analysis of gap junctions in nonpregnant and term pregnant human myometrium. Acta Obstet Gynecol Scand 74: 497504, 1995.[ISI][Medline]
9. Couet J, Li S, Okamoto T, Ikezu T, and Lisanti MP. Identification of peptide and protein ligands for the caveolin-scaffolding domain: implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272: 65256533, 1997.
10. Darby PJ, Kwan CY, and Daniel EE. Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca2+ handling. Am J Physiol Lung Cell Mol Physiol 279: L1226L1235, 2000.
11. Deng L, Fairbank NJ, Fabry B, Smith PG, and Maksym GN. Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells. Am J Physiol Cell Physiol 287: C440C448, 2004.
12. Doyle DD, Upshaw-Earley J, Bell E, and Palfrey HC. Expression of caveolin-3 in rat aortic vascular smooth muscle cells is determined by developmental state. Biochem Biophys Res Commun 304: 2225, 2003.[CrossRef][ISI][Medline]
13. Ehrhardt AG, Frankish N, and Isenberg G. A large-conductance K+ channel that is inhibited by the cytoskeleton in the smooth muscle cell line DDT1 MF-2. J Physiol 496: 663676, 1996.[Abstract]
14. El Hadri K, Moldes M, Mercier N, Andreani M, Pairault J, and Feve B. Semicarbazide-sensitive amine oxidase in vascular smooth muscle cells: differentiation-dependent expression and role in glucose uptake. Arterioscler Thromb Vasc Biol 22: 8994, 2002.
15. Fucini RV, Navarrete A, Vadakkan C, Lacomis L, Erdjument-Bromage H, Tempst P, and Stamnes M. Activated ADP-ribosylation factor assembles distinct pools of actin on Golgi membranes. J Biol Chem 275: 1884218849, 2000.
16. Gabella G. Structure of smooth muscles. In: Smooth Muscle: An Assessment of Current Knowledge, edited by Bulbring E, Brading AF, Jones AW, and Tomita T. London: Arnold, 1981, p. 146.
17. Gokina NI and Osol G. Actin cytoskeletal modulation of pressure-induced depolarization and Ca2+ influx in cerebral arteries. Am J Physiol Heart Circ Physiol 282: H1410H1420, 2002.
18. Hagiwara Y, Nishina Y, Yorifuji H, and Kikuchi T. Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal, cardiac and uterine smooth muscles. Cell Struct Funct 27: 785794, 2002.
19. Harder T, Scheiffele P, Verkade P, and Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 141: 929942, 1998.
20. Huang H, Rao Y, Sun P, and Gong LW. Involvement of actin cytoskeleton in modulation of Ca2+-activated K+ channels from rat hippocampal CA1 pyramidal neurons. Neurosci Lett 332: 141145, 2002.[CrossRef][ISI][Medline]
21. Jackson WF. Ion channels and vascular tone. Hypertension 35: 173178, 2000.
22. Kao CY. Electrophysiological properties of uterine muscle. In: Biology of the Uterus (2nd ed.), edited by Wynn RM and Jollie WP. New York: Plenum, 1989, p. 403453.
23. Khan RN, Smith SK, Morrison JJ, and Ashford ML. Properties of large-conductance K+ channels in human myometrium during pregnancy and labour. Proc R Soc Lond B Biol Sci 251: 915, 1993.[ISI][Medline]
24. Khan RN, Smith SK, Morrison JJ, and Ashford MLJ. Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes. Am J Physiol Cell Physiol 273: C1721C1731, 1997.
25. Liu P, Rudick M, and Anderson RG. Multiple functions of caveolin-1. J Biol Chem 277: 4129541298, 2002.
26. Löhn M, Fürstenau M, Sagach V, Elger M, Schulze W, Luft FC, Haller H, and Gollasch M. Ignition of calcium sparks in arterial and cardiac muscle through caveolae. Circ Res 87: 10341039, 2000.
27. Marshall JM. Relation between membrane potential and spontaneous contraction of the uterus. In: Uterine Contractility: Mechanisms of Control, edited by Garfield RE. Norwell, MA: Sereno Symposia, 1990, p. 37.
28. Martens JR, O'Connell K, and Tamkun M. Targeting of ion channels to membrane microdomains: localization of KV channels to lipid rafts. Trends Pharmacol Sci 25: 1621, 2004.[CrossRef][ISI][Medline]
29. Matharoo-Ball B, Ashford ML, Arulkumaran S, and Khan RN. Down-regulation of the and
-subunits of the calcium-activate potassium channel in human myometrium during parturition. Biol Reprod 68: 21352141, 2003.
30. Mundy DI, Machleidt T, Ying Y, Anderson RGW, and Bloom GS. Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton. J Cell Sci 115: 43274339, 2002.
31. Nakashima S. Protein kinase C (PKC
): regulation and biological function. J Biochem (Tokyo) 132: 669675, 2002.[Abstract]
32. Oh P, McIntosh DP, and 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 141: 101114, 1998.
33. Parkington HC and Coleman HA. The role of membrane potential in the control of uterine motility. In: Uterine Function: Molecular and Cellular Aspects, edited by Carston ME and Miller JD. New York: Plenum, 1990, p. 195248.
34. Pérez G and Toro L. Differential modulation of large-conductance KCa channels by PKA in pregnant and nonpregnant myometrium. Am J Physiol Cell Physiol 266: C1459C1463, 1994.
35. Petrecca K, Miller DM, and Shrier A. Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin-binding protein filamin. J Neurosci 20: 87368744, 2000.
36. Razani B, Woodman SE, and Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431467, 2002.
37. Sampson LJ, Leyland ML, and Dart C. Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1. J Biol Chem 278: 19881997, 2003.
38. Shogomori H and Brown DA. Use of detergents to study membrane rafts: the good, the bad and the ugly. Biol Chem 384: 12591263, 2003.[CrossRef][ISI][Medline]
39. Stahlhut M and van Deurs B. Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton. Mol Biol Cell 11: 325337, 2000.
40. Turi A, Kiss AL, and Müllner N. Estrogen downregulates the number of caveolae and the level of caveolin in uterine smooth muscle. Cell Biol Int 25: 785794, 2001.[CrossRef][ISI][Medline]
41. Van Deurs B, Roepstorff K, Hommelgaard AM, and Sandvig K. Caveolae: anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol 13: 92100, 2003.[CrossRef][ISI][Medline]
42. Wang SX, Ikeda M, and Guggino WB. The cytoplasmic tail of large conductance, voltage- and Ca2+-activated K+ (maxiK) channel is necessary for its cell surface expression. J Biol Chem 278: 27132722, 2003.
43. Wickelgren I. Premature labor: resetting pregnancy's clock. Science 304: 666668, 2004.
44. Williams TM and Lisanti MP. The caveolin proteins. Genome Biol 5: 214, 2004.[CrossRef][Medline]
45. Wray S. Uterine contraction and physiological mechanisms of modulation. Am J Physiol Cell Physiol 264: C1C18, 1993.
46. Zhou XB, Arntz C, Kamm S, Motejlek K, Sausbier U, Wang GX, Ruth P, and Korth M. A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase. J Biol Chem 276: 4323943245, 2001.
47. Zhou XB, Wang GX, Hüneke B, Wieland T, and Korth M. Pregnancy switches adrenergic signal transduction in rat and human uterine myocytes as probed by BKCa channel activity. J Physiol 524: 339352, 2000.