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• W2009959265 abstract "The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K+channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K+ (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K+ channel function. The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K+channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K+ (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K+ channel function. voltage-gated K+ postsynaptic density-95, discs large, zonula occludens 4-morpholineethanesulfonic acid Ion channel regulation within neuronal and muscle membranes is an important determinant of electrical excitability in the nervous and cardiovascular systems, skeletal muscle, gastrointestinal tract, and uterus. Voltage-gated K+(Kv)1 channels play an important role in setting the resting membrane potential and determining repolarization in these functionally diverse systems. In addition, Kv channels are involved in the regulation of membrane potential in both T-lymphocytes and pancreatic β cells (1Roe M.W. Worley III, J.F. Mittal A.A. Kuznetsov A. DasGupta S. Mertz R.J. Witherspoon III, S.M. Blair N. Lancaster M.E. McIntyre M.S. Shehee W.R. Dukes I.D. Philipson L.H. J. Biol. Chem. 1996; 271: 32241-32246Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 2Cahalan M.D. Chandy K.G. Curr. Opin. Biotechnol. 1997; 8: 749-756Crossref PubMed Scopus (140) Google Scholar). Given their diverse role, it is not surprising that Kv channels comprise the largest and most diverse class of ion channels. Multiple Kv channel gene families (Kv1–Kv9) have been identified that give rise to more than 40 mammalian isoforms. Most tissues, and even single cells, express multiple channel types belonging to one or more subfamilies. Importantly, the subcellular localization of these different channel isoforms is often necessary for proper cell function and signaling. Although progress has been made in identifying elements involved in channel targeting, clustering, and anchoring, it is not yet clear how the number and location of channel complexes within the plane of the membrane are determined or how this compartmentalization affects channel function. Historically, K+ channel targeting and localization is believed to primarily involve protein-protein interactions between channels and PDZ domain containing scaffolding proteins or the actin cytoskeleton (3Sheng M. Kim E. Curr. Opin. Neurobiol. 1996; 6: 602-608Crossref PubMed Scopus (100) Google Scholar, 4Johnson B.D. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 203-228Crossref PubMed Google Scholar). Recently, we reported that Kv channels differentially target to specialized microdomains, termed lipid rafts, within the plane of the plasma membrane, suggesting that protein-lipid interactions should be considered as a mechanism of Kv channel localization (5Martens J.R. Navarro-Polanco R. Coppock E.A. Nishiyama A. Parshley L. Grobaski T.D. Tamkun M.M. J. Biol. Chem. 2000; 275: 7443-7446Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The term “lipid raft” is used to describe membrane microdomains rich in tightly packed cholesterol and sphingolipids. Two defining characteristics of these lipid microdomains are resistance to detergent solubilization and a low buoyant density (6Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2610) Google Scholar). Biochemical isolation of raft domains has demonstrated that certain proteins target to these subcellular compartments, whereas others are excluded. The presence or absence of proteins is used to define different types of lipid rafts (7Roper K. Corbeil D. Huttner W.B. Nat. Cell Biol. 2000; 2: 582-592Crossref PubMed Scopus (483) Google Scholar). Caveolae represent one well studied subpopulation of lipid rafts that contain the scaffolding protein, caveolin. Caveolin can bind, organize, and sometimes functionally regulate proteins within the lipid raft complex (8Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1725) Google Scholar, 9Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). The emerging picture of the coexistence of multiple raft populations, containing diverse proteins or different ratios of similar proteins, within a single membrane provides a unique mechanism of functional regulation based on spatial organization. Here, we demonstrate isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. The polyclonal anti-Kv2.1 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-caveolin polyclonal, which recognizes caveolin isoforms 1, 2, and 3, was obtained from Transduction Laboratories (Lexington, KY). Rabbit antiserum against the NH2-terminal region of Kv1.5 was produced in the Tamkun laboratory using a glutathione transferase fusion protein containing 112 amino acids of the NH2-terminal Kv1.5 channel sequence (10Mays D.J. Foose J.M. Philipson L.H. Tamkun M.M. J. Clin. Invest. 1995; 96: 282-292Crossref PubMed Scopus (192) Google Scholar). Antiserum against the S1-S2 epitope of Kv1.5 was produced in the Philipson laboratory using a synthetic peptide, LLRHPPAPHQPPAPAPGANGSGVMA, as previously described (11Philipson L.H. Malayev A. Kuznetsov A. Chang C. Nelson D.J. Biochim. Biophys. Acta. 1993; 1153: 111-121Crossref PubMed Scopus (33) Google Scholar). Fumonisin B was purchased from Sigma. Low density, Triton-insoluble complexes were isolated as described by Lisanti and co-workers (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar) from mouse L-cells stably expressing either rat Kv1.5 or Kv2.1 channels under the control of a dexamethasone-inducible promoter (13Hulme J.T. Coppock E.A. Felipe A. Martens J.R. Tamkun M.M. Circ. Res. 1999; 85: 489-497Crossref PubMed Scopus (152) Google Scholar, 14Snyders D.J. Knoth K.M. Roberds S.L. Tamkun M.M. Mol. Pharm. 1992; 41: 322-330PubMed Google Scholar). Briefly, cells from ten 100-mm near-confluent culture dishes were homogenized in 1 ml of 1% Triton X-100, and sucrose was added to a final concentration of 40%. A 5–30% linear sucrose gradient was layered on top of this detergent extract followed by ultracentrifugation (39,000 rpm) for 18–20 h at 4 °C in a Beckman SW41 rotor. Gradient fractions (600 μl) were collected from the top and analyzed by Western blot. Sucrose gradient fractions were fractionated on a 10% polyacrylamide SDS gel. After electrophoretic transfer to nitrocellulose, the membrane was incubated with the Kv1.5, Kv2.1, or caveolin antibodies (1:500, 1:500, and 1:1000 dilutions, respectively). Bound primary antibody was detected with a 1:3,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG and the Renaissance Western blot chemiluminescence reagent according to manufacturer's protocol (PerkinElmer Life Sciences). Immune purification of caveolae was performed as previously described with a few modifications (15Badizadegan K. Dickinson B.L. Wheeler H.E. Blumberg R.S. Holmes R.K. Lencer W.I. Am. J. Physiol. 2000; 278: G895-G904Crossref PubMed Google Scholar). Briefly, Triton X-100 was used to solubilize Kv1.5-containing rafts from stably expressing L-cells. Following sucrose gradient sedimentation, floating membranes were collected and pooled. Sample was diluted with Mes-buffered saline (25 mm Mes, pH 6.5, 0.15m NaCl) containing 0.05% Triton X-100 and precleared with 25 μl of protein A-Sepharose beads (Sigma) for 2 h at 4 °C with gentle mixing. The beads were then removed by centrifugation at 200 × g for 2 min at 4 °C. The sample was then incubated overnight with anti-caveolin antibodies (5 ng/ml) at 4 °C with gently mixing. 25 μl of protein A-Sepharose were then added to each sample for 2 h at 4 °C. The beads were then removed by centrifugation at 200 × g for 2 min at 4 °C, washed three times in phosphate-buffered saline (pH 7.5), and resuspended in 50 μl of SDS sample buffer. Immunostaining of cells was performed using Kv1.5 antiserum and/or anti-caveolin antibodies as described previously (10Mays D.J. Foose J.M. Philipson L.H. Tamkun M.M. J. Clin. Invest. 1995; 96: 282-292Crossref PubMed Scopus (192) Google Scholar). Briefly, cells grown on gelatin-coated coverslips were fixed with methanol and incubated with primary antibody followed by biotin-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA). The bound Kv1.5 antibody was detected with BODIPY-conjugated streptavidin, and the anti-caveolin antibody was detected with CY3-conjugated streptavidin. Fluorescent signals were collected using a Nikon E800 microscope equipped with standard epifluorescence and a Princeton Instruments CCD camera. Demecolcine (0.1 μg/ml; Sigma) was dissolved in the culture medium. For antibody-induced patching experiments, gelatin-coated coverslips with nonfixed cells were incubated in their culture dishes with the S1-S2 Kv1.5 antiserum (diluted 1:1000 in HEPES-based culture medium) for 1 h at room temperature. The cells were taken through three 2-min washes in phosphate-buffered saline and then incubated for 1.5 h with biotin-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500 in culture medium. Another series of washes with phosphate-buffered saline was followed by incubation for 30 min with CY3-conjugated streptavidin (Jackson ImmunoResearch Laboratories) diluted 1:500 in culture medium. Following three 2-min washes in phosphate-buffered saline, cells were fixed with 4% paraformaldehyde for 5 min and stained with monoclonal caveolin-1 antibodies. A BODIPY-conjugated goat anti-mouse IgG was used for visualization. Electrophysiological recordings and data analysis were made as described previously using the whole cell configuration of the patch clamp technique (13Hulme J.T. Coppock E.A. Felipe A. Martens J.R. Tamkun M.M. Circ. Res. 1999; 85: 489-497Crossref PubMed Scopus (152) Google Scholar). The steady-state inactivation was measured using a 10-s conditioning pulse to various potentials followed by 250-ms test steps to +50 mV. Because Kv1.5 shows incomplete inactivation, data were normalized after subtraction of the non-inactivating fraction at the test potential (16Snyders D.J. Tamkun M.M. Bennett P.B. J. Gen. Physiol. 1993; 101: 513-543Crossref PubMed Scopus (237) Google Scholar). Additional details are presented in the figure legends. A defining characteristic of lipid rafts is their resistance to solubilization in nonionic detergents and relative buoyancy because of an enriched lipid content (6Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2610) Google Scholar). We isolated low density, Triton X-100-insoluble complexes from mouse Ltk− cells (L-cells) stably expressing Kv1.5 channel protein. Western blot analysis of sucrose gradient fractions probed with anti-Kv1.5 antibodies demonstrates that Kv1.5 floats in a low density, 1% Triton-insoluble fraction together with caveolin (Fig.1 A). As previously reported, this is also true for L-cells stably expressing Kv2.1, a member of theShab family of voltage-gated K+ channels (5Martens J.R. Navarro-Polanco R. Coppock E.A. Nishiyama A. Parshley L. Grobaski T.D. Tamkun M.M. J. Biol. Chem. 2000; 275: 7443-7446Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). To facilitate comparison, these data have been reproduced in thelower two panels of Fig. 1 A. Although cells stably expressed channel protein, the extent of cell surface expression varied between individual cells. In the case of the Kv1.5 cell line, channel expression was often very high and resulted in the intracellular accumulation of protein (Fig. 1 B, left panel). The percentage of channel in the nonraft fractions at the bottom of the gradient most likely represents overexpressed intracellular protein. The percentage of nonraft channel varied between clonal cell lines and induction times and correlates with the amount of intracellular protein as determined by immunostaining (Fig.1 B, top panels). However, cell surface channel expression was easily detectable, and nanoampere currents could be recorded from both the Kv1.5- and Kv2.1-expressing cell lines (Fig.1 B, bottom panels). The localization of Kv1.5 and caveolin to light membrane fractions was also confirmed using a detergent-free method of raft isolation (data not shown and Ref.17Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). The detergent-to-protein ratio affects recovery of raft-localized proteins (18Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and has been exploited to describe the nature of raft association (19Parkin E.T. Hussain I. Turner A.J. Hooper N.M. J. Neurochem. 1997; 69: 2179-2188Crossref PubMed Scopus (36) Google Scholar). We examined the effect of increasing Triton X-100 concentrations on Kv1.5 and Kv2.1 raft association. As shown in Fig.1 C, Kv1.5 remains raft-associated together with caveolin following extraction with 2.5% Triton X-100. Even though the signals were detected in a greater number of floating fractions, the two protein peaks were symmetrical within the sucrose gradient. In contrast, the Kv2.1 channel protein is partially solubilized by the increased detergent concentration and no longer cofractionates with caveolin (Fig. 1, compare A and C). The differential sensitivity to detergent extraction suggests that the two proteins may exist in different raft populations. We recently reported that Kv2.1 associates with a noncaveolar lipid raft (5Martens J.R. Navarro-Polanco R. Coppock E.A. Nishiyama A. Parshley L. Grobaski T.D. Tamkun M.M. J. Biol. Chem. 2000; 275: 7443-7446Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). In that report, it was shown that Kv2.1 channel protein does not internalize with caveolin after microtubule disruption. In Fig. 2, we show that Kv1.5 channel protein redistributes together with caveolin after treatment with colcemide (100 ng/ml). Fig. 2 C shows a DIC image of morphologically altered L-cells following disruption of the microtubules. In these cells, the Kv1.5 was retracted from the cell surface (Fig. 2 D). This was also true for the caveolin protein (Fig. 2 H) in which colcemide treatment disrupted the normal cell surface distribution and resulted in internalization of the protein. Although this experiment suggests an association of Kv1.5 and caveolae, it may simply reflect a dependence of both proteins on microtubule organization. Therefore, additional experiments were performed to demonstrate association. Overexpression of channel protein often results in a uniform distribution of cell surface protein, which is often difficult to resolve with conventional microscopic techniques. To improve resolution, we performed antibody-induced patching experiments using an antibody directed against an extracellular epitope of the Kv1.5 channel. L-cells stably expressing Kv1.5 channel protein were incubated at room temperature with polyclonal anti-Kv1.5 antibody directed against the S1-S2 linker region for 1 h. Cross-linking was performed using biotin-conjugated goat anti-rabbit secondary antibody and detected with fluorophor-conjugated streptavidin. Punctate cell surface patches of Kv1.5 protein were observed (Fig.3 B). The cells were then fixed and immunostained with monoclonal anti-caveolin antibodies (Fig.3 C). An overlay of the two images (Fig. 3 D) shows complete overlap of Kv1.5 and caveolin. The enlarged area displayed in Fig. 3 E shows the extent of colocalization of the two proteins (yellow). Immunoprecipitation experiments using membranes from stably transfected L-cells solubilized with n-octyl-glucoside failed to demonstrate direct protein-protein interaction between Kv1.5 and caveolin (data not shown). This is not surprising because Kv1.5 lacks a caveolin-binding motif (20Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). Therefore, we performed immunoprecipitations under nonsolubulizing conditions (see “Experimental Procedures”) with anti-caveolin antibodies as described by other investigators (21Stan R.V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (176) Google Scholar, 22Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 23Oh P. Schnitzer J.E. J. Biol. Chem. 1999; 274: 23144-23154Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Raft-containing gradient fractions from L-cells stably expressing Kv1.5 protein were pooled and incubated with polyclonal anti-caveolin antibodies. Protein A-Sepharose beads were then used to isolate caveolar rafts. After extensive washing, bound material was eluted with SDS sample buffer. Samples were then analyzed for Kv1.5 content via Western blot analysis. Western analysis of membranes probed with Kv1.5 antiserum from nontransfected and stably transfected L-cells (Fig. 4,lanes 1 and 2) were used as controls for the Kv1.5 signal. Lane 3 of Fig. 4 shows the presence of Kv1.5 in the starting raft fraction from which immunoisolation was performed, and lane 4 shows Kv1.5 immunoprecipitation with the caveolin antibody. The arrow points to bands representing Kv1.5. Lipid rafts containing Kv1.5 did not nonspecifically bind to the beads as indicated in lane 5, where the anti-caveolin antibody was omitted. In addition, the caveolin antibodies specifically detected raft-associated caveolin protein (data not shown). Note the absence of the high molecular weight band in the starting material. This was characteristic of all experiments performed and suggests that post-translational processing of the channel protein in the fibroblasts affects caveolae association. These results suggest that although there does not exist a direct protein-protein interaction between Kv1.5 and caveolin, the channel protein does immunoisolate with protein-lipid complexes that contain caveolin. This result raises the issue initially put forth by Johnson (4Johnson B.D. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 203-228Crossref PubMed Google Scholar) and confirmed here experimentally that what are now considered direct protein interactions should be assumed to include the possibility of a lipid intermediary where direct binding has not been clearly demonstrated. The structure of lipid rafts is dependent on the tight packing of cholesterol and sphingolipids (24Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar). Agents that bind and/or extract raft lipids have been shown to alter the function of raft-associated proteins (24Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar). We have shown that depletion of cellular cholesterol modifies noncaveolar, raft-associated Kv2.1 channel function but does not affect Kv4.2, which is not localized to lipid rafts (5Martens J.R. Navarro-Polanco R. Coppock E.A. Nishiyama A. Parshley L. Grobaski T.D. Tamkun M.M. J. Biol. Chem. 2000; 275: 7443-7446Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Therefore, we tested whether disruption of raft lipids affects caveolar-associated Kv1.5 function. Treatment of cells stably expressing Kv1.5 with 2% 2-hydroxypropyl-β-cyclodextrin, a cholesterol-modifying agent, for 2 h altered the steady-state activation and inactivation properties of Kv1.5 (Fig. 5). Although current density was not affected, the activation and inactivation curves were shifted in the hyperpolarizing direction. TheV 12for activation (−17.8 ± 1.3 mV,k = 11.4 ± 1.1) and inactivation (−27.8 ± 0.8 mV, k = −9.1 ± 0.7) under control conditions were shifted by ∼10 mV (−27.8 ± 1.1 mV, k = 8.0 ± 0.9, and 37.2 ± 0.5 mV, k = −6.0 ± 0.4, respectively) following cyclodextrin treatment. Treatment with fumonisin B, which inhibits steps in the biosynthetic pathway of sphingomyelin, has been used to deplete sphingolipids (25Ledesma M.D. Simons K. Dotti C.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar). Treatment of cells for 72 h with 25 μm fumonisin B produced results similar to those observed with cholesterol depletion (Fig. 5). The V 12for steady-state activation was shifted from −16.2 ± 1.3 mV, k = 11.9 ± 1.1 to −26.9 ± 1.1 mV, k = 8.7 ± 1.0 after treatment with fumonisin B. Similarly, the steady-state inactivation curve was also shifted in the hyperpolarizing direction fromV 12equal to −28.3 ± 0.7 mV,k = −8.8 ± 0.6 to −40.4 ± 0.4 mV,k = −5.4 ± 0.3 with depletion of sphingolipids. Acute application of either cyclodextrin or fumonisin B in the bath solution did not affect channel function. In addition, neither intervention affected the properties of Kv4.2, a nonraft associated channel (data not shown). These data show that altering raft structure affects the function of caveolae-associated Kv channels. A role for the Kv channel COOH terminus in functional cell surface expression has been suggested (26Bentley G.N. Brooks M.A. O'Neill C.A. Findlay J.B. Biochim. Biophys. Acta. 1999; 1418: 176-184Crossref PubMed Scopus (17) Google Scholar, 27Li D. Takimoto K. Levitan E.S. J. Biol. Chem. 2000; 275: 11597-11602Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Recently, a COOH-terminal motif that influences processing and cell surface expression of Kv1.5 was identified at residues 593–598 (27Li D. Takimoto K. Levitan E.S. J. Biol. Chem. 2000; 275: 11597-11602Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). We tested the importance of the Kv1.5 COOH terminus in lipid raft association using a deletion mutant in which the last 57 COOH-terminal amino acids were removed. Thus, this mutant lacks both the PDZ binding motif and the motif at residues 593–596. Stable expression of the Δ57 Kv1.5 mutant in mouse L-cells produced nanoampere currents with wild-type kinetics and amplitude (28Uebele V.N. Yeola S.W. Snyders D.J. Tamkun M.M. FEBS Lett. 1994; 340: 104-108Crossref PubMed Scopus (16) Google Scholar). As predicted, the electrophoretic mobility of this truncation mutant (Fig.6 A, lane 1) is altered compared with wild-type Kv1.5 (Fig. 6 A, lane 2). Importantly, the COOH-terminal truncation did not affect lipid raft association (Fig. 6 B). These results suggest that the signal for Kv1.5 raft association does not reside in the COOH terminus of the channel. Previously, we demonstrated that Kv2.1, a member of theShab family of voltage-gated K+ channels, targeted to noncaveolar lipid rafts, whereas Kv4.2 is not raft associated in mouse L-cells. Here we report for the first time that a member of the mammalian Shaker family, Kv1.5, targets to caveolar rafts in this same cell type. Biochemical and immunohistochemical evidence demonstrate that Kv1.5 is localized in caveolae in the transfected fibroblasts. Kv1.5 cofractionates with caveolin in low density Triton X-100 insoluble raft fractions, but unlike Kv2.1 expressed in the same cell system, the symmetrical peak of Kv1.5 and caveolin proteins is maintained with increased stringency of membrane solubilization. In addition, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption. Finally, immunoisolation of caveolae using caveolin antibodies demonstrates that Kv1.5 channel protein copurifies with intact caveolae. Our results suggest that within a single cell type there exist mechanisms for the differential sorting of Kv channel isoforms to unique lipid microdomains. Given that the protein content of a lipid raft is currently used to distinguish between raft subtypes, it follows that protein-protein interactions may be a mechanism of isoform-specific recruiting of proteins to distinct raft populations. Caveolin, for example, is a scaffolding protein that can bind and assemble multiple signaling molecules in a large complex (9Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). One recent example is the caveolin-mediated association of Ca2+-signaling proteins and the human Trp1 channel (29Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2000; 275: 11934-11942Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). However, to the best of our knowledge, no work has demonstrated that caveolin binding is a prerequisite for lipid raft association. Regardless, the results of our immunoisolation experiments indicate that Kv1.5 is associated with intact caveolae, but we cannot demonstrate a direct protein-protein interaction between caveolin and channel protein (see “Results”). In addition, Kv1.5 sequence does not contain any known caveolin binding motifs (20Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). Therefore, it is unlikely that the caveolin protein is responsible for targeting Kv1.5 to caveolae. Much of the past work on the spatial organization of ion channels has focused on interactions between ion channels and scaffolding proteins. Great emphasis has been placed on the role of PDZ domain-containing membrane-associated guanylate kinases (MAGUK), such as PSD-95, in the targeting and localization of ion channels (30Sheng M. Neuron. 1996; 17: 575-578Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Recently, it was suggested that palmitoylated PSD-95 and PSD-93 may function as a lipid raft scaffolding protein, similar to caveolin (31El Husseini A.E. Topinka J.R. Lehrer-Graiwer J.E. Firestein B.L. Craven S.E. Aoki C. Bredt D.S. J. Biol. Chem. 2000; 275: 23904-23910Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The fact that Kv1.5 sequence contains a consensus PDZ binding domain, but the Kv2.1 channel does not, makes the possibility of channel binding to endogenous PDZ proteins an attractive candidate for the isoform-specific localization of the two channels to different lipid microdomains. However, the Kv1.5 COOH-terminal truncation shown in Fig. 6 removes the only known PDZ-binding site on the channel but does not affect lipid raft association. Therefore, PDZ proteins are not likely to be responsible for raft localization. In addition, this truncation removes the 5-amino acid COOH-terminal motif identified in Kv1 subunits that reportedly influences membrane localization (27Li D. Takimoto K. Levitan E.S. J. Biol. Chem. 2000; 275: 11597-11602Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Together, these results suggest that the mechanism of lipid raft association is independent of this amino acid sequence. The phosphorylation of ion channels is thought to be one of the key regulatory mechanisms of membrane excitability. Not surprisingly, channel complexes are basally and transiently phosphorylated/dephosphorylated by a variety of modulatory enzymes (32Huang X.Y. Morielli A.D. Peralta E.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 624-628Crossref PubMed Scopus (64) Google Scholar). Because rafts localize a number of signaling proteins such as protein kinase C, tyrosine kinases, nitrogen-oxide synthase, Ha-Ras, mitogen-activated protein kinase, and G-proteins (33Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: L843-L851PubMed Google Scholar), channel/raft association could serve primarily to cluster signaling molecules with their K+ channel substrates. The isoform-specific targeting of Kv channels to distinct lipid raft subpopulations, with unique protein compositions, would allow for differential regulation while preventing cross-talk between competing pathways. Several recent reports emphasize the importance of compartmentalization of ion channels and signaling molecules (34An R. Heath B.M. Higgins J.P. Koch W.J. Lefkowitz R.J. Kass R.S. J. Physiol. ( Lond. ). 1999; 516: 19-30Crossref PubMed Scopus (25) Google Scholar, 35Rich T.C. Fagan K.A. Nakata H. Schaack J. Cooper D.M. Karpen J.W. J. Gen. Physiol. 2000; 116: 147-161Crossref PubMed Scopus (232) Google Scholar). For example, An et al. (34An R. Heath B.M. Higgins J.P. Koch W.J. Lefkowitz R.J. Kass R.S. J. Physiol. ( Lond. ). 1999; 516: 19-30Crossref PubMed Scopus (25) Google Scholar) have reported that in developing cardiac muscle, the electrophysiological consequences of β2-adrenergic receptor-induced stimulation of cAMP is dependent on compartmentalization with L-type Ca2+ channels. Although not yet shown for the cardiac Ca2+ channel, both β2-adrenergic receptors and adenylyl cyclase localize to caveolae in cardiac myocytes (36Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Clearly, the partitioning of K+ channels and regulatory signaling elements within the same subcellular microdomains provides for efficient coupling and rapid signaling. It is possible that the functional effects of raft disruption reported here are a consequence of altered signaling,i.e. kinase/phosphatase disruption. Although fumonisin B has been used in the raft literature to deplete sphingolipids, its mechanism of action is to inhibit ceramide synthase. Lipid rafts/caveolae are believed to be highly localized sites for the initiation of ceramide signaling pathways (37Dobrowsky R.T. Cell Signal. 2000; 12: 81-90Crossref PubMed Scopus (100) Google Scholar). In fact, it has been reported that in fibroblasts ∼50% of cellular ceramide is localized in caveolae fractions (38Liu P. Anderson R.G. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Interestingly, ceramide has been shown to decrease Kv1.3 channel current in T-lymphocytes (39Gulbins E. Szabo I. Baltzer K. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7661-7666Crossref PubMed Scopus (168) Google Scholar). Therefore, it is possible that the observed functional effects on Kv1.5 are mediated through caveolae-associated ceramide signaling pathways. Furthermore, tyrosine kinase signaling cascades are also believed to initiate from lipid raft domains (37Dobrowsky R.T. Cell Signal. 2000; 12: 81-90Crossref PubMed Scopus (100) Google Scholar). Not surprisingly, Kv1.5 channel activity is modulated by tyrosine phosphorylation in heterologous expression systems and cultured Schwann cells (40Holmes T.C. Fadool D.A. Ren R.B. Levitan I.B. Science. 1996; 274: 2089-2091Crossref PubMed Scopus (231) Google Scholar, 41Sobko A. Peretz A. Attali B. EMBO J. 1998; 17: 4723-4734Crossref PubMed Scopus (75) Google Scholar). Alternatively, the functional changes illustrated in Fig. 5 could be due to direct effects of altered lipid on channel activity, for charged lipids produce a surface potential. Disruption of raft lipid via cholesterol depletion or inhibition of sphingolipid synthesis could in theory alter the surface charge and affect channel function. However, the magnitude of an effect of altered bilayer charge on potential-dependent channel parameters remains unknown. Given that the Debye length for electrostatic interactions under physiological conditions (estimated at 9 Å) is shorter than the predicted dimensions of the channel protein (42Elinder F. Liu Y. Arhem P. J. Membr. Biol. 1998; 165: 183-189Crossref PubMed Scopus (17) Google Scholar, 43Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A.L. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5747) Google Scholar), the gating charges of the channel appear to be partially insulated from surrounding bilayer charge. Therefore, the channel may not sense the full value of the surface potential in the charged lipid. According to Arhem, a pure surface charge effect predicts that thet 12(V), time to half-maximum currentversus potential, and the G(V), conductance versus potential, curves shift equally (44Elinder F. Madeja M. Arhem P. J. Gen. Physiol. 1996; 108: 325-332Crossref PubMed Scopus (43) Google Scholar). This was not the case for the effects of cyclodextrin on Kv1.5 (data not shown). Regardless of the mechanism, the effects are raft-specific, for neither cyclodextrin nor fumonisin B had functional effects on Kv4.2, which is not raft-localized. Future experiments will address the mechanism of lipid disruption on the function of raft-associated channels. In conclusion, we show for the first time localization of a voltage-dependent K+ channel, Kv1.5, to caveolar membranes. These results, together with our previous published work (5Martens J.R. Navarro-Polanco R. Coppock E.A. Nishiyama A. Parshley L. Grobaski T.D. Tamkun M.M. J. Biol. Chem. 2000; 275: 7443-7446Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), demonstrate that voltage-dependent K+channels from different families target to distinct lipid microdomains. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within the same membrane represents a mechanism of isoform-specific compartmentalization, thus allowing modulation of K+ channel function within well defined membrane microdomains. We thank Barbara Birks for technical assistance and review of this manuscript." @default.
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• W2009959265 title "Isoform-specific Localization of Voltage-gated K+Channels to Distinct Lipid Raft Populations" @default.
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