Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (α1H) T-type Ca2+ Channels

Abstract

Voltage-gated T-type Ca2+ channel Cav3.2 (α1H) subunit, responsible for T-type Ca2+ current, is expressed in different tissues and participates in Ca2+ entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Cav3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca2+ in different cells. We examined the localization and regulation of the Cav3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Cav3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Cav3.1 and Cav3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Cav3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Cav3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Cav3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (ICa,T) and adenovirus (AdCav3.2)-mediated increase in peak Cav3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of ICa,T by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Cav3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca2+ signaling during normal and pathological processes. Voltage-gated T-type Ca2+ channel Cav3.2 (α1H) subunit, responsible for T-type Ca2+ current, is expressed in different tissues and participates in Ca2+ entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Cav3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca2+ in different cells. We examined the localization and regulation of the Cav3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Cav3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Cav3.1 and Cav3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Cav3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Cav3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Cav3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (ICa,T) and adenovirus (AdCav3.2)-mediated increase in peak Cav3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of ICa,T by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Cav3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca2+ signaling during normal and pathological processes. IntroductionT-type Ca2+ channels (TTCC) 2The abbreviations used are: TTCCvoltage-gated T-type Ca2+ channelCav-3caveolin-3AdCav3.2adenovirus expressing Cav3.2 channel proteinAdGFPadenovirus expressing green fluorescent proteinTACtransthoracic aortic constrictionPKAcAMP-dependent protein kinase AHCN4hyperpolarization activated cyclic nucleotide-gated potassium channel 4ICa,TT-type calcium currentsICav3.1Cav3.1 channel calcium currentsICav3.2Cav3.2 channel calcium currents8-Br-CAMP8-bromo-cyclic AMP. are low voltage-activated Ca2+ channels, expressed in various tissues including brain and heart and contribute to a variety of physiological functions such as neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity (1Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1319) Google Scholar, 2Iftinca M.C. Zamponi G.W. Trends Pharmacol. Sci. 2009; 30: 32-40Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Huc S. Monteil A. Bidaud I. Barbara G. Chemin J. Lory P. Biochim. Biophys. Acta. 2009; 1793: 947-952Crossref PubMed Scopus (55) Google Scholar). Molecular cloning studies have identified three different TTCC isoforms, CaV3.1 (α1G), CaV3.2 (α1H), and Cav3.3 (α1I), which functionally can be distinguished by their electrophysiological properties (4Lee J.H. Daud A.N. Cribbs L.L. Lacerda A.E. Pereverzev A. Klöckner U. Schneider T. Perez-Reyes E. J. Neurosci. 1999; 19: 1912-1921Crossref PubMed Google Scholar, 5Klöckner U. Lee J.H. Cribbs L.L. Daud A. Hescheler J. Pereverzev A. Perez-Reyes E. Schneider T. Eur. J. Neurosci. 1999; 11: 4171-4178Crossref PubMed Scopus (151) Google Scholar, 6Kozlov A.S. McKenna F. Lee J.H. Cribbs L.L. Perez-Reyes E. Feltz A. Lambert R.C. Eur. J. Neurosci. 1999; 11: 4149-4158Crossref PubMed Scopus (72) Google Scholar, 7Chemin J. Monteil A. Perez-Reyes E. Bourinet E. Nargeot J. Lory P. J. Physiol. 2002; 540: 3-14Crossref PubMed Scopus (184) Google Scholar). Cav3.1 and Cav3.2 are the most commonly expressed subunits generating T-type Ca2+ current (ICa,T) in brain and heart. Cav3.2 T-type Ca2+ channels are involved in neurological disorders such as epilepsy and pain (8McGivern J.G. Drug Discov. Today. 2006; 11: 245-253Crossref PubMed Scopus (113) Google Scholar). In the heart, the ICa,T participates in Ca2+ entry and Ca2+-dependent hormonal secretion, pacemaker activity, and arrhythmia (9Vassort G. Talavera K. Alvarez J.L. Cell Calcium. 2006; 40: 205-220Crossref PubMed Scopus (100) Google Scholar, 10Ono K. Iijima T. J. Mol. Cell Cardiol. 2010; 48: 65-70Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The Cav3.1 and Cav3.2 isoforms are normally expressed in embryonic hearts (11Nuss H.B. Marban E. J. Physiol. 1994; 479: 265-279Crossref PubMed Scopus (118) Google Scholar, 12Cribbs L.L. Martin B.L. Schroder E.A. Keller B.B. Delisle B.P. Satin J. Circ. Res. 2001; 88: 403-407Crossref PubMed Scopus (86) Google Scholar), but postnatal expression of these isoforms diminishes with almost no expression in normal adult ventricular myocytes. However, the TTCCs are re-expressed during conditions of cardiac hypertrophy and heart failure and are reported to be associated with decreased cardiac function (13Nuss H.B. Houser S.R. Circ. Res. 1993; 73: 777-782Crossref PubMed Scopus (276) Google Scholar, 14Martínez M.L. Heredia M.P. Delgado C. J. Mol. Cell Cardiol. 1999; 31: 1617-1625Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 15Ferron L. Capuano V. Ruchon Y. Deroubaix E. Coulombe A. Renaud J.F. Circ. Res. 2003; 93: 1241-1248Crossref PubMed Scopus (75) Google Scholar). Ca2+ influx through the re-expressed voltage-gated Cav3.2 (α1H) TTCC is indicated to be responsible for inducing pathological cardiac hypertrophy in a pressure overload model (16Chiang C.S. Huang C.H. Chieng H. Chang Y.T. Chang D. Chen J.J. Chen Y.C. Chen Y.H. Shin H.S. Campbell K.P. Chen C.C. Circ. Res. 2009; 104: 522-530Crossref PubMed Scopus (129) Google Scholar).Although we are beginning to understand the physiological role of Cav3.2 channels in Ca2+ cycling, the precise regulation of these channels has not been defined. To clearly understand the regulation and physiological function of these proteins in normal and diseased stages it is important to define the exact subcellular localization of this protein. Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids, contain the signature scaffolding protein caveolins, and provide temporal and spatial regulation of intracellular Ca2+ in many cell types (17Insel P.A. Patel H.H. Curr. Opin. Nephrol. Hypertens. 2009; 18: 50-56Crossref PubMed Scopus (54) Google Scholar, 18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar, 19Pani B. Singh B.B. Cell Calcium. 2009; 45: 625-633Crossref PubMed Scopus (209) Google Scholar). Three different caveolin isoforms are identified, of which Cav-1 and Cav-2 are ubiquitously expressed (20Glenney Jr., J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 21Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar), whereas Cav-3 is widely expressed in muscle (cardiac, skeletal, and smooth muscle) cells (22Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar) and neuronal tissue (23Boulware M.I. Kordasiewicz H. Mermelstein P.G. J. Neurosci. 2007; 27: 9941-9950Crossref PubMed Scopus (165) Google Scholar). A number of ion channels and transporters have been localized to caveolae and associate with Cav-3, including L-type Ca2+ channels (Cav1.2) (24Balijepalli R.C. Foell J.D. Hall D.D. Hell J.W. Kamp T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7500-7505Crossref PubMed Scopus (320) Google Scholar), the Na+ channel (Nav1.5) (25Yarbrough T.L. Lu T. Lee H.C. Shibata E.F. Circ. Res. 2002; 90: 443-449Crossref PubMed Scopus (192) Google Scholar), pacemaker channels (HCN4) (26Ye B. Balijepalli R.C. Foell J.D. Kroboth S. Ye Q. Luo Y.H. Shi N.Q. Biochemistry. 2008; 47: 12312-12318Crossref PubMed Scopus (44) Google Scholar), Na+/Ca2+ exchanger (27Bossuyt J. Taylor B.E. James-Kracke M. Hale C.C. Ann. N.Y. Acad. Sci. 2002; 976: 197-204Crossref PubMed Scopus (46) Google Scholar), and others (18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar). Closely associated with these channels are specific macromolecular signaling complexes containing a variety of regulatory proteins including the G protein-coupled receptors and kinases such as PKC and PKA that provide highly localized regulation of the channels (18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar). The present study therefore was designed to determine the precise subcellular localization and regulation of the Cav3.2 subunit of the T-type Ca2+ channels. Our results demonstrate that the Cav3.2 channel protein is localized to caveolar microdomains in the ventricular myocytes and Cav-3 interacts with Cav3.2 channel and regulates its function.DISCUSSIONIn the present study we describe regulation of the Cav3.2 subunit of T-type Ca2+ channels by Cav-3 in the mouse ventricular myocytes. Double immunogold labeling and electron microscopy imaging data clearly demonstrate co-localization of the Cav3.2 channel with Cav-3 relative to caveolae in the mouse ventricular myocytes. Co-immunoprecipitation analysis suggested an association of Cav3.1 and Cav3.2 channel isoforms with Cav-3, the GST pulldown assay confirmed a close interaction between the Cav3.2 channel protein and Cav-3. Functional analysis using whole cell patch clamp in the heterologous expression system of HEK293 cells demonstrated Cav-3 inhibition of the Cav3.2 channel but not Cav3.1 channel. In addition, we show that the N terminus region of Cav-3 interacts with the Cav3.2 channel and modulates the ICav3.2. Whole cell patch clamp studies using the neonatal cardiomyocytes demonstrated specific inhibition of ICav3.2 by Cav-3. On the other hand, siRNA-mediated knockdown of Cav-3 eliminated PKA regulation of the ICa,T.T-type Ca2+ channels are known to modulate Ca2+ influx, membrane potential and hormone secretion. Of the three different T-type Ca2+ channel isoforms reported, alternative splicing of the Cav3.2 channels results in functional diversity of the channel during cardiac development (47David L.S. Garcia E. Cain S.M. Thau E.M. Tyson J.R. Snutch T.P. Channels. 2010; 4: 375-389Crossref PubMed Scopus (44) Google Scholar). Recent findings using a heterologous expression system of HEK293 cells suggest a complex regulation for the Cav3.2 channels. It is well known that G-protein-dependent signaling pathways modulate native T-type Ca2+ channels (2Iftinca M.C. Zamponi G.W. Trends Pharmacol. Sci. 2009; 30: 32-40Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Extensive studies from the laboratory of P. Berrett have demonstrated Cav3.2 channel regulation by PKA (48Hu C. Depuy S.D. Yao J. McIntire W.E. Barrett P.Q. J. Biol. Chem. 2009; 284: 7465-7473Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), Ca2+/calmodulin-dependent protein kinase II (49Wolfe J.T. Wang H. Perez-Reyes E. Barrett P.Q. J. Physiol. 2002; 538: 343-355Crossref PubMed Scopus (68) Google Scholar, 50Welsby P.J. Wang H. Wolfe J.T. Colbran R.J. Johnson M.L. Barrett P.Q. J. Neurosci. 2003; 23: 10116-10121Crossref PubMed Google Scholar), and inhibition by Gβγ (51Wolfe J.T. Wang H. Howard J. Garrison Barrett P.Q. 2003; PubMed Scopus Google Scholar, S.D. Yao J. C. McIntire Bidaud I. Lory P. F. C. Garrison Barrett P.Q. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: PubMed Scopus Google Scholar). study demonstrated a inhibition of the Cav3.2 channel by is by J. P. M.C. G. Li S. Snutch T.P. Mol. Pharmacol. 2008; 73: PubMed Scopus Google Scholar). On the other hand, a study demonstrated and protein kinase modulation of the Cav3.2 channel by in a and Gβγ A. S. U. Mol. Pharmacol. 2010; PubMed Scopus Google Scholar). In in was to Cav3.2 channels H. C. Zamponi G.W. Mol. Pharmacol. 2010; PubMed Scopus Google Scholar). The present that Cav-3 modulates transiently expressed Cav3.2 in HEK293 cells and native Cav3.2 current in neonatal mouse cardiomyocytes. of Cav-3 inhibited ICav3.2 the properties of the Cav3.2 that Cav-3 modulates Cav3.2 current in a Our data demonstrate that Cav-3 co-expression did not alter membrane expression of the Cav3.2 channels, which the of of the Cav3.2 channels. Cav-3 inhibition of ICav3.2 through different signaling associated with it is from pulldown that the N terminus region of Cav-3 interacts with the Cav3.2 channel and modulates the a of interaction between the Cav3.2 channel and Cav-3. The intracellular region between II and for the Cav3.2 channel of and and has been identified as an important region for interaction and regulation of the channel by kinases and signaling proteins S. Monteil A. Bidaud I. Barbara G. Chemin J. Lory P. Biochim. Biophys. Acta. 2009; 1793: 947-952Crossref PubMed Scopus (55) Google Scholar). In this it not be to an interaction between the N terminus region of Cav-3 and the intracellular between II and of the Cav3.2 channel. studies are to the of Cav3.2 channel involved in the interaction with Cav-3, which understand regulation of the Cav3.2 that siRNA-mediated knockdown of Cav-3 did not affect the ICa,T density in the mouse neonatal cardiomyocytes but abolished PKA regulation of the to this in an study we demonstrated that a of the L-type Ca2+ channels are localized to caveolae and siRNA-mediated Cav-3 knockdown did not affect the density in neonatal ventricular myocytes. However, Cav-3 knockdown inhibited the regulation of the channels (24Balijepalli R.C. Foell J.D. Hall D.D. Hell J.W. Kamp T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7500-7505Crossref PubMed Scopus (320) Google Scholar). contain scaffolding that with signaling including and regulatory proteins such as Ca2+/calmodulin-dependent protein kinase and and ion channels, temporal and spatial regulation of (18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar, J. Mol. Cell Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar, H.H. F. P.A. Pharmacol. 2008; Scopus Google Scholar). The subcellular localization of ion channels to caveolae the of these channels specific macromolecular signaling complexes in a for their precise localization and Cav-3 inhibition of Cav3.2 channels in neonatal cardiomyocytes the compartmentalized and regulation of the Cav3.2 channels by PKA and other kinases in the native cells. caveolar localization and Cav-3 interaction of the Cav3.2 are for precise modulation of this channel not by PKA but by different signaling during normal and pathological of Cav-3 is as it and by by a to expression in the adult cardiomyocytes B. I. Physiol. Rev. 1999; PubMed Scopus Google Scholar, P. P. F. F. B. J.L. J. Physiol. PubMed Scopus Google Scholar). The cardiac T-type Ca2+ channels are expressed during cardiac development but their expression is not in normal adult ventricular myocytes. It is well that to ventricular tissue with associated the during pathological such as cardiac hypertrophy and heart failure R.C. H.H. Kamp T.J. Res. 2003; PubMed Scopus Google Scholar, Y. Garcia E. J. Proc. Natl. Acad. Sci. U.S.A. 2009; PubMed Scopus (276) Google Scholar, S. A. Chen B. K. H. L.S. Circ. Res. 2010; PubMed Scopus Google Scholar). cardiac Cav3.1 and Cav3.2 channels are known to be in the ventricular myocytes. Caveolae and expression of Cav-3 is known to be during these conditions L. B. H. S. Circ. Res. PubMed Scopus Google Scholar, T. A. H. H. T. J. Pharmacol. 45: PubMed Scopus Google Scholar). It be that during the ventricular Cav-3 and caveolae expression alter subcellular localization and expression of the Ca2+ channels, including the Cav3.2 channels that in and regulation of the channels. The Cav3.2 T-type Ca2+ channels are involved in various such as epilepsy H. C. B. K.S. Snutch T.P. J. Zamponi G.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. C. Snutch T.P. Zamponi G.W. Ann. PubMed Scopus Google Scholar, J. D. T. M. Chen C.C. Campbell K.P. S. Y. H. J. Neurosci. 2008; PubMed Scopus Google Scholar, Cain S.M. C. S. L.S. M. Garcia E. Tyson J.R. M. Snutch T.P. T.J. J. Neurosci. 2009; PubMed Scopus Google Scholar), pain Y. Y. F. M. T. H. A. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar), J.F. 2009; PubMed Scopus Google Scholar), and cardiac hypertrophy (16Chiang C.S. Huang C.H. Chieng H. Chang Y.T. Chang D. Chen J.J. Chen Y.C. Chen Y.H. Shin H.S. Campbell K.P. Chen C.C. Circ. Res. 2009; 104: 522-530Crossref PubMed Scopus (129) Google Scholar). In using of which the Cav3.2 it was demonstrated that the Cav3.2 T-type Ca2+ channel is for of pathological cardiac hypertrophy (16Chiang C.S. Huang C.H. Chieng H. Chang Y.T. Chang D. Chen J.J. Chen Y.C. Chen Y.H. Shin H.S. Campbell K.P. Chen C.C. Circ. Res. 2009; 104: 522-530Crossref PubMed Scopus (129) Google Scholar). other indicated that overexpression of Cav-3 is against in the neonatal cardiomyocytes A. T. H. S. T. 2003; PubMed Scopus Google Scholar). In studies overexpression of Cav-3 inhibited the T-type Ca2+ current the L-type Ca2+ which are known to localize to caveolae and associate with Cav-3 (24Balijepalli R.C. Foell J.D. Hall D.D. Hell J.W. Kamp T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7500-7505Crossref PubMed Scopus (320) Google Scholar). Our data demonstrate that Cav-3 inhibited the overexpression of ICav3.2. In this have important to the T-type Ca2+ channel T-type Ca2+ channel have been to be in the of a variety of conditions including and heart failure J.F. 2009; PubMed Scopus Google Scholar, H. K. M. Y. Y. S. Y. M. M. M. M. K. K. 2009; PubMed Scopus Google Scholar). studies are to understand the role of Cav-3 and caveolae in the ventricular during cardiac conditions such as hypertrophy and heart of the T-type Ca2+ channels and Ca2+ signaling by caveolae is important for understanding the of pathological in cardiac hypertrophy and heart results demonstrate for the a precise caveolar localization of the Cav3.2 T-type Ca2+ channel in cardiomyocytes. We show that Cav-3 interacts with the Cav3.2 channels and modulates PKA regulation of ICa,T in the ventricular myocytes. Our studies provide the for understanding the compartmentalized regulation of the T-type Ca2+ channel in cardiomyocytes and other cell types in normal and pathological IntroductionT-type Ca2+ channels (TTCC) 2The abbreviations used are: TTCCvoltage-gated T-type Ca2+ channelCav-3caveolin-3AdCav3.2adenovirus expressing Cav3.2 channel proteinAdGFPadenovirus expressing green fluorescent proteinTACtransthoracic aortic constrictionPKAcAMP-dependent protein kinase AHCN4hyperpolarization activated cyclic nucleotide-gated potassium channel 4ICa,TT-type calcium currentsICav3.1Cav3.1 channel calcium currentsICav3.2Cav3.2 channel calcium currents8-Br-CAMP8-bromo-cyclic AMP. are low voltage-activated Ca2+ channels, expressed in various tissues including brain and heart and contribute to a variety of physiological functions such as neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity (1Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1319) Google Scholar, 2Iftinca M.C. Zamponi G.W. Trends Pharmacol. Sci. 2009; 30: 32-40Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Huc S. Monteil A. Bidaud I. Barbara G. Chemin J. Lory P. Biochim. Biophys. Acta. 2009; 1793: 947-952Crossref PubMed Scopus (55) Google Scholar). Molecular cloning studies have identified three different TTCC isoforms, CaV3.1 (α1G), CaV3.2 (α1H), and Cav3.3 (α1I), which functionally can be distinguished by their electrophysiological properties (4Lee J.H. Daud A.N. Cribbs L.L. Lacerda A.E. Pereverzev A. Klöckner U. Schneider T. Perez-Reyes E. J. Neurosci. 1999; 19: 1912-1921Crossref PubMed Google Scholar, 5Klöckner U. Lee J.H. Cribbs L.L. Daud A. Hescheler J. Pereverzev A. Perez-Reyes E. Schneider T. Eur. J. Neurosci. 1999; 11: 4171-4178Crossref PubMed Scopus (151) Google Scholar, 6Kozlov A.S. McKenna F. Lee J.H. Cribbs L.L. Perez-Reyes E. Feltz A. Lambert R.C. Eur. J. Neurosci. 1999; 11: 4149-4158Crossref PubMed Scopus (72) Google Scholar, 7Chemin J. Monteil A. Perez-Reyes E. Bourinet E. Nargeot J. Lory P. J. Physiol. 2002; 540: 3-14Crossref PubMed Scopus (184) Google Scholar). Cav3.1 and Cav3.2 are the most commonly expressed subunits generating T-type Ca2+ current (ICa,T) in brain and heart. Cav3.2 T-type Ca2+ channels are involved in neurological disorders such as epilepsy and pain (8McGivern J.G. Drug Discov. Today. 2006; 11: 245-253Crossref PubMed Scopus (113) Google Scholar). In the heart, the ICa,T participates in Ca2+ entry and Ca2+-dependent hormonal secretion, pacemaker activity, and arrhythmia (9Vassort G. Talavera K. Alvarez J.L. Cell Calcium. 2006; 40: 205-220Crossref PubMed Scopus (100) Google Scholar, 10Ono K. Iijima T. J. Mol. Cell Cardiol. 2010; 48: 65-70Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The Cav3.1 and Cav3.2 isoforms are normally expressed in embryonic hearts (11Nuss H.B. Marban E. J. Physiol. 1994; 479: 265-279Crossref PubMed Scopus (118) Google Scholar, 12Cribbs L.L. Martin B.L. Schroder E.A. Keller B.B. Delisle B.P. Satin J. Circ. Res. 2001; 88: 403-407Crossref PubMed Scopus (86) Google Scholar), but postnatal expression of these isoforms diminishes with almost no expression in normal adult ventricular myocytes. However, the TTCCs are re-expressed during conditions of cardiac hypertrophy and heart failure and are reported to be associated with decreased cardiac function (13Nuss H.B. Houser S.R. Circ. Res. 1993; 73: 777-782Crossref PubMed Scopus (276) Google Scholar, 14Martínez M.L. Heredia M.P. Delgado C. J. Mol. Cell Cardiol. 1999; 31: 1617-1625Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 15Ferron L. Capuano V. Ruchon Y. Deroubaix E. Coulombe A. Renaud J.F. Circ. Res. 2003; 93: 1241-1248Crossref PubMed Scopus (75) Google Scholar). Ca2+ influx through the re-expressed voltage-gated Cav3.2 (α1H) TTCC is indicated to be responsible for inducing pathological cardiac hypertrophy in a pressure overload model (16Chiang C.S. Huang C.H. Chieng H. Chang Y.T. Chang D. Chen J.J. Chen Y.C. Chen Y.H. Shin H.S. Campbell K.P. Chen C.C. Circ. Res. 2009; 104: 522-530Crossref PubMed Scopus (129) Google Scholar).Although we are beginning to understand the physiological role of Cav3.2 channels in Ca2+ cycling, the precise regulation of these channels has not been defined. To clearly understand the regulation and physiological function of these proteins in normal and diseased stages it is important to define the exact subcellular localization of this protein. Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids, contain the signature scaffolding protein caveolins, and provide temporal and spatial regulation of intracellular Ca2+ in many cell types (17Insel P.A. Patel H.H. Curr. Opin. Nephrol. Hypertens. 2009; 18: 50-56Crossref PubMed Scopus (54) Google Scholar, 18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar, 19Pani B. Singh B.B. Cell Calcium. 2009; 45: 625-633Crossref PubMed Scopus (209) Google Scholar). Three different caveolin isoforms are identified, of which Cav-1 and Cav-2 are ubiquitously expressed (20Glenney Jr., J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 21Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar), whereas Cav-3 is widely expressed in muscle (cardiac, skeletal, and smooth muscle) cells (22Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar) and neuronal tissue (23Boulware M.I. Kordasiewicz H. Mermelstein P.G. J. Neurosci. 2007; 27: 9941-9950Crossref PubMed Scopus (165) Google Scholar). A number of ion channels and transporters have been localized to caveolae and associate with Cav-3, including L-type Ca2+ channels (Cav1.2) (24Balijepalli R.C. Foell J.D. Hall D.D. Hell J.W. Kamp T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7500-7505Crossref PubMed Scopus (320) Google Scholar), the Na+ channel (Nav1.5) (25Yarbrough T.L. Lu T. Lee H.C. Shibata E.F. Circ. Res. 2002; 90: 443-449Crossref PubMed Scopus (192) Google Scholar), pacemaker channels (HCN4) (26Ye B. Balijepalli R.C. Foell J.D. Kroboth S. Ye Q. Luo Y.H. Shi N.Q. Biochemistry. 2008; 47: 12312-12318Crossref PubMed Scopus (44) Google Scholar), Na+/Ca2+ exchanger (27Bossuyt J. Taylor B.E. James-Kracke M. Hale C.C. Ann. N.Y. Acad. Sci. 2002; 976: 197-204Crossref PubMed Scopus (46) Google Scholar), and others (18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar). Closely associated with these channels are specific macromolecular signaling complexes containing a variety of regulatory proteins including the G protein-coupled receptors and kinases such as PKC and PKA that provide highly localized regulation of the channels (18Balijepalli R.C. Kamp T.J. Prog. Biophys. Mol. Biol. 2008; 98: 149-160Crossref PubMed Scopus (127) Google Scholar). The present study therefore was designed to determine the precise subcellular localization and regulation of the Cav3.2 subunit of the T-type Ca2+ channels. Our results demonstrate that the Cav3.2 channel protein is localized to caveolar microdomains in the ventricular myocytes and Cav-3 interacts with Cav3.2 channel and regulates its