Skip to main content

Advertisement

SpringerLink
Log in

Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand-gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to buoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native-PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in the membranes of murine cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 proteins in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2X7R and caveolins may serve an important regulatory control point for disease pathology in the heart.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alvarez JL, Mongo K, Scamps F, Vassort G (1990) Effects of purinergic stimulation on the Ca current in single frog cardiac cells. Pflugers Arch 416:189–195

    Article  PubMed  CAS  Google Scholar 

  • Balijepalli RC, Foell JD, Hall DD, Hell JW, Kamp TJ (2006) Localization of cardiac L-type Ca(2 +) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation. Proc Natl Acad Sci USA 103:7500–7505

    Article  PubMed  CAS  Google Scholar 

  • Barbuti A, Terragni B, Brioschi C, DiFrancesco D (2007) Localization of f-channels to caveolae mediates specific beta2-adrenergic receptor modulation of rate in sinoatrial myocytes. J Mol Cell Cardiol 42:71–78

    Article  PubMed  CAS  Google Scholar 

  • Barth K, Weinhold K, Guenther A, Linge A, Gereke M, Kasper M (2008) Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells. Int J Biochem Cell Biol 40:2230–2239

    Article  PubMed  CAS  Google Scholar 

  • Barth K, Blasche R, Kasper M (2010a) T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells. Cell Physiol Biochem 25:103–112

    Article  PubMed  CAS  Google Scholar 

  • Barth K, Pfleger C, Linge A, Sim JA, Surprenant A, Steinbronn N, Strasser RH, Kasper M (2010b) Increased P2X7R expression in atrial cardiomyocytes of caveolin-1 deficient mice. Histochem Cell Biol 134:31–38

    Article  PubMed  CAS  Google Scholar 

  • Bauernfeind F, Ablasser A, Bartok E, Kim S, Schmid-Burgk J, Cavlar T, Hornung V (2010) Inflammasomes: current understanding and open questions. Cell Mol Life Sci 68:765–783

    Article  PubMed  Google Scholar 

  • Bossuyt J, Taylor BE, James-Kracke M, Hale CC (2002) The cardiac sodium–calcium exchanger associates with caveolin-3. Ann NY Acad Sci 976:197–204

    Article  PubMed  CAS  Google Scholar 

  • Boumechache M, Masin M, Edwardson JM, Gorecki DC, Murrell-Lagnado R (2009) Analysis of assembly and trafficking of native P2X4 and P2X7 receptor complexes in rodent immune cells. J Biol Chem 284:13446–13454

    Article  PubMed  CAS  Google Scholar 

  • Cho WJ, Chow AK, Schulz R, Daniel EE (2007) Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium. J Cell Mol Med 11:1069–1086

    Article  PubMed  CAS  Google Scholar 

  • Cho WJ, Chow AK, Schulz R, Daniel EE (2010) Caveolin-1 exists and may function in cardiomyocytes. Can J Physiol Pharmacol 88:73–76

    Article  PubMed  CAS  Google Scholar 

  • Dubyak GR (2007) Go it alone no more—P2X7 joins the society of heteromeric ATP-gated receptor channels. Mol Pharmacol 72:1402–1405

    Article  PubMed  CAS  Google Scholar 

  • Faria RX, Defarias FP, Alves LA (2005) Are second messengers crucial for opening the pore associated with P2X7 receptor? Am J Physiol Cell Physiol 288:C260–C271

    Article  PubMed  CAS  Google Scholar 

  • Galbiati F, Razani B, Lisanti MP (2001) Caveolae and caveolin-3 in muscular dystrophy. Trends Mol Med 7:435–441

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Marcos M, Pochet S, Tandel S, Fontanils U, Astigarraga E, Fernandez-Gonzalez JA, Kumps A, Marino A, Dehaye JP (2006) Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells. Biochim Biophys Acta 1758:796–806

    Article  PubMed  CAS  Google Scholar 

  • Garg V, Jiao J, Hu K (2009a) Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes. Cardiovasc Res 82:51–58

    Article  PubMed  CAS  Google Scholar 

  • Garg V, Sun W, Hu K (2009b) Caveolin-3 negatively regulates recombinant cardiac K(ATP) channels. Biochem Biophys Res Commun 385:472–477

    Article  PubMed  CAS  Google Scholar 

  • Garlanda C, Parravicini C, Sironi M, De Rossi M, Wainstok de Calmanovici R, Carozzi F, Bussolino F, Colotta F, Mantovani A, Vecchi A (1994) Progressive growth in immunodeficient mice and host cell recruitment by mouse endothelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors. Proc Natl Acad Sci USA 91:7291–7295

    Article  PubMed  CAS  Google Scholar 

  • Hagiwara Y, Sasaoka T, Araishi K, Imamura M, Yorifuji H, Nonaka I, Ozawa E, Kikuchi T (2000) Caveolin-3 deficiency causes muscle degeneration in mice. Hum Mol Genet 9:3047–3054

    Article  PubMed  CAS  Google Scholar 

  • Head BP, Patel HH, Roth DM, Lai NC, Niesman IR, Farquhar MG, Insel PA (2005) G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes. J Biol Chem 280:31036–31044

    Article  PubMed  CAS  Google Scholar 

  • Kahlenberg JM, Lundberg KC, Kertesy SB, Qu Y, Dubyak GR (2005) Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals and requires NF-kappaB-driven protein synthesis. J Immunol 175:7611–7622

    PubMed  CAS  Google Scholar 

  • Lapidos KA, Kakkar R, McNally EM (2004) The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma. Circ Res 94:1023–1031

    Article  PubMed  CAS  Google Scholar 

  • Li J, Liu D, Ke HZ, Duncan RL, Turner CH (2005) The P2X7 nucleotide receptor mediates skeletal mechanotransduction. J Biol Chem 280:42952–42959

    Article  PubMed  CAS  Google Scholar 

  • Lin E, Hung VH, Kashihara H, Dan P, Tibbits GF (2009) Distribution patterns of the Na+-Ca2+ exchanger and caveolin-3 in developing rabbit cardiomyocytes. Cell Calcium 45:369–383

    Article  PubMed  CAS  Google Scholar 

  • Markandeya YS, Fahey JM, Pluteanu F, Cribbs LL, Balijepalli RC (2011) Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels. J Biol Chem 286:2433–2444

    Article  PubMed  CAS  Google Scholar 

  • Matsuura H, Ehara T (1997) Selective enhancement of the slow component of delayed rectifier K+ current in guinea-pig atrial cells by external ATP. J Physiol 503(Pt 1):45–54

    PubMed  CAS  Google Scholar 

  • Merkle S, Frantz S, Schon MP, Bauersachs J, Buitrago M, Frost RJ, Schmitteckert EM, Lohse MJ, Engelhardt S (2007) A role for caspase-1 in heart failure. Circ Res 100:645–653

    Article  PubMed  CAS  Google Scholar 

  • Musa H, Tellez JO, Chandler NJ, Greener ID, Maczewski M, Mackiewicz U, Beresewicz A, Molenaar P, Boyett MR, Dobrzynski H (2009) P2 purinergic receptor mRNA in rat and human sinoatrial node and other heart regions. Naunyn Schmiedebergs Arch Pharmacol 379:541–549

    Article  PubMed  CAS  Google Scholar 

  • Nichols CB, Rossow CF, Navedo MF, Westenbroek RE, Catterall WA, Santana LF, McKnight GS (2010) Sympathetic stimulation of adult cardiomyocytes requires association of AKAP5 with a subpopulation of L-type calcium channels. Circ Res 107:747–756

    Article  PubMed  CAS  Google Scholar 

  • Nicke A (2008) Homotrimeric complexes are the dominant assembly state of native P2X7 subunits. Biochem Biophys Res Commun 377:803–808

    Article  PubMed  CAS  Google Scholar 

  • Parton RG, Simons K (2007) The multiple faces of caveolae. Nat Rev Mol Cell Biol 8:185–194

    Article  PubMed  CAS  Google Scholar 

  • Patel HH, Tsutsumi YM, Head BP, Niesman IR, Jennings M, Horikawa Y, Huang D, Moreno AL, Patel PM, Insel PA, Roth DM (2007) Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1. Faseb J 21:1565–1574

    Article  PubMed  CAS  Google Scholar 

  • Razani B, Lisanti MP (2001) Caveolins and caveolae: molecular and functional relationships. Exp Cell Res 271:36–44

    Article  PubMed  CAS  Google Scholar 

  • Razani B, Woodman SE, Lisanti MP (2002) Caveolae: from cell biology to animal physiology. Pharmacol Rev 54:431–467

    Article  PubMed  CAS  Google Scholar 

  • Schwab W, Harada H, Goetz W, Nowicki M, Witt M, Kasper M, Barth K (2007) Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells. Histochem Cell Biol 128:195–203

    Article  PubMed  CAS  Google Scholar 

  • Sharma V, Sharma A, Saran V, Bernatchez PN, Allard MF, McNeill JH (2011) β-receptor antagonist treatment prevents activation of cell death signaling in the diabetic heart independent of its metabolic actions. Eur J Pharmacol 657:117–125

    Article  PubMed  CAS  Google Scholar 

  • Shen JB, Pappano AJ, Liang BT (2006) Extracellular ATP-stimulated current in wild-type and P2X4 receptor transgenic mouse ventricular myocytes: implications for a cardiac physiologic role of P2X4 receptors. Faseb J 20:277–284

    Article  PubMed  CAS  Google Scholar 

  • Shen JB, Cronin C, Sonin D, Joshi BV, Gongora Nieto M, Harrison D, Jacobson KA, Liang BT (2007) P2X purinergic receptor-mediated ionic current in cardiac myocytes of calsequestrin model of cardiomyopathy: implications for the treatment of heart failure. Am J Physiol Heart Circ Physiol 292:H1077–H1084

    Article  PubMed  CAS  Google Scholar 

  • Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA (2001) Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 276:125–132

    Article  PubMed  CAS  Google Scholar 

  • Sun W, Hu K (2010) Role for SUR2A in coupling cardiac K(ATP) channels to caveolin-3. Cell Physiol Biochem 25:409–418

    Article  PubMed  CAS  Google Scholar 

  • Vacca F, Amadio S, Sancesario G, Bernardi G, Volonte C (2004) P2X3 receptor localizes into lipid rafts in neuronal cells. J Neurosci Res 76:653–661

    Article  PubMed  CAS  Google Scholar 

  • Venema VJ, Ju H, Zou R, Venema RC (1997) Interaction of neuronal nitric-oxide synthase with caveolin-3 in skeletal muscle. Identification of a novel caveolin scaffolding/inhibitory domain. J Biol Chem 272:28187–28190

    Article  PubMed  CAS  Google Scholar 

  • Volonte D, McTiernan CF, Drab M, Kasper M, Galbiati F (2008) Caveolin-1 and caveolin-3 form heterooligomeric complexes in atrial cardiac myocytes that are required for doxorubicin-induced apoptosis. Am J Physiol Heart Circ Physiol 294:H392–H401

    Article  PubMed  CAS  Google Scholar 

  • Weinhold K, Krause-Buchholz U, Rodel G, Kasper M, Barth K (2010) Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells. Cell Mol Life Sci 67:2631–2642

    Article  PubMed  CAS  Google Scholar 

  • Woodman SE, Park DS, Cohen AW, Cheung MW, Chandra M, Shirani J, Tang B, Jelicks LA, Kitsis RN, Christ GJ, Factor SM, Tanowitz HB, Lisanti MP (2002) Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade. J Biol Chem 277:38988–38997

    Article  PubMed  CAS  Google Scholar 

  • Ye B, Balijepalli RC, Foell JD, Kroboth S, Ye Q, Luo YH, Shi NQ (2008) Caveolin-3 associates with and affects the function of hyperpolarization-activated cyclic nucleotide-gated channel 4. Biochemistry 47:12312–12318

    Article  PubMed  CAS  Google Scholar 

  • Young MT, Pelegrin P, Surprenant A (2007) Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP. Mol Pharmacol 71:92–100

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy, Medical Faculty, Institute of Anatomy, University of Technology Dresden, TU Dresden, Fetscherstr. 76, 01307, Dresden, Germany

    Claudia Pfleger, Georg Ebeling, Robert Bläsche, Michael Kasper & Kathrin Barth

  2. Department of Anesthesiology, University of California, La Jolla, San Diego, CA, 92093, USA

    Miranda Patton & Hemal H. Patel

  3. VA San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA, 92161, USA

    Hemal H. Patel

Authors
  1. Claudia Pfleger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Georg Ebeling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert Bläsche
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miranda Patton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hemal H. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Kasper
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kathrin Barth
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kathrin Barth.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pfleger, C., Ebeling, G., Bläsche, R. et al. Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro. Histochem Cell Biol 138, 231–241 (2012). https://doi.org/10.1007/s00418-012-0961-0

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-012-0961-0

Keywords

Search

Navigation