Skip to main content
SpringerLink
Log in

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies

  • Invited Review
  • Published:
Pflügers Archiv Aims and scope Submit manuscript

Abstract

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the “full-length” TRPC4 protein and a slightly smaller variant, called TRPC4Δ761-864, which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.

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. 1a,b
Fig. 2a,b
Fig. 3a–c
Fig. 4a–c

Similar content being viewed by others

References

  1. Corey DP, Garcia-Añoveros J, Holt JR, Kwan KY, Lin SY, Vollrath MA, Amalfitano A, Cheung ELM, Derfler BH, Duggan A, Géléoc GSG, Gray PA, Hoffmann MP, Rehm HL, Tamasauskas D, Zhang DS (2004) TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells. Nature 432:723–730

    Article  CAS  PubMed  Google Scholar 

  2. Montell C, Birnbaumer L, Flockerzi V (2002) The TRP channels, a remarkably functional family. Cell 108:595–598

    CAS  PubMed  Google Scholar 

  3. Wissenbach U, Niemeyer BA, Himmerkus N, Fixemer T, Bonkhoff H, Flockerzi V (2004) TRPV6 and prostate cancer: cancer growth beyond the prostate correlates with increased TRPV6 Ca2+ channel expression. Biophys Biochem Res Com 322:359–1363

    Google Scholar 

  4. Birnbaumer L, Boulay G, Brown D, Jiang M, Dietrich A, Mikoshiba K, Zhu X, Qin N (2000) Mechanism of capacitative Ca2+ entry (CCE): interaction between IP3 receptor and TRP links the internal calcium storage compartment to plasma membrane CCE channels. Recent Prog Horm Res 55:127–162

    CAS  PubMed  Google Scholar 

  5. Kiselyov KI, Shin DM, Wang Y, Pessah IN, Allen PD, Muallem S (2000) Gating of store-operated channels by conformational coupling to ryanodine receptors. Mol Cell 6:421–431

    CAS  PubMed  Google Scholar 

  6. Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G (1999) Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397:259–263

    Article  CAS  PubMed  Google Scholar 

  7. Philipp S, Cavalié A, Freichel M, Wissenbach U, Zimmer S, Trost C, Marquart A, Murakami M, Flockerzi V (1996) A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J 15:6166–6171

    CAS  PubMed  Google Scholar 

  8. Warnat J, Philipp S, Zimmer S, Flockerzi V, Cavalié A (1999) Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+. J Physiol (Lond) 518:631–638

    Google Scholar 

  9. Philipp S, Trost C, Warnat J, Rautmann J, Himmerkus N, Schroth G, Kretz O, Nastainczyk W, Cavalié A, Hoth M, Flockerzi V (2000) TRP4 (CCE1) Protein is part of native calcium release-activated Ca2+-like channels in adrenal cells. J Biol Chem 275:23965–23972

    Article  CAS  PubMed  Google Scholar 

  10. Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weißgerber P, Biel M, Philipp S, Freise D, Droogmans G, Hofmann F, Flockerzi V, Nilius B (2001) Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4−/− mice. Nat Cell Biol 3:121–127

    Article  CAS  PubMed  Google Scholar 

  11. Vazquez G, Lievremont JP, Bird GSJ, Putney JW (2001) Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci 98:11777–11782

    Article  CAS  PubMed  Google Scholar 

  12. Zitt C, Zobel A, Obukhov AG, Harteneck C, Kalkbrenner F, Lückhoff A, Schultz G (1996) Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion. Neuron 16:1189–1196

    Article  CAS  PubMed  Google Scholar 

  13. van Rossum DB, Patterson RL, Sharma S, Barrow RK, Kornberg M, Gill DL, Snyder SH (2005) Phospholipase Cγ1 controls surface expression of TRPC3 through an intermolecular PH domain. Nature 434:99–104

    Google Scholar 

  14. Singh BB, Lockwich TP, Bandyopadhyay BC, Liu X, Bollimuntha S, Brazer S, Combs C, Das S, Leenders M, Sheng ZH, Knepper MA, Ambudkar SV, Ambudkar IS (2004) VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx. Mol Cell 15:635–646

    Article  CAS  PubMed  Google Scholar 

  15. Bezzerides VJ, Ramsey IS, Kotecha S, Greka A, Clapham DE (2004) Rapid vesicular translocation and insertion of TRP channels. Nat Cell Biol 6:709–720

    Article  CAS  PubMed  Google Scholar 

  16. Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP (2005) TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol 7:179–185

    Article  CAS  PubMed  Google Scholar 

  17. Trost C, Bergs C, Himmerkus N, Flockerzi V (2001) The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins. Biochem J 355:663–670

    CAS  PubMed  Google Scholar 

  18. Tang J, Lin Y, Zhang Z, Tikunova S, Birnbaumer L, Zhu MX (2001) Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels. J Biol Chem 276:21303–21310

    Article  CAS  PubMed  Google Scholar 

  19. Mery L, Magnino F, Schmidt K, Krause KH, Dufour JF (2001) Alternative splice variants of hTrp4 differentially interact with the C-terminal portion of the inositol 1,4,5-trisphosphate receptors. FEBS Lett 487:377–383

    Article  CAS  PubMed  Google Scholar 

  20. Zhang Z, Tang J, Tikunova S, Johnson JD, Chen Z, Qin N, Dietrich A, Stefani E, Birnbaumer L, Zhu MX (2000) Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain. Proc Natl Acad Sci 98:3168–3173

    Article  Google Scholar 

  21. Vandebrouck C, Martin D, Colson-Van Schorr M, Debaix H, Gailly P (2002) Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers. J Cell Biol 158:1089–1096

    Article  CAS  PubMed  Google Scholar 

  22. Weigl L, Zidar A, Gscheidlinger R, Karel A, Hohenegger M (2003) Store-operated Ca2+ influx by selective depletion of ryanodine-sensitive Ca2+ pools in primary human skeletal muscle cells. Naunyn Schmiedeberg’s Arch Pharmacol 367:353–363

    CAS  Google Scholar 

  23. Freichel M, Wissenbach U, Philipp S, Flockerzi V (1998) Alternative splicing and tissue specific expression of the 5′ truncated bCCE 1 variant bCCE 1Δ 514. FEBS Lett 422:354–358

    Article  CAS  PubMed  Google Scholar 

  24. Maurer P, Moratzky A, Fecher-Trost C, Flockerzi V, Lenk U, Sommer T, Völzing C, Zimmermann R (2003) Cell-free synthesis of membrane proteins on a preparative scale. In: Swartz JR (ed) Cell-free protein expression. Springer, Berlin Heidelberg New York, pp 133–139

    Google Scholar 

  25. Yellen G (2002) The voltage-gated potassium channels and their relatives. Nature 419:35–42

    Google Scholar 

  26. Ong HL, Chen J, Chataway T, Brereton H, Zhang L, Downs T, Tsiokas L, Barritt G (2002) Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis. Biochem J 364:641–648

    Article  CAS  PubMed  Google Scholar 

  27. Reuter H (1983) Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301:569–574

    Google Scholar 

  28. Bosse E, Bottlender R, Kleppisch T, Hescheler J, Welling A, Hofmann F, Flockerzi V (1992) Stable and functional expression of the calcium channel α1 subunit from smooth muscle in somatic cell lines. EMBO J 11:2033–2038

    CAS  PubMed  Google Scholar 

  29. Flockerzi V, Oeken HJ, Hofmann F, Pelzer D, Cavalié A, Trautwein W (1986) Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel. Nature 323:66–68

    Google Scholar 

  30. Lockwich TP, Liu X, Singh BB, Jadlowiec J, Weiland S, Ambudkar IS (2000) Assembly of Trp1 in a signalling complex associated with caveolin-scaffolding lipid raft domains. J Biol Chem 275:11934–11942

    CAS  PubMed  Google Scholar 

  31. Murakami M, Fleischmann B, DeFelipe C, Freichel M, Trost C, Ludwig A, Wissenbach U, Schwegler H, Hofmann F, Hescheler J, Flockerzi V, Cavalié A (2002) Pain perception in mice lacking the β3 subunit of voltage-activated calcium channels. J Biol Chem 277:40342–40351

    Google Scholar 

  32. Berggren PO, Yang SN, Murakami M, Efanov AM, Uhles S, Köhler M, Moede T, Fernström A, Appelskog IB, Aspinwall CA, Zaitsev SV, Larsson O, de Vargas LM, Fecher-Trost C, Weißgerber P, Ludwig A, Leibiger B, Juntti-Berggren L, Barker CJ, Gromada J, Freichel M, Leibiger IB, Flockerzi V (2004) Removal of Ca2+ channel β3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis. Cell 119: 273–284

    Article  CAS  PubMed  Google Scholar 

  33. Niemeyer BA (2005) Structure-function analysis of TRPV channels. Naunyn Schmiedeberg’s Arch Pharmacol DOI: 10.1007/s00210-005-1053-7

  34. Nicke A, Rettinger J, Schmalzing G (2003) Monomeric and dimeric byproducts are the principal functional elements of higher order P2X1 concatamers. Mol Pharmacol 63:243–252

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Philippe Gailly for providing the skeletal muscle tissue from dystrophic (mdx) and wild-type (C57) mice. This work was supported, in part, by the DFG, the Fonds der Chemischen Industrie, the BMBF and HOMFOR.

Author information

Authors and Affiliations

  1. Experimentelle und Klinische Pharmakologie und Toxikologie, Medizinische Fakultät, Universität des Saarlandes, 66421, Homburg, Deutschland

    Veit Flockerzi, Christine Jung, Thomas Aberle, Marcel Meissner, Marc Freichel & Stephan E. Philipp

  2. Medizinische Biochemie und Molekularbiologie, Medizinische Fakultät Universität des Saarlandes, 66421, Homburg, Deutschland

    Wolfgang Nastainczyk, Patrick Maurer & Richard Zimmermann

Authors
  1. Veit Flockerzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christine Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Aberle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcel Meissner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marc Freichel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephan E. Philipp
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wolfgang Nastainczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Patrick Maurer
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Richard Zimmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Veit Flockerzi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Flockerzi, V., Jung, C., Aberle, T. et al. Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies. Pflugers Arch - Eur J Physiol 451, 81–86 (2005). https://doi.org/10.1007/s00424-005-1443-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-005-1443-1

Keywords

Search

Navigation