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Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytes

Abstract

Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is already known to be highly expressed in the brain, and is found in many other tissues, including the atrium of the heart. Although the complete primary structure of IP3R1 in the rat brain has been reported, the complete sequence of an IP3R1 clone from atrial myocytes has not been reported. We isolated an IP3R1 complementary DNA (cDNA) clone from isolated adult rat atrial myocytes, and found a new splice variant of IP3R1 that was different from a previously reported IP3R1 cDNA clone obtained from a rat brain (NCBI GenBank accession number: NM_001007235). Our clone had 99% similarity with the rat brain IP3R1 sequence; the exceptions were 39 amino acid deletions at the position of 1693–1731, and the deletion of phenylalanine at position 1372 that lay in the regulatory region. Compared with the rat brain IP3R1, in our clone proline was replaced with serine at residue 2439, and alanine was substituted for valine at residue 2445. These changes lie adjacent to or within the fifth transmembrane domain (2440–2462). Although such changes in the amino acid sequences were different from the rat brain IP3R1 clone, they were conserved in human or mouse IP3R1. We produced a plasmid construct expressing the atrial IP3R1 together with green fluorescent protein (GFP), and successfully overexpressed the atrial IP3R1 in the adult atrial cell line HL-1. Further investigation is needed on the physiological significance of the new splice variant in atrial cell function.

Abbreviations

AcGFP:

Aequorea coerulescens green fluorescent protein

IP3R1:

inositol 1,4,5-trisphosphate receptor type 1

PCR:

polymerase chain reaction

RT:

reverse transcription

References

  1. 1.

    Berridge, M.J. Unlocking the secrets of cell signaling. Annu. Rev. Physiol. 67 (2005) 1–21.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Macara, B. and Rico, J.M. Effect of Ca2+ modulators on acetylcholineinduced phasic and tonic contractions and A23187-induced contractions in ileal longitudinal muscle and IP3 production. Eur. J. Pharmacol. 218 (1992) 27–33.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Hilal-Dandan, R., Urasawa, K. and Brunton, L.L. Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes. J. Biol. Chem. 267 (1992) 10620–10624.

    PubMed  CAS  Google Scholar 

  4. 4.

    Pucéat, M. and Vassort, G. Purinergic stimulation of rat cardiomyocytes induces tyrosine phosphorylation and membrane association of phospholipase C gamma: a major mechanism for InsP3 generation. Biochem. J. 318 (1996) 723–728.

    PubMed  Google Scholar 

  5. 5.

    Goutsouliak, V. and Rabkin, S.W. Angiotensin II-induced inositol phosphate generation is mediated through tyrosine kinase pathways in cardiomyocytes. Cell. Signal. 9 (1997) 505–512.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Furuichi, T., Kohda, T.K., Miyawaki, A. and Mikoshiba, K. Intracellular channels. Curr. Opin. Neurobiol. 4 (1994) 294–303.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Patel, S., Joseph, S.K. and Thomas, A.P. Molecular properties of inositol 1, 4, 5-trisphosphate receptors. Cell Calcium 25 (1999) 247–264.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Furuichi, T., Simon-Chazottes, D., Fujino, I., Yamada, N., Hasegawa, M., Miyawaki, A., Yoshikawa, S., Guenet, J.L. and Mikoshiba, K. Widespread expression of inositol 1, 4, 5-trisphosphate receptor type 1 gene (InsP3R1) in the mouse central nervous system. Receptors Channels 1 (1993) 11–24.

    PubMed  CAS  Google Scholar 

  9. 9.

    Mourey, R.J., Verma, A., Supattapone, S. and Snyder, S.H. Purification and characterization of the inositol 1, 4, 5-trisphosphate receptor protein from rat vas deferens. Biochem. J. 272 (1990) 383–389.

    PubMed  CAS  Google Scholar 

  10. 10.

    Vermassen, E., Van Acker, K., Annaert, W.G., Himpens, B., Callewaert, G., Missiaen, L., De Smedt, H. and Parys, J.B. Microtubule-dependent redistribution of the type-1 inositol 1, 4, 5 trisphosphate receptor in A7r5 smooth muscle cells. J. Cell Sci. 116 (2003) 1269–1277.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Monkawa, T., Hayashi, M., Miyawaki, A., Sugiyama, T., Yamamoto-Hino, M., Hasegawa, M., Furuichi, T., Mikoshiba, K. and Saruta, T. Localization of inositol 1, 4, 5-trisphophate receptors in the rat kidney. Kidney Int. 53 (1998) 296–301.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Kim, J.C., Son, M.J., Subedi, K.P., Kim, D.H. and Woo, S.H. IP3-induced cytosolic and nuclear Ca2+ signals in HL-1 atrial myocytes: possible role of IP3 receptor subtypes. Mol. Cells 29 (2010) 387–395.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Slavikova, J., Dvorakova, M., Reischig, J., Palkovits, M., Ondrias, K., Tarabova, B., Lacinova, L., Kvetnansky, R., Marks, A. and Krizanova, O. IP3 type 1 receptors in the heart: their predominance in atrial walls with ganglion cells. Life Sci. 78 (2006) 1598–1602.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Supattapone, S., Worley, P.F., Baraban, J.M. and Snyder, S.H. Solubilization, purification, and characterization of an inositol trisphosphate receptor. J. Biol. Chem. 263 (1988) 1530–1534.

    PubMed  CAS  Google Scholar 

  15. 15.

    Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiba, K. Primary structure and functional expression of the inositol 1, 4, 5-trisphosphate-binding protein P400. Nature 342 (1989) 32–38.

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Mignery, G.A., Newton, C.L., Archer, B.T. and Südhof, T.C. Structure and expression of the rat inositol 1, 4, 5-trisphosphate receptor. J. Biol. Chem. 265 (1990) 12679–12685.

    PubMed  CAS  Google Scholar 

  17. 17.

    Mignery, G.A. and Sudhof, T.C. The ligand binding site and transduction mechanism in the inositol-1, 4, 5-triphosphate receptor. EMBO J. 9 (1990) 3893–3898.

    PubMed  CAS  Google Scholar 

  18. 18.

    Nakagawa, T., Shiota, C., Okano, H. and Mikoshiba, K. Differential localization of alternative spliced transcripts encoding inositol 1, 4, 5-trisphosphate receptors in mouse cerebellum and hippocampus — in situ hybridization study. J. Neurochem. 57 (1991) 1807–1810.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Nakagawa, T., Okano, H., Furuichi, T., Aruga, J. and Mikoshiba, K. The subtypes of the mouse inositol 1, 4, 5-trisphosphate receptor are expressed in a tissue-specific and developmentally specific manner. Proc. Natl. Acad. Sci. USA. 88 (1991) 6244–6248.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Lee, S., Kim, J.C., Li, Y., Son, M.J. and Woo, S.H. Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes. Am. J. Physiol. Cell Physiol. 294 (2008) C966–C976.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Thompson, J.D., Higgins, D. G. and Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 (1994) 4673–4680.

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Claycomb, W.C., Lanson, N.A. Jr., Stallworth, B.S., Egeland, D.B., Delcarpio, J.B., Bahinski, A. and Izzo, N.J. Jr. HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 2979–2984.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Sudhof, T.C., Newton, C.L., Archer, B.T. 3rd., Ushkaryov, Y.A. and Mignery, G.A. Structure of a novel InsP3 receptor. EMBO J. 10 (1991) 3199–3206.

    PubMed  CAS  Google Scholar 

  24. 24.

    Blondel, O., Takeda, J., Janssen, H., Seino, S. and Bell, G. I. Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues. J. Biol. Chem. 268 (1993) 11356–11363.

    PubMed  CAS  Google Scholar 

  25. 25.

    Danoff, S.K., Ferris, C.D., Donath, C., Fischer, G.A., Munemitsu, S., Ullrich, A., Snyder, S.H. and Ross, C.A. Inositol 1, 4, 5-trisphosphate receptors: distinct neuronal and nonneuronal forms derived by alternative splicing differ in phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 2951–2955.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Kim, J.C., Son, M.J., Subedi, K.P., Li, Y., Ahn, J.R., and Woo, S.H. Atrial local Ca2+ signaling and inositol 1, 4, 5-trisphosphate receptors. Prog. Biophys. Mol. Biol. 103 (2010) 59–70.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Bare, D.J., Kettlun, C.S., Liang, M., Bers, D.M. and Mignery, G.A. Cardiac type 2 inositol 1, 4, 5-trisphosphate receptor. Interaction and modulation by calcium/calmodulin-dependent protein kinase II. J. Biol. Chem. 280 (2005) 15912–15920.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Luo, D., Yang, D., Lan, X., Li, K., Chen, J., Zhang, Y., Xiao, R.P., Han, Q. and Cheng, H. Nuclear Ca2+ sparks and waves mediated by inositol 1, 4, 5-trisphosphate receptors in neonatal rat cardiomyocytes. Cell Calcium 43 (2008) 165–174.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Lipp, P., Laine, M., Tovey, S.C., Burrell, K.M., Berridge, M.J., Li, W. and Bootman, M.D. Functional InsP3 receptors that may modulate excitationcontraction coupling in the heart. Curr. Biol. 10 (2000) 939–942.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Yamada, J., Ohkusa, T., Nao, T., Ueyama, T., Yano, M., Kobayashi, S., Hamano, K., Esato, K. and Matsuzaki, M. Up-regulation of inositol 1, 4, 5 trisphosphate receptor expression in atrial tissue in patients with chronic atrial fibrillation. J. Am. Coll. Cardiol. 37 (2001) 1111–1119.

    Article  CAS  Google Scholar 

  31. 31.

    Gorza, L., Schiaffino, S. and Volpe, P. Inositol 1, 4, 5-trisphosphate receptor in heart: evidence for its concentration in Purkinje myocytes of the conduction system. J. Cell Biol. 121 (1993) 345–353.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Go, L.O., Moschella, M.C., Watras, J., Handa, K.K., Fyfe, B.S. and Marks, A.R. Differential regulation of two types of intracellular calcium release channels during end-stage heart failure. J. Clin. Invest. 95 (1995) 888–894.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Ferris, C.D., Cameron, A.M., Bredt, D.S., Huganir, R.L. and Snyder, S.H. Inositol 1, 4, 5-trisphosphate receptor is phosphorylated by cyclic AMPdependent protein kinase at serines 1755 and 1589. Biochem. Biophys. Res. Commun. 175 (1991) 192–198.

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Tu, H., Miyakawa, T., Wang, Z., Glouchankova, L., Iino, M. and Bezprozvanny, I. Functional characterization of the type 1 inositol 1, 4, 5-trisphosphate receptor coupling domain SII(+/−) splice variants and the Opisthotonos mutant form. Biophys. J. 82 (2002) 1995–2004.

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Sun-Hee Woo.

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K. P. Subedi and T. D. Singh contributed equally to this work.

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Subedi, K.P., Singh, T.D., Kim, J. et al. Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytes. Cell Mol Biol Lett 17, 124–135 (2012). https://doi.org/10.2478/s11658-011-0043-7

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Key words

  • Type 1 inositol 1,4,5-trisphosphate receptor type
  • Splice variant
  • Rat atrial myocytes
  • Cloning
  • Expression