Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Dec;75(6):2783–2793. doi: 10.1016/S0006-3495(98)77721-5

Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants.

J Ramos-Franco 1, S Caenepeel 1, M Fill 1, G Mignery 1
PMCID: PMC1299951  PMID: 9826600

Abstract

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.

Full Text

The Full Text of this article is available as a PDF (282.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  2. Bezprozvanny I., Ehrlich B. E. The inositol 1,4,5-trisphosphate (InsP3) receptor. J Membr Biol. 1995 Jun;145(3):205–216. doi: 10.1007/BF00232713. [DOI] [PubMed] [Google Scholar]
  3. Bezprozvanny I., Watras J., Ehrlich B. E. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 1991 Jun 27;351(6329):751–754. doi: 10.1038/351751a0. [DOI] [PubMed] [Google Scholar]
  4. Danoff S. K., Ferris C. D., Donath C., Fischer G. A., Munemitsu S., Ullrich A., Snyder S. H., 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. 1991 Apr 1;88(7):2951–2955. doi: 10.1073/pnas.88.7.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Smedt H., Missiaen L., Parys J. B., Henning R. H., Sienaert I., Vanlingen S., Gijsens A., Himpens B., Casteels R. Isoform diversity of the inositol trisphosphate receptor in cell types of mouse origin. Biochem J. 1997 Mar 1;322(Pt 2):575–583. doi: 10.1042/bj3220575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Furuichi T., Mikoshiba K. Inositol 1, 4, 5-trisphosphate receptor-mediated Ca2+ signaling in the brain. J Neurochem. 1995 Mar;64(3):953–960. doi: 10.1046/j.1471-4159.1995.64030953.x. [DOI] [PubMed] [Google Scholar]
  7. Furuichi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N., Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 1989 Nov 2;342(6245):32–38. doi: 10.1038/342032a0. [DOI] [PubMed] [Google Scholar]
  8. Joseph S. K. The inositol triphosphate receptor family. Cell Signal. 1996 Jan;8(1):1–7. doi: 10.1016/0898-6568(95)02012-8. [DOI] [PubMed] [Google Scholar]
  9. Kaznacheyeva E., Lupu V. D., Bezprozvanny I. Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells. J Gen Physiol. 1998 Jun;111(6):847–856. doi: 10.1085/jgp.111.6.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mignery G. A., Johnston P. A., Südhof T. C. Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor. J Biol Chem. 1992 Apr 15;267(11):7450–7455. [PubMed] [Google Scholar]
  11. Mignery G. A., Newton C. L., Archer B. T., 3rd, Südhof T. C. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1990 Jul 25;265(21):12679–12685. [PubMed] [Google Scholar]
  12. Mignery G. A., Südhof T. C., Takei K., De Camilli P. Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor. Nature. 1989 Nov 9;342(6246):192–195. doi: 10.1038/342192a0. [DOI] [PubMed] [Google Scholar]
  13. Mignery G. A., Südhof T. C. The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor. EMBO J. 1990 Dec;9(12):3893–3898. doi: 10.1002/j.1460-2075.1990.tb07609.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nakagawa T., Okano H., Furuichi T., Aruga J., 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 U S A. 1991 Jul 15;88(14):6244–6248. doi: 10.1073/pnas.88.14.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nakanishi S., Fujii A., Nakade S., Mikoshiba K. Immunohistochemical localization of inositol 1,4,5-trisphosphate receptors in non-neural tissues, with special reference to epithelia, the reproductive system, and muscular tissues. Cell Tissue Res. 1996 Aug;285(2):235–251. doi: 10.1007/s004410050641. [DOI] [PubMed] [Google Scholar]
  16. Newton C. L., Mignery G. A., Südhof T. C. Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J Biol Chem. 1994 Nov 18;269(46):28613–28619. [PubMed] [Google Scholar]
  17. Perez P. J., Ramos-Franco J., Fill M., Mignery G. A. Identification and functional reconstitution of the type 2 inositol 1,4,5-trisphosphate receptor from ventricular cardiac myocytes. J Biol Chem. 1997 Sep 19;272(38):23961–23969. doi: 10.1074/jbc.272.38.23961. [DOI] [PubMed] [Google Scholar]
  18. Ramos-Franco J., Fill M., Mignery G. A. Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels. Biophys J. 1998 Aug;75(2):834–839. doi: 10.1016/S0006-3495(98)77572-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Takei K., Mignery G. A., Mugnaini E., Südhof T. C., De Camilli P. Inositol 1,4,5-trisphosphate receptor causes formation of ER cisternal stacks in transfected fibroblasts and in cerebellar Purkinje cells. Neuron. 1994 Feb;12(2):327–342. doi: 10.1016/0896-6273(94)90275-5. [DOI] [PubMed] [Google Scholar]
  20. Watras J., Bezprozvanny I., Ehrlich B. E. Inositol 1,4,5-trisphosphate-gated channels in cerebellum: presence of multiple conductance states. J Neurosci. 1991 Oct;11(10):3239–3245. doi: 10.1523/JNEUROSCI.11-10-03239.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES