Abstract
Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-dependent channel activity in only 1.5% of patches. With K(+) (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.
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- Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
- Blondel O., Takeda J., Janssen H., Seino S., 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. 1993 May 25;268(15):11356–11363. [PubMed] [Google Scholar]
- Boehning D., Joseph S. K. Functional properties of recombinant type I and type III inositol 1, 4,5-trisphosphate receptor isoforms expressed in COS-7 cells. J Biol Chem. 2000 Jul 14;275(28):21492–21499. doi: 10.1074/jbc.M001724200. [DOI] [PubMed] [Google Scholar]
- 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]
- Hagar R. E., Burgstahler A. D., Nathanson M. H., Ehrlich B. E. Type III InsP3 receptor channel stays open in the presence of increased calcium. Nature. 1998 Nov 5;396(6706):81–84. doi: 10.1038/23954. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joseph S. K., Bokkala S., Boehning D., Zeigler S. Factors determining the composition of inositol trisphosphate receptor hetero-oligomers expressed in COS cells. J Biol Chem. 2000 May 26;275(21):16084–16090. doi: 10.1074/jbc.M000506200. [DOI] [PubMed] [Google Scholar]
- Joseph S. K., Lin C., Pierson S., Thomas A. P., Maranto A. R. Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells. J Biol Chem. 1995 Oct 6;270(40):23310–23316. doi: 10.1074/jbc.270.40.23310. [DOI] [PubMed] [Google Scholar]
- 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]
- Lin C., Widjaja J., Joseph S. K. The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor. J Biol Chem. 2000 Jan 28;275(4):2305–2311. doi: 10.1074/jbc.275.4.2305. [DOI] [PubMed] [Google Scholar]
- Mak D. O., Foskett J. K. Effects of divalent cations on single-channel conduction properties of Xenopus IP3 receptor. Am J Physiol. 1998 Jul;275(1 Pt 1):C179–C188. doi: 10.1152/ajpcell.1998.275.1.C179. [DOI] [PubMed] [Google Scholar]
- Mak D. O., Foskett J. K. Single-channel inositol 1,4,5-trisphosphate receptor currents revealed by patch clamp of isolated Xenopus oocyte nuclei. J Biol Chem. 1994 Nov 25;269(47):29375–29378. [PubMed] [Google Scholar]
- Mak D. O., Foskett J. K. Single-channel kinetics, inactivation, and spatial distribution of inositol trisphosphate (IP3) receptors in Xenopus oocyte nucleus. J Gen Physiol. 1997 May;109(5):571–587. doi: 10.1085/jgp.109.5.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mak D. O., McBride S., Foskett J. K. ATP regulation of type 1 inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of Ca(2+) activation. J Biol Chem. 1999 Aug 6;274(32):22231–22237. doi: 10.1074/jbc.274.32.22231. [DOI] [PubMed] [Google Scholar]
- Mak D. O., McBride S., Foskett J. K. Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15821–15825. doi: 10.1073/pnas.95.26.15821. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mak D. O., McBride S., Foskett J. K. Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors. J Gen Physiol. 2001 May;117(5):435–446. doi: 10.1085/jgp.117.5.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mak D. O., McBride S., Raghuram V., Yue Y., Joseph S. K., Foskett J. K. Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor. J Gen Physiol. 2000 Mar;115(3):241–256. doi: 10.1085/jgp.115.3.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marks A. R. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol. 1997 Feb;272(2 Pt 2):H597–H605. doi: 10.1152/ajpheart.1997.272.2.H597. [DOI] [PubMed] [Google Scholar]
- 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]
- Miyakawa T., Maeda A., Yamazawa T., Hirose K., Kurosaki T., Iino M. Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. EMBO J. 1999 Mar 1;18(5):1303–1308. doi: 10.1093/emboj/18.5.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Monkawa T., Miyawaki A., Sugiyama T., Yoneshima H., Yamamoto-Hino M., Furuichi T., Saruta T., Hasegawa M., Mikoshiba K. Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits. J Biol Chem. 1995 Jun 16;270(24):14700–14704. doi: 10.1074/jbc.270.24.14700. [DOI] [PubMed] [Google Scholar]
- 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]
- Nucifora F. C., Jr, Sharp A. H., Milgram S. L., Ross C. A. Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers. Mol Biol Cell. 1996 Jun;7(6):949–960. doi: 10.1091/mbc.7.6.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Patel S., Joseph S. K., Thomas A. P. Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium. 1999 Mar;25(3):247–264. doi: 10.1054/ceca.1999.0021. [DOI] [PubMed] [Google Scholar]
- Ramos-Franco J., Bare D., Caenepeel S., Nani A., Fill M., Mignery G. Single-channel function of recombinant type 2 inositol 1,4, 5-trisphosphate receptor. Biophys J. 2000 Sep;79(3):1388–1399. doi: 10.1016/S0006-3495(00)76391-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramos-Franco J., Caenepeel S., Fill M., Mignery G. Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants. Biophys J. 1998 Dec;75(6):2783–2793. doi: 10.1016/S0006-3495(98)77721-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Sigel E., Affolter H. Preparation and utilization of an ion-specific calcium minielectrode. Methods Enzymol. 1987;141:25–36. doi: 10.1016/0076-6879(87)41053-7. [DOI] [PubMed] [Google Scholar]
- Südhof T. C., Newton C. L., Archer B. T., 3rd, Ushkaryov Y. A., Mignery G. A. Structure of a novel InsP3 receptor. EMBO J. 1991 Nov;10(11):3199–3206. doi: 10.1002/j.1460-2075.1991.tb04882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taylor C. W., Genazzani A. A., Morris S. A. Expression of inositol trisphosphate receptors. Cell Calcium. 1999 Dec;26(6):237–251. doi: 10.1054/ceca.1999.0090. [DOI] [PubMed] [Google Scholar]
- Taylor C. W. Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+ channels. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):19–33. doi: 10.1016/s0005-2760(98)00122-2. [DOI] [PubMed] [Google Scholar]
- Thomas A. P., Bird G. S., Hajnóczky G., Robb-Gaspers L. D., Putney J. W., Jr Spatial and temporal aspects of cellular calcium signaling. FASEB J. 1996 Nov;10(13):1505–1517. [PubMed] [Google Scholar]
- 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]
- Wojcikiewicz R. J., He Y. Type I, II and III inositol 1,4,5-trisphosphate receptor co-immunoprecipitation as evidence for the existence of heterotetrameric receptor complexes. Biochem Biophys Res Commun. 1995 Aug 4;213(1):334–341. doi: 10.1006/bbrc.1995.2134. [DOI] [PubMed] [Google Scholar]
- Wojcikiewicz R. J. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J Biol Chem. 1995 May 12;270(19):11678–11683. doi: 10.1074/jbc.270.19.11678. [DOI] [PubMed] [Google Scholar]