ATP Binding to a Unique Site in the Type-1 S2- Inositol 1,4,5-Trisphosphate Receptor Defines Susceptibility to Phosphorylation by Protein Kinase A
2006; Elsevier BV; Volume: 281; Issue: 25 Linguagem: Inglês
10.1074/jbc.m601340200
ISSN1083-351X
AutoresLarry E. Wagner, Matthew J. Betzenhauser, David I. Yule,
Tópico(s)Cellular transport and secretion
ResumoThe subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2- InsP3R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors. The S2- InsP3R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2- InsP3R-1 or receptors containing a similar (Gly → Ala) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2- InsP3R or Gly → Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the G1690A mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2- InsP3R-1 and ultimately the sensitivity of cells to agonist stimulation. The subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2- InsP3R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors. The S2- InsP3R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2- InsP3R-1 or receptors containing a similar (Gly → Ala) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2- InsP3R or Gly → Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the G1690A mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2- InsP3R-1 and ultimately the sensitivity of cells to agonist stimulation. The release of Ca2+ from intracellular stores mediated by inositol 1,4,5-trisphosphate binding to inositol 1,4,5-trisphosphate receptors is a ubiquitous process important for control over a diverse array of physiological processes (1Bezprozvanny I. Cell Calcium. 2005; 38: 261-272Crossref PubMed Scopus (184) Google Scholar, 2Patterson R.L. 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The inositol 1,4,5-trisphosphate receptor (InsP3R) 3The abbreviations used are: InsP3R, inositol 1,4,5-trisphosphate receptor; InsP3, inositol 1,4,5-trisphosphate; CCh, carbamylcholine (carbachol); PKA, cAMP-dependent protein kinase; aa, amino acids; GST, glutathione S-transferase; TNP-ATP, trinitrophenyl-ATP; WT, wild type; TEV, tobacco etch virus. 3The abbreviations used are: InsP3R, inositol 1,4,5-trisphosphate receptor; InsP3, inositol 1,4,5-trisphosphate; CCh, carbamylcholine (carbachol); PKA, cAMP-dependent protein kinase; aa, amino acids; GST, glutathione S-transferase; TNP-ATP, trinitrophenyl-ATP; WT, wild type; TEV, tobacco etch virus. family is encoded by three genes, resulting in the expression of three distinct proteins with molecular mass of ∼300 kDa, named InsP3R-1, InsP3R-2, and InsP3R-3 (4Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (817) Google Scholar, 5Maranto A.R. J. Biol. 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Additional diversity exists at the protein level by the expression of several splice variants, particularly of the InsP3R-1 and InsP3R-2 (10Danoff S.K. Ferris C.D. Donath C. Fischer G.A. Munemitsu S. Ullrich A. Snyder S.H. Ross C.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2951-2955Crossref PubMed Scopus (213) Google Scholar, 11Iwai M. Tateishi Y. Hattori M. Mizutani A. Nakamura T. Futatsugi A. Inoue T. Furuichi T. Michikawa T. Mikoshiba K. J. Biol. Chem. 2005; 280: 10305-10317Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Nakagawa T. Shiota C. Okano H. Mikoshiba K. J. Neurochem. 1991; 57: 1807-1810Crossref PubMed Scopus (46) Google Scholar). The functional protein consists of four individual InsP3R monomers and can exist in both homo- and heterotetrameric forms (13Joseph S.K. Boehning D. Pierson S. Nicchitta C.V. J. Biol. Chem. 1997; 272: 1579-1588Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 14Wojcikiewicz R.J. He Y. Biochem. Biophys. Res. Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar, 15Galvan D.L. Borrego-Diaz E. Perez P.J. Mignery G.A. J. Biol. Chem. 1999; 274: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The domain organization for the InsP3R family is well established, with the linear structure divided into three general regions. The InsP3 binding core is toward the N terminus and was originally identified by mutagenesis studies to consist of a stretch of dispersed, positively charged amino acids critical for InsP3 binding (16Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 17Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 328-334Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This observation indicated the importance of the three-dimensional structure of the binding pocket in coordinating the negatively charged phosphate groups of InsP3, a proposal that was recently supported by solving the crystal structure of the InsP3R binding core (18Bosanac I. Alattia J.R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. Michikawa T. Mikoshiba K. Ikura M. Nature. 2002; 420: 696-700Crossref PubMed Scopus (272) Google Scholar). The ion-conducting pore of the InsP3R is toward the extreme C terminus of the protein. This region, termed the "channel domain," is predicted to span the endoplasmic reticulum membrane six times (19Ramos-Franco J. Galvan D. Mignery G.A. Fill M. J. Gen. Physiol. 1999; 114: 243-250Crossref PubMed Scopus (70) Google Scholar, 20Boehning D. Mak D.O. Foskett J.K. Joseph S.K. J. Biol. Chem. 2001; 276: 13509-13512Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and contains a motif between the fifth and sixth putative transmembrane span, which is conserved in potassium and calcium channels (GVGD) and has subsequently been demonstrated to constitute the ion-conducting pore of the InsP3R (20Boehning D. Mak D.O. Foskett J.K. Joseph S.K. J. Biol. Chem. 2001; 276: 13509-13512Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Whereas the InsP3R binding core and channel domain are highly conserved between InsP3R family members, the intervening ∼1700-amino acid region is appreciably more variable and has been called the "modulatory" or "regulatory and coupling domain." This designation is based on the presence of numerous loci for interaction with regulatory factors that influence the Ca2+ release properties of the receptor. Moreover, because of the variability in sequence in this region, modulation can potentially occur in an InsP3R-specific manner. The most important regulator of InsP3R activity is undoubtedly Ca2+ itself. Ca2+ acts as a co-agonist to facilitate Ca2+ release at low concentrations and inhibit channel activity at high concentrations (21Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1419) Google Scholar, 22Taylor C.W. Laude A.J. Cell Calcium. 2002; 32: 321-334Crossref PubMed Scopus (181) Google Scholar). Numerous additional factors, such as the binding of proteins, interaction with adenine nucleotides, and phosphorylation by numerous kinases, can also significantly influence the activity of the receptor (for reviews, see Refs. 1Bezprozvanny I. Cell Calcium. 2005; 38: 261-272Crossref PubMed Scopus (184) Google Scholar and 2Patterson R.L. Boehning D. Snyder S.H. Annu. Rev. Biochem. 2004; 73: 437-465Crossref PubMed Scopus (366) Google Scholar). For example, our laboratory has recently demonstrated that phosphorylation by cyclic nucleotide-dependent protein kinases can greatly augment the activity of the InsP3R-1 (23Wagner L.E. II Li Yule W. H.D.I. J. Biol. Chem. 2003; 278: 45811-45817Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 24Wagner L.E. II Li Joseph W.H. Yule S. K.D.I. J. Biol. Chem. 2004; 279: 46242-46252Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) (see Refs. 25Tu H. Miyakawa T. Wang Z. Glouchankova L. Iino M. Bezprozvanny I. Biophys. J. 2002; 82: 1995-2004Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 26Tang T.S. Tu H. Wang Z. Bezprozvanny I. J. Neurosci. 2003; 23: 403-415Crossref PubMed Google Scholar, 27Wojcikiewicz R.J. Luo S.G. J. Biol. Chem. 1998; 273: 5670-5677Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 28Nakade S. Rhee S.K. Hamanaka H. Mikoshiba K. J. Biol. Chem. 1994; 269: 6735-6742Abstract Full Text PDF PubMed Google Scholar, 29Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar). This event results in enhanced Ca2+ release and serves as a potentially important point of interaction between Ca2+ and cAMP-mediated signaling. Interestingly, whereas both the peripherally expressed S2- and neuronal S2+InsP3R-1 variant contain two identical consensus motifs for phosphorylation by cAMP-dependent protein kinase (PKA) (10Danoff S.K. Ferris C.D. Donath C. Fischer G.A. Munemitsu S. Ullrich A. Snyder S.H. Ross C.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2951-2955Crossref PubMed Scopus (213) Google Scholar, 30Soulsby M.D. Alzayady K. Xu Q. Wojcikiewicz R.J. FEBS Lett. 2004; 557: 181-184Crossref PubMed Scopus (26) Google Scholar) (at Ser-1589 and Ser-1755), one of which is also a motif predicted to be phosphorylated by cGMP-dependent protein kinase (Ser-1755), mutagenesis studies indicated that the particular functionally important phosphorylation sites were different in the individual splice variants. Specifically, PKA-dependent phosphorylation of only Ser-1755 led to enhanced Ca2+ release in the S2+ form, whereas phosphorylation of both sites was functionally relevant in the S2+ form was modulated following phosphorylation by cGMP-dependent protein kinase (23Wagner L.E. II Li Yule W. H.D.I. J. Biol. Chem. 2003; 278: 45811-45817Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The S2- (23Wagner L.E. II Li Yule W. H.D.I. J. Biol. Chem. 2003; 278: 45811-45817Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 24Wagner L.E. II Li Joseph W.H. Yule S. K.D.I. J. Biol. Chem. 2004; 279: 46242-46252Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Furthermore, only the S2+ InsP3R differs from the S2+ form by the excision of 39 amino acids (Glu-1693 to Arg-1731) between the two phosphorylation sites (see Fig. 1A). The structural or mechanistic grounds for this and other differences between the two splice variants, however, are presently unknown. Of interest, sequence analysis of the S2- InsP3R-1 indicates that the only striking difference from the S2+ InsP3R-1 is the presence of a glycinerich motif (Gly-X-Gly-X-X-Gly) at aa 1688-1732 (Fig. 1A) (31Ferris C.D. Snyder S.H. Annu. Rev. Physiol. 1992; 54: 469-488Crossref PubMed Scopus (196) Google Scholar, 32Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (367) Google Scholar, 33Maes K. Missiaen L. Parys J.B. Sienaert I. Bultynck G. Zizi M. De Smet P. Casteels R. De Smedt H. Cell Calcium. 1999; 25: 143-152Crossref PubMed Scopus (39) Google Scholar). This motif could potentially serve as a nucleotide binding fold and thus is suggestive of a role for S2 --specific effects mediated by the binding of adenine nucleotides. Indeed, adenine nucleotides, in particular ATP, have marked effects on InsP3R activity. Low concentrations of ATP have been shown to enhance the activity of both InsP3R-1 and InsP3R-3 but interestingly not InsP3R-2 (34Tu H. Wang Z. Nosyreva E. De Smedt H. Bezprozvanny I. Biophys. J. 2005; 88: 1046-1055Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 35Maes K. Missiaen L. De Smet P. Vanlingen S. Callewaert G. Parys J.B. De Smedt H. Cell Calcium. 2000; 27: 257-267Crossref PubMed Scopus (58) Google Scholar, 36Bezprozvanny I. Ehrlich B.E. Neuron. 1993; 10: 1175-1184Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 37Mak D.O. McBride S. Foskett J.K. J. Biol. Chem. 1999; 274: 22231-22237Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 38Miyakawa T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Iino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (336) Google Scholar). High concentrations of ATP inhibit activity of all InsP3R. The latter observation reflects ATP competing with InsP3 for binding to the receptor (39Maeda N. Kawasaki T. Nakade S. Yokota N. Taguchi T. Kasai M. Mikoshiba K. J. Biol. Chem. 1991; 266: 1109-1116Abstract Full Text PDF PubMed Google Scholar, 40Iino M. J. Gen. Physiol. 1991; 98: 681-698Crossref PubMed Scopus (79) Google Scholar), whereas the former is thought to occur by binding of ATP to specific sites in the receptor. The effect of ATP to enhance Ca2+ release has been proposed to occur by allosterically tuning the Ca2+ sensitivity of the receptor to favor activation (37Mak D.O. McBride S. Foskett J.K. J. Biol. Chem. 1999; 274: 22231-22237Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Nucleotide binding folds, based on the Gly-X-Gly-X-X-Gly motif (41Wierenga R.K. Hol W.G. Nature. 1983; 302: 842-844Crossref PubMed Scopus (268) Google Scholar), have been identified in individual InsP3R. A site spanning aa 2016-2021 in S2+InsP3R-1 is present in all InsP3R types, whereas a site at aa 1773-1780 is unique to the InsP3R-1 (see Fig. 1B). Both of these sites, termed ATPB and ATPA, respectively (1Bezprozvanny I. Cell Calcium. 2005; 38: 261-272Crossref PubMed Scopus (184) Google Scholar, 2Patterson R.L. Boehning D. Snyder S.H. Annu. Rev. Biochem. 2004; 73: 437-465Crossref PubMed Scopus (366) Google Scholar, 32Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (367) Google Scholar), have been shown to bind ATP in both the full-length receptor and when expressed individually as fusion proteins (42Maes K. Missiaen L. Parys J.B. De Smet P. Sienaert I. Waelkens E. Callewaert G. De Smedt H. J. Biol. Chem. 2001; 276: 3492-3497Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). It is, however, not known whether ATP binds to the unique putative site in the S2- form (termed ATPC by Tu et al. (25Tu H. Miyakawa T. Wang Z. Glouchankova L. Iino M. Bezprozvanny I. Biophys. J. 2002; 82: 1995-2004Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar)) or what, if any, functional consequences for InsP3R-1 activity this may have. In this study, we have constructed a GST fusion protein encompassing the putative nucleotide binding site corresponding to the ATPC site and confirmed that the motif indeed binds ATP with high affinity. In addition, by constructing a mutation within this motif, which fails to bind ATP, we have investigated any role this unique site plays in the specific properties of the peripheral S2- InsP3R-1 splice variant. Our studies demonstrate that binding of ATP to this site results in enhanced sensitivity of the InsP3R to InsP3. Surprisingly, our studies also demonstrate that the ATPC site plays a crucial role in defining the susceptibility of the S2-InsP3R-1 to phosphorylation by PKA. Thus, binding of ATP to the ATPC site in S2-InsP3R-1 is important in defining the functional sensitivity of the receptor when the InsP3R pathway is activated concurrently with PKA activation. Mutagenesis—potential ATP binding sites of the rat InsP R-1 S1-3/S2- isoform were mutated by sequential PCR. All numbering is based on the S2+InsP3R-1. The addition of an amino acid with a side chain on the second Gly of the ATP binding recognition motif Gly-X-Gly-X-X-ly is predicted to prevent the pyrophosphate moiety of ATP from interacting with the binding site. Mutation of the ATPC site in S2- InsP3R-1 (amino acids 1688-1732) was created by mutation of Gly-1690 to Ala (construct ΔATPC). ATPA was altered by mutating both Gly-1775 and Gly-1777 to Ala (construct ΔATPA). The ATPB site in S2-InsP3R-1 (aa 2016-2021) was altered by mutating Gly-2018 to Ala (construct ΔATPB). Creation of GST Fusion Proteins—GST fusion proteins were created using the pFN2A (GST) Flexi Vector (Promega, Madison, WI). Nucleotides corresponding to amino acids 1574-1765 (ATPC), 1756-1850 (ATPA), and 1944-2040 (ATPB) of the rat InsP3 R-1 S2- splice variant were amplified by PCR. SgfI and PmeI restriction sites were incorporated into the oligonucleotides used for PCR amplification. The PCR products were restriction enzyme-digested and ligated into pFN2A at the SgfI and PmeI sites. This creates a fusion construct with GST at the N terminus of the InsP3R-1 and a TEV protease recognition sequence in between to allow cleavage and removal of GST. WT S2-InsP3R-1 and a construct with G1690A mutation in this region were created and were verified by sequencing. BL21 (DE3) pLysS cells (Promega, Madison, WI) were transformed with each DNA construct. Individual colonies were picked and grown overnight in 10 ml of Luria broth supplemented with ampicillin. The overnight cultures were added to 990 ml of Luria broth. The 1000-ml cultures were grown for 2 h at 37°C and200rpm. Protein production was induced by the addition of 1 ml of 0.1 m isopropyl 1-thio-β-d-galactopyranoside (EMD Biosciences, San Diego, CA), and cultures were further incubated for 2 h. Cells were pelleted and lysed, and GST fusion protein was purified using a GST purification kit (BD Biosciences). This purified protein was then cleaved with TEV protease (Invitrogen), and GST was removed by running through a GST purification column. The GST fraction was bound to the glutathione column, whereas the InsP3R-1 protein immediately eluted off. Purified InsP3R-1 protein was concentrated using an Amicon Ultra-15 centrifugal filter device (Millipore Corp., Bedford, MA) and brought up to a final con centration of 0.5 mg/ml in 50 mm Tris buffer. Purified proteins were separated on 15% polyacrylamide gels and were either detected by Bio-Safe Coomassie Blue staining (Bio-Rad) or by immunoblotting with a monoclonal α-GST (Rockland, Gilbertsville, PA) antibody or a polyclonal antibody directed against the nonphosphorylated sequence flanking Ser-1755 (43Pieper A.A. Brat D.J. O'Hearn E. Krug D.K. Kaplin A.I. Takahashi K. Greenberg J.H. Ginty D. Molliver M.E. Snyder S.H. Neuroscience. 2001; 102: 433-444Crossref PubMed Scopus (33) Google Scholar) (kindly provided by Dr. S. Snyder). ATP Binding Assay—The fluorescent ATP analogue TNP-ATP (Molecular Probes, Inc., Carlsbad, CA) was used to measure binding of ATP to the purified InsP3R-1 protein fragments. TNP-ATP fluorescence increases upon binding to protein (44Hiratsuka T. Eur. J. Biochem. 2003; 270: 3479-3485Crossref PubMed Scopus (47) Google Scholar, 45Dong K. Tang L.Q. MacGregor G.G. Leng Q. Hebert S.C. EMBO J. 2005; 24: 1318-1329Crossref PubMed Scopus (13) Google Scholar) (excitation, 403 nm; emission, 546 nm). 1 mg of protein in 2 ml of 50 mm Tris-HCl was used for each assay. Increases in TNP-ATP fluorescence were detected using a PerkinElmer LS-5B luminescence spectrometer (Wellesley, MA). Increasing concentrations of TNP-ATP were added sequentially to cuvettes containing protein, and fluorescence was measured every second. Background fluorescence was determined by measuring TNP-ATP emission at various concentrations in Tris buffer alone. Net fluorescence for binding to protein was determined by subtracting background fluorescence. Normalized concentration-response relationships were created and fit with a logistic equation, and apparent EC50 values were calculated using OriginPro software (OriginLab, Northampton, MA). ATP competition assays were performed identically to the standard TNP-ATP binding assay, except protein-induced fluorescence was measured in the presence of 10 mm Na-ATP. Creation of Stable Muscarinic M3 Receptor-expressing DT40-3KO Cells—3× HA-tagged human M3R cDNA in pCDNA3.1 was obtained from the UMR cDNA resource center (available on the World Wide Web at www.cdna.org). MfeI-digested plasmid was introduced into DT40 cells lacking expression of all InsP3R types (38Miyakawa T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Iino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (336) Google Scholar, 46Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (373) Google Scholar) (DT40-3K0 cells) by nucleofection using program B23 and solution T as per the manufacturer's instructions (Amaxa, Inc.) to create the DT40-3KO-M3 stable cell line. After nucleofection, the cells were incubated in growth medium for 24 h prior to dilution in selection medium containing 2 mg/ml Geneticin (Invitrogen). Cells were then seeded into 96-well tissue culture plates at ∼1000 cells/well and incubated in selection medium for at least 7 days. Wells exhibiting growth after the selection period were picked for expansion. Transient Transfection of DT40-3KO Cells—For permeabilized cell studies, DT40-3KO cells were transfected by nucleofection, as described above. For concentration-response relationships in intact DT40-3KO-M3 cells, cells were transfected by electroporation at 350 V and 950 microfarads (4-mm gap cuvette). 2 × 107 cells were co-transfected with 25 μg of the InsP3R-1 cDNA, and 4 μg of the red fluorescent protein plasmid pHcRed1-N1 (BD Biosciences). For forskolin-induced PKA potentiation assays, DT40-3KO cells were co-transfected with 25 μg of the InsP3R-1 cDNA, 25 μg of the mouse type 3 muscarinic receptor, and 4 μg of the red fluorescent protein plasmid pHcRed1-N1 (BD Biosciences). Cells were incubated with DNA in 500 μl of Opti-MEM medium (Invitrogen) on ice for 10 min. The cell/DNA mixture was electroporated, incubated on ice for 30 min, increased to 5 ml with Opti-MEM, and placed in a 5% CO2 incubator at 39 °C for 5 h. The cells were then centrifuged and resuspended in 12 ml of complete RPMI medium (Invitrogen). Transfection efficiency was typically ∼20%. Experiments were performed within 32 h of transfection. Transfection of HEK-293 Cells and Assessment of Phosphorylation— HEK-293 cells were plated onto 25-cm2 culture flasks and allowed to grow to near confluence. Cells were transfected with 5 μg of each S2- InsP3R-1 DNA construct by using the Lipofectamine reagent (Invitrogen) as per the manufacturer's instructions. The following day, batches of cells were treated in the presence or absence of 20 μm forskolin for 10 min, aspirated from flasks, lysed in a buffer containing 50 mm Tris-HCl, 250 mm NaCl, 50 mm NaF, 5 mm EDTA, 0.1% Triton, and 1 mm Complete Protease inhibitor tablet (Roche Applied Science) at pH 7.4. InsP3R were immunoprecipitated with a polyclonal α-InsP3R-1 antibody that recognizes amino acids 2731-2749 of InsP3R-1 (α-InsP3R-1). Immunoprecipitates were separated on 5% SDS gels, transferred to nitrocellulose, and then probed with either a polyclonal antibody raised against the sequence flanking Ser-1755 of InsP3R-1 (α-S1755) or a polyclonal antibody raised against a similar region that specifically recognizes the phosphorylated state of Ser-1755 (α-S1755P). Blots that were probed with α-S1755P were stripped and reprobed with the α-S1755 antiserum to confirm the presence and relative quantity of the InsP3R-1. Digital Imaging of [Ca2+]i in Intact Cells—Transfected DT40-3KO cells were washed once in a HEPES-buffered physiological saline solution (HEPES-PSS) containing 5.5 mm glucose, 137 mm NaCl, 0.56 mm MgCl2, 4.7 mm KCl, 1 mm Na2HPO4, 10 mm HEPES (pH 7.4), 1.2 mm CaCl2, and 1% (w/v) bovine serum albumin. Cells were then resuspended in bovine serum albumin HEPES-PSS with 1 μm Fura-2 (AM) (Teflabs Inc., Austin, TX), placed on a 15-mm glass coverslip in a low volume perfusion chamber, and allowed to adhere for 30 min at room temperature. Cells were perfused continuously for 10 min with HEPES-PSS before experimentation to allow complete Fura-2 de-esterification. A field of cells for each experiment was chosen that contained a wide range of transfection efficiency based upon the intensity of red fluorescence emitted when excited at 560 nm. Individual cells that had emission gray levels between 1500 and 2500 were subsequently chosen to standardize expression levels. [Ca2+]i imaging was performed essentially as described previously, using an inverted epifluorescence Nikon microscope with a ×40 oil immersion objective lens (numerical aperture, 1.3). Cells were excited alternately with light at 340-nm and 380 ± 10-nm bandpass filters (Chroma, Rockingham, VT) using a monochrometer (TILL Photonics, Pleasanton, CA). Fluorescence images were captured and digitized with a digital camera driven by TILL Photonics software. Images were captured every 2 s withan exposure of 10 ms and 4 × 4 binning. 340/380 ratio images were calculated online and stored immediately to a hard disk. Permeabilized Cell Ca2+ Measurements—InsP3R expression constructs along with a nuclear targeted HcRed expression construct (pHcRed-nuc; BD Biosciences, Palo Alto, CA) were introduced into DT40-3KO cells using nucleofector protocol B23 and solution T (Amaxa Biosystems, Gaithersburg, MD). 8 μg of InsP3R DNA and 2 μg of pHcRed-nuc DNA were used in each nucleofection. Cells were loaded with 20 μm Furaptra-AM (Teflabs, Austin, TX) at 39 °C for1hin HEPES-PSS supplemented with 1% bovine serum albumin 16-20 h after nucleofection. Furaptra-loaded cells were permeabilized by superfusion for 1-2 min with 40 μm β-escin in intracellular medium (ICM) containing 125 mm KCl, 19 mm NaCl, 1.4 mm MgCl2, 0.33 mm CaCl2,10 mm HEPES, 3 mm ATP, 1 mm EGTA (pH 7.3). The free [Ca2+] was estimated to be 50 nm (MaxChelator). Permeabilized cells were then washed in ICM without β-escin for 5 min to facilitate removal of cytosolic dye. Transfected cells were identified by the presence of nuclear localized red fluorescence. The cells were then superfused in ICM containing 0.650 mm CaCl2 (free [Ca2+] of 200 nm) to load the intracellular stores. The experimental recordings presented are taken after establishment of a new stable base line following loading of the stores. The free [Ca2+] was subsequently maintained at a constant 200 nm throughout all experimental mane
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