Protein Kinase A Increases Type-2 Inositol 1,4,5-Trisphosphate Receptor Activity by Phosphorylation of Serine 937
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.010132
ISSN1083-351X
AutoresMatthew J. Betzenhauser, Jenna L. Fike, Larry E. Wagner, David I. Yule,
Tópico(s)Receptor Mechanisms and Signaling
ResumoProtein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) represents a mechanism for shaping intracellular Ca2+ signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca2+ release in cells that express predominantly InsP3R2. PKA is known to phosphorylate InsP3R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP3R2 in DT40-3KO cells that are devoid of endogenous InsP3R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca2+ signals and augmented the single channel open probability of InsP3R2. A PKA phosphorylation site unique to the InsP3R2 was identified at Ser937. The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser937, since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca2+ signaling following PKA activation in cells that express predominantly InsP3R2. Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) represents a mechanism for shaping intracellular Ca2+ signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca2+ release in cells that express predominantly InsP3R2. PKA is known to phosphorylate InsP3R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP3R2 in DT40-3KO cells that are devoid of endogenous InsP3R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca2+ signals and augmented the single channel open probability of InsP3R2. A PKA phosphorylation site unique to the InsP3R2 was identified at Ser937. The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser937, since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca2+ signaling following PKA activation in cells that express predominantly InsP3R2. Hormones, neurotransmitters, and growth factors stimulate the production of InsP3 3The abbreviations used are:InsP3inositol 1,4,5-trisphosphateInsP3RInsP3 receptorCChcarbacholM3Rhuman muscarinic M3RPKAprotein kinase AcBIMPs5,6-dichloro-1-β-d-ribofuranosylbenzylimadazole-3′,5′-cyclic monophosphorothioateGFPgreen fluorescent proteinEGFPenhanced GFPIBMXisobutylmethylxanthineBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. and Ca2+ signals in virtually all cell types (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). The ubiquitous nature of this mode of signaling dictates that this pathway does not exist in isolation; indeed, a multitude of additional signaling pathways can be activated simultaneously. A prime example of this type of "cross-talk" between independently activated signaling systems results from the parallel activation of cAMP and Ca2+ signaling pathways (2Bruce J.I. Straub S.V. Yule D.I. Cell Calcium. 2003; 34: 431-444Crossref PubMed Scopus (103) Google Scholar, 3Natarajan M. Lin K.M. Hsueh R.C. Sternweis P.C. Ranganathan R. Nat. Cell Biol. 2006; 8: 571-580Crossref PubMed Scopus (198) Google Scholar). Interactions between these two systems occur in numerous distinct cell types with various physiological consequences (3Natarajan M. Lin K.M. Hsueh R.C. Sternweis P.C. Ranganathan R. Nat. Cell Biol. 2006; 8: 571-580Crossref PubMed Scopus (198) Google Scholar, 4Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 5Straub S.V. Wagner 2nd, L.E. Bruce J.I. Yule D.I. Biol. Res. 2004; 37: 593-602Crossref PubMed Scopus (25) Google Scholar, 6Kang G. Joseph J.W. Chepurny O.G. Monaco M. Wheeler M.B. Bos J.L. Schwede F. Genieser H.G. Holz G.G. J. Biol. Chem. 2003; 278: 8279-8285Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Given the central role of InsP3R in Ca2+ signaling, a major route of modulating the spatial and temporal features of Ca2+ signals following cAMP production is potentially through PKA phosphorylation of the InsP3R isoform(s) expressed in a particular cell type. inositol 1,4,5-trisphosphate InsP3 receptor carbachol human muscarinic M3R protein kinase A 5,6-dichloro-1-β-d-ribofuranosylbenzylimadazole-3′,5′-cyclic monophosphorothioate green fluorescent protein enhanced GFP isobutylmethylxanthine 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. There are three InsP3R isoforms (InsP3R1, InsP3R2, and InsP3R3) expressed to varying degrees in mammalian cells (7Taylor C.W. Genazzani A.A. Morris S.A. Cell Calcium. 1999; 26: 237-251Crossref PubMed Scopus (245) Google Scholar, 8Wojcikiewicz R.J. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). InsP3R1 is the major isoform expressed in the nervous system, but it is less abundant compared with other subtypes in non-neuronal tissues (8Wojcikiewicz R.J. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Ca2+ release via InsP3R2 and InsP3R3 predominate in these tissues. InsP3R2 is the major InsP3R isoform in many cell types, including hepatocytes (7Taylor C.W. Genazzani A.A. Morris S.A. Cell Calcium. 1999; 26: 237-251Crossref PubMed Scopus (245) Google Scholar, 8Wojcikiewicz R.J. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), astrocytes (9Holtzclaw L.A. Pandhit S. Bare D.J. Mignery G.A. Russell J.T. Glia. 2002; 39: 69-84Crossref PubMed Scopus (94) Google Scholar, 10Petravicz J. Fiacco T.A. McCarthy K.D. J. Neurosci. 2008; 28: 4967-4973Crossref PubMed Scopus (253) Google Scholar), cardiac myocytes (11Li X. Zima A.V. Sheikh F. Blatter L.A. Chen J. Circ. Res. 2005; 96: 1274-1281Crossref PubMed Scopus (270) Google Scholar), and exocrine acinar cells (8Wojcikiewicz R.J. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 12Zhang X. Wen J. Bidasee K.R. Besch Jr., H.R. Wojcikiewicz R.J. Lee B. Rubin R.P. Biochem. J. 1999; 340: 519-527Crossref PubMed Google Scholar). Activation of PKA has been demonstrated to enhance InsP3-induced Ca2+ signaling in hepatocytes (13Burgess G.M. Bird G.S. Obie J.F. Putney Jr., J.W. J. Biol. Chem. 1991; 266: 4772-4781Abstract Full Text PDF PubMed Google Scholar) and parotid acinar cells (4Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 14Hirono C. Sugita M. Furuya K. Yamagishi S. Shiba Y. J. Membr. Biol. 1998; 164: 197-203Crossref PubMed Scopus (26) Google Scholar). Although PKA phosphorylation of InsP3R2 is a likely causal mechanism underlying these effects, the functional effects of phosphorylation have not been determined in cells unambiguously expressing InsP3R2 in isolation. Furthermore, the molecular determinants of PKA phosphorylation of this isoform are not known. PKA-mediated phosphorylation is an efficient means of transiently and reversibly regulating the activity of the InsP3R. InsP3R1 was identified as a major substrate of PKA in the brain prior to its identification as the InsP3R (15Walaas S.I. Nairn A.C. Greengard P. J. Neurosci. 1986; 6: 954-961Crossref PubMed Google Scholar, 16Supattapone S. Danoff S.K. Theibert A. Joseph S.K. Steiner J. Snyder S.H. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 8747-8750Crossref PubMed Scopus (291) Google Scholar). However, until recently, the functional consequences of phosphorylation were unresolved. Initial conflicting results were reported indicating that phosphoregulation of InsP3R1 could result in either inhibition or stimulation of receptor activity (16Supattapone S. Danoff S.K. Theibert A. Joseph S.K. Steiner J. Snyder S.H. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 8747-8750Crossref PubMed Scopus (291) Google Scholar, 17Volpe P. Alderson-Lang B.H. Am. J. Physiol. 1990; 258: C1086-C1091Crossref PubMed Google Scholar). Mutagenic strategies were employed by our laboratory to clarify this discrepancy. These studies unequivocally assigned phosphorylation-dependent enhanced Ca2+ release and InsP3R1 activity at the single channel level, through phosphorylation at canonical PKA consensus motifs at Ser1589 and Ser1755. The sites responsible were also shown to be specific to the particular InsP3R1 splice variant (18Wagner 2nd, L.E. Li W.H. Yule D.I. J. Biol. Chem. 2003; 278: 45811-45817Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These data were also corroborated by replacing the relevant serines with glutamates in a strategy designed to construct "phosphomimetic" InsP3R1 by mimicking the negative charge added by phosphorylation (19Wagner 2nd, L.E. Li W.H. Joseph S.K. Yule D.I. J. Biol. Chem. 2004; 279: 46242-46252Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Wagner 2nd, L.E. Joseph S.K. Yule D.I. J. Physiol. 2008; 586: 3577-3596Crossref PubMed Scopus (73) Google Scholar). Of particular note, however, although all three isoforms are substrates for PKA, neither of the sites phosphorylated by PKA in InsP3R1 are conserved in the other two isoforms (21Wojcikiewicz R.J. Luo S.G. J. Biol. Chem. 1998; 273: 5670-5677Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Recently, three distinct PKA phosphorylation sites were identified in InsP3R3 that were in different regions of the protein when compared with InsP3R1 (22Soulsby M.D. Wojcikiewicz R.J. Biochem. J. 2005; 392: 493-497Crossref PubMed Scopus (55) Google Scholar). To date, no PKA phosphorylation sites have been identified in InsP3R2. Interactions between Ca2+ and cAMP signaling pathways are evident in exocrine acinar cells of the parotid salivary gland. In these cells, both signals are important mediators of fluid and protein secretion (23Melvin J.E. Yule D. Shuttleworth T. Begenisich T. Annu. Rev. Physiol. 2005; 67: 445-469Crossref PubMed Scopus (335) Google Scholar). Multiple components of the [Ca2+]i signaling pathway in these cells are potential substrates for modulation by PKA. Previous work from this laboratory established that activation of PKA potentiates muscarinic acetylcholine receptor-induced [Ca2+]i signaling in mouse and human parotid acinar cells (4Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 24Giovannucci D.R. Groblewski G.E. Sneyd J. Yule D.I. J. Biol. Chem. 2000; 275: 33704-33711Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25Straub S.V. Giovannucci D.R. Bruce J.I. Yule D.I. J. Biol. Chem. 2002; 277: 31949-31956Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A likely mechanism to explain this effect is that PKA phosphorylation increases the activity of InsP3R expressed in these cells. Consistent with this idea, activation of PKA enhanced InsP3-induced Ca2+ release in permeabilized mouse parotid acinar cells and also resulted in the phosphorylation of InsP3R2 (4Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Invariably, prior work examining the functional effects of PKA phosphorylation on InsP3R2 has been performed using cell types expressing multiple InsP3R isoforms. For example, AR4-2J cells are the preferred cell type for examining InsP3R2 in relative isolation, because this isoform constitutes more than 85% of the total InsP3R population (8Wojcikiewicz R.J. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). InsP3R1, however, contributes up to ∼12% of the total InsP3R in AR4-2J cells. An initial report using InsP3-mediated 45Ca2+ flux suggested that PKA activation increased InsP3R activity in AR4-2J cells (21Wojcikiewicz R.J. Luo S.G. J. Biol. Chem. 1998; 273: 5670-5677Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). A similar conclusion was made in a later study, which documented the effects of PKA activation on agonist stimulated Ca2+ signals in AR4-2J cells (26Regimbald-Dumas Y. Arguin G. Fregeau M.O. Guillemette G. J. Cell. Biochem. 2007; 101: 609-618Crossref PubMed Scopus (17) Google Scholar). Any effects of phosphorylation observed in these experiments could plausibly have resulted from phosphorylation of the residual InsP3R1. Although PKA enhances InsP3-induced calcium release in cells expressing predominantly InsP3R2, including hepatocytes, parotid acinar cells, and AR4-2J cells (4Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 13Burgess G.M. Bird G.S. Obie J.F. Putney Jr., J.W. J. Biol. Chem. 1991; 266: 4772-4781Abstract Full Text PDF PubMed Google Scholar, 21Wojcikiewicz R.J. Luo S.G. J. Biol. Chem. 1998; 273: 5670-5677Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 26Regimbald-Dumas Y. Arguin G. Fregeau M.O. Guillemette G. J. Cell. Biochem. 2007; 101: 609-618Crossref PubMed Scopus (17) Google Scholar, 27Hajnóczky G. Gao E. Nomura T. Hoek J.B. Thomas A.P. Biochem. J. 1993; 293: 413-422Crossref PubMed Scopus (75) Google Scholar), InsP3R2 is not phosphorylated at stoichiometric levels by PKA (21Wojcikiewicz R.J. Luo S.G. J. Biol. Chem. 1998; 273: 5670-5677Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). This observation has called into question the physiological significance of PKA phosphorylation of InsP3R2 (28Soulsby M.D. Wojcikiewicz R.J. Cell Calcium. 2007; 42: 261-270Crossref PubMed Scopus (27) Google Scholar). The apparent low levels of InsP3R2 phosphorylation are clearly at odds with the augmented Ca2+ release observed in cells expressing predominantly this isoform. The equivocal nature of these findings probably stems from the fact that, to date, all of the studies demonstrating positive effects of PKA activation on Ca2+ release were conducted in cells that also express InsP3R1. The purpose of the current experiments was to analyze the functional effects of phosphorylation on InsP3R2 expressed in isolation on a null background. We report that InsP3R2 activity is increased by PKA phosphorylation under these conditions, and furthermore, we have identified a unique phosphorylation site in InsP3R2 at Ser937. In total, these results provide a direct mechanism for the cAMP-induced activation of InsP3R2 via PKA phosphorylation of InsP3R2. Mouse InsP3R2 cDNA, a kind gift of Dr. Katsuhiko Mikoshiba (Riken, Japan), was used as the template for creation of EGFP-tagged subclones (29Iwai 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 (93) Google Scholar). All fragments were amplified by PCR with MluI and NotI restriction sites incorporated into the N termini and C termini, respectively. A BssHII site was incorporated into the N terminus of fragment 3 because of an internal MluI site in this fragment. PCR products were ligated with MluI- and NotI-digested pCI-Neo-EGFP (a kind gift from Dr. Sundeep Malik, University of Rochester) to create the mammalian expression vectors. The full-length rat InsP3R2 in the expression vector pCMV5 was a kind gift of Dr. Suresh Joseph (Thomas Jefferson University). A Kozak initiation motif was engineered into the receptor DNA using PCR. The modified receptor DNA was then cloned into the pEF6/V5-His Topo TA expression vector (Invitrogen). All point mutations in the InsP3R2 fragments were created using QuikChange XL or QuikChange multisite mutagenesis (Stratagene, La Jolla, CA). Point mutations in the full-length InsP3R2 expression constructs to allow S937A and S2633A amino acid substitutions were constructed using a two-step QuikChange mutagenesis strategy (30Wang W. Malcolm B.A. Methods Mol. Biol. 2002; 182: 37-43PubMed Google Scholar). The cDNA construct for 3× hemagglutinin-tagged human type-3 muscarinic receptor (M3R) and wild type and mutated InsP3R2 constructs were linearized with MfeI. Linearized constructs were introduced into DT40-3KO cells, which are devoid of InsP3R, by nucleofection using an Amaxa nucleofector as described previously (31Betzenhauser M.J. Wagner 2nd, L.E. Iwai M. Michikawa T. Mikoshiba K. Yule D.I. J. Biol. Chem. 2008; 283: 21579-21587Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 32Betzenhauser M.J. Wagner 2nd, L.E. Won J.H. Yule D.I. Methods. 2008; 46: 177-182Crossref PubMed Scopus (24) Google Scholar, 33Wagner 2nd, L.E. Betzenhauser M.J. Yule D.I. J. Biol. Chem. 2006; 281: 17410-17419Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). After nucleofection, the cells were incubated in growth medium for 24 h prior to dilution in selection medium containing 2 mg/ml Geneticin. 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. COS-7 cells were transfected with InsP3R2 expression constructs 36–40 h prior to labeling with 32PO4− for 2 h in phosphate-free Dulbecco's modified Eagle's medium. ∼150 μCi were added to 1 ml of phosphate-free media in each well of a 6-well culture dish. After labeling, cells were washed three times in Tris-buffered saline, treated with forskolin for 15 min, and lysed in lysis buffer (10 mm Tris, 150 mm NaCl, 100 mm NaF, 1 EDTA, 1% Nonidet P-40, pH 7.4) supplemented with protease inhibitor tablets (Roche Applied Science). Lysates were cleared with pansorbin prior to incubation with immunoprecipitation antibody for 2 h. Protein A/G beads were then added for 1 h. The beads were washed three times in lysis buffer and resuspended in Laemmli sample buffer. Samples were separated by PAGE on 5% polyacrylamide, and the gels were dried for phosphorimaging using a phosphor storage screen (Amersham Biosciences) and Amersham Biosciences PhosphorImager. COS-7 cells were transfected with wild type or mutated InsP3R2 expression constructs 36–40 h before harvest. Cells were washed twice in phosphate-buffered saline prior to suspension in lysis buffer supplemented with protease inhibitor mixture tablets (Roche Applied Science). Cells were dispersed using a cell scraper (Corning Glass) and allowed to incubate on ice for 20 min. Lysates were cleared with pansorbin prior to incubation with immunoprecipitation antibody for 2 h. Protein A/G beads were then added for 1 h. The beads were washed three times in lysis buffer and three times in PKA phosphorylation buffer (120 mm KCl, 50 mm Tris, 0.1% Triton X-100, 0.3 mm MgCl2, pH 7.2) and resuspended in phosphorylation buffer. 20 units of purified recombinant PKA (Promega) were added to the samples along with [γ-32P]ATP (∼5 μCi/reaction) and 0.5 μm unlabeled ATP. Kinase reactions were incubated for 0–15 min, and the beads were washed six times in lysis buffer prior to resuspension and SDS-PAGE. Gels were stained with BioSafe Coomassie (Bio-Rad) and subsequently dried down for phosphorimaging. 32P signals were detected by phosphorimaging as described above. Rabbit polyclonal antibodies designed against a specific sequence in the rat InsP3R2 extreme C terminus (α-InsP3R2-CT; 2686GFLGSNTPHENHHMPPH2702) were generated by Pocono Rabbit Farms & Laboratories (Canadensis, PA). A rabbit polyclonal antibody against the region surrounding phosphorylated Ser937 of mouse InsP3R2 (934SRGpSIFPVSVPDAC946, where pS represents phosphoserine) was generated by Quality Controlled Biochemicals (Hopkinton, MA). Mouse parotid acinar cells were isolated by collagenase digestion, as described previously (4Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). COS-7 cells or mouse parotid acinar cells were treated with 10 μm forskolin and 100 μm IBMX for 15 min. Cell lysates were harvested in lysis buffer supplemented with 100 nm okadaic acid. InsP3R2 was immunoprecipitated from cell lysates using α-InsP3R2-CT. Where noted, some immunoprecipitated samples were treated with 10 units of calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) at 37 °C for 1 h. Samples were separated on SDS-PAGE, and phosphorylated serine 937 was detected with α-Ser(P)937 by Western blotting. Blots were stripped and reprobed with α-InsP3R2-CT to verify equal amounts of total immunoprecipitated InsP3R2. DT40 cells were loaded with 2 μm of the Ca2+-sensitive dye Fura-2 AM at room temperature for 15–30 min. Fura-2-loaded cells were allowed to adhere to a glass coverslip at the bottom of a perfusion chamber. Cells were perfused in HEPES-buffered physiological saline containing 137 mm NaCl, 0.56 mm MgCl2, 4.7 mm KCl, 1 mm Na2HPO4, 10 mm HEPES, 5.5 mm glucose, and 1.26 mm CaCl2, pH 7.4. Imaging was performed using an inverted Nikon microscope through a ×40 oil immersion objective lens (numerical aperture, 1.3). Fura-2-loaded cells were excited alternately with light at 340 and 380 nm by using a monochrometer-based illumination system (TILL Photonics), and the emission at 510 nm was captured by using a digital frame transfer CCD camera. In experiments where InsP3R or M3R were transiently expressed, cDNA encoding HcRed was included to indicate transfected cells. HcRed fluorescence was detected by excitation at 560 nm and observing the emission at >600 nm. Whole cell patch clamp recordings of single InsP3R2 channel activity present in the plasma membrane (34Dellis O. Dedos S.G. Tovey S.C. Taufiq-Ur-Rahman Dubel S.J. Taylor C.W. Science. 2006; 313: 229-233Crossref PubMed Scopus (167) Google Scholar, 35Dellis O. Rossi A.M. Dedos S.G. Taylor C.W. J. Biol. Chem. 2008; 283: 751-755Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) were made from DT40-3KO cells stably expressing mouse InsP3R2. K+ was utilized as the charge carrier in all experiments, and free Ca2+ was clamped at 200 nm to favor activation of InsP3R (bath: 140 mm KCl, 10 mm HEPES, 500 μm BAPTA, free Ca2+ 250 nm (pH 7.1); pipette: 140 mm KCl, 10 mm HEPES, 100 μm BAPTA, 200 nm free Ca2+, 5 mm Na2-ATP unless otherwise noted (pH 7.1)). Borosilicate glass pipettes were pulled and fire-polished to resistances of about 20 megaohms. Following establishment of stable high resistance seals, the membrane patches were ruptured to form the whole cell configuration with resistances >5 gigaohms and capacitances of >8 picofarads. Currents were recorded under voltage clamp conditions at −100 mV using an Axopatch 200B amplifier and pClamp 9. Channel recordings were digitized at 20 kHz and filtered at 5 kHz with a −3 dB, 4-pole Bessel filter. Activity was typically evident essentially immediately following breakthrough with InsP3 in the pipette. Analyses were performed using the event detection protocol in Clampfit 9. Channel openings were detected by half-threshold crossing criteria. We assumed that the number of channels in any particular cell is represented by the maximum number of discrete stacked events observed during the experiment. The single channel open probability (Po) was calculated using the multimodal distribution for the open and closed current levels. Analyzing subtype-specific regulation of individual InsP3R isoforms in native tissue is hampered by the fact that most mammalian cell types express multiple isoforms and that the functional receptors can form heterotetrameric channels (36Wojcikiewicz R.J. He Y. Biochem. Biophys. Res. Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar, 37Swatton J.E. Taylor C.W. J. Biol. Chem. 2002; 277: 17571-17579Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 38Joseph S.K. Lin C. Pierson S. Thomas A.P. Maranto A.R. J. Biol. Chem. 1995; 270: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Given these limitations, the functional properties of specific homotetrameric receptors must be determined in a defined system. Kurosaki and colleagues (40Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar) developed such a system based on the DT40 chicken B-cell precursor line by creating a null background (DT40-3KO cells) following elimination of all three InsP3R isoforms through homologous recombination (39Maes 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, 40Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar). In order to examine the effects of PKA phosphorylation on InsP3R2, we transiently expressed this isoform in a DT40-3KO cell line (DT40-M3) stably expressing the human M3R (33Wagner 2nd, L.E. Betzenhauser M.J. Yule D.I. J. Biol. Chem. 2006; 281: 17410-17419Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). DT40-M3 cells transfected with mouse InsP3R2 cDNA, identified by expression of HcRed, responded to the muscarinic agonist carbachol (CCh) stimulation in a concentration-dependent manner (Fig. 1A). Treatment with 1 μm CCh produced Ca2+ signals with a range of amplitudes, presumably as a consequence of a range of InsP3R2 expression levels following transient transfection (Fig. 1B). Cells that responded to 1 μm CCh with amplitudes no greater than 0.2 340 nm/380 nm ratio units were used for the purposes of analyzing the effects of raising cAMP. An example of a DT40-M3 cell expressing mouse InsP3R2 and treated three times with 1 μm CCh is shown in Fig. 1C. Treatment of cells with 20 μm forskolin after the first CCh treatment resulted in Ca2+ transients with >5-fold larger amplitudes as shown in Fig. 1C, indicating that activation of PKA enhances Ca2+ release from InsP3R2. This effect was only evident in cells that responded submaximally to 1 μm CCh. As previously observed, forskolin induced a rise in [Ca2+]i in some cells (27Hajnóczky G. Gao E. Nomura T. Hoek J.B. Thomas A.P. Biochem. J. 1993; 293: 413-422Crossref PubMed Scopus (75) Google Scholar). This effect was evident in ∼50% of cells from six separate experiments, and these cells were excluded from analysis. The mechanism underlying this effect is presently unknown; however, it is independent of InsP3R as it occurs in DT40-3KO cells at approximately the same frequency. The activation of PKA by this treatment had clear enhancing effects on the Ca2+ signal (Fig. 1D). We also obtained additional evidence that PKA-induced phosphorylation results in increased InsP3R2 activity by examining the effects of activating PKA on the single channel activity of InsP3R2. Single InsP3R2 measurements were conducted using whole cell recordings of plasma membrane-resident InsP3R2 in DT40 cells stably expressing InsP3R2 (20Wagner 2nd, L.E. Joseph S.K. Yule D.I. J. Physiol. 2008; 586: 3577-3596Crossref PubMed Scopus (73) Google Scholar, 31Betzenhauser M.J. Wagner 2nd, L.E. Iwai M. Michikawa T. Mikoshiba K. Yule D.I. J. Biol. Chem. 2008; 283: 21579-21587Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 34Dellis O. Dedos S.G. Tovey S.C. Taufiq-Ur-Rahman Dubel S.J. Taylor C.W. Science. 2006; 313: 229-233Crossref PubMed Scopus (167) Google Scholar, 35Dellis O. Rossi A.M. Dedos S.G. Taylor C.W. J. Biol. Chem. 2008; 283: 751-755Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 41Schug Z.T. da Fonseca P.C. Bhanumathy C.D. Wagner 2nd, L. Zhang X. Bailey B. Morris E.P. Yule D.I. Joseph S.K. J. Biol. Chem. 2008; 283: 2939-2948Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This configuration allows the monitoring of single InsP3R channels during the activation of endogenous PKA following exposure to forskolin (20Wagner 2nd, L.E. Joseph S.K. Yule D.I. J. Physiol. 2008; 586: 3577-3596Crossref PubMed Scopus (73) Google Scholar). Fig. 2A shows an example of channel activity when low concentrations of InsP3 (100 nm) were included in the recording pipette. No channel activity is observed in this preparation in the absence of InsP3 or in DT40-3KO cells devoid of InsP3R (31Betzenhauser M.J. Wagner 2nd, L.E. Iwai M. Michikawa T. Mikoshiba K. Yule D.I. J. Biol. Chem. 2008; 283: 21579-21587Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The open probability of the channel was markedly enhanced following exposure to forskolin (Fig. 2, B (diary plot for representative cell) and C (pooled data)). InsP3R2 channel activity returned to pre-PKA activation levels following washout of forskolin. Enhanced channel activity was readily evident at threshold [InsP3] but not observed at higher levels of InsP3 (1 μm; Fig. 2C). In total, these data provide clear evidence that activation of PKA results in enhanced Ca2+ release through increased activity of InsP3R2. A likely mechanism for the positive effects of increasing cAMP on the Ca2+ signal is through direct phosphorylation of InsP3R2 by PKA. Because this signaling system is a rich source of potential PKA substrates, we cannot, however, discount other effects of PKA on the M3R-induced Ca2+ signals. Potential loci might include effects on InsP3 levels, Ca2+ clearance, or Ca2+ entry. In addition, cAMP at millimolar concentrations was recently reported to enhance Ca2+ release from InsP3R2 by a mechanism that did not require PKA activity (42Tovey S.C. Dedos S.G. Taylor E.J. Church J.E. Taylor C.W. J. Cell Biol. 2008; 183: 297-311Crossref PubMed Scopus (79) Google Scholar). Establishing the P
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