Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m305378200
ISSN1083-351X
AutoresAyako Miyakawa-Naito, Per Uhlén, Mark Lal, Oleg Aizman, Katsuhiko Mikoshiba, Hjalmar Brismar, Sergey Zelenin, Anita Aperia,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoRecent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic α1-subunit abolished Ca2+ oscillations and downstream activation of NF-κB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor. Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic α1-subunit abolished Ca2+ oscillations and downstream activation of NF-κB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor. Na,K-ATPase is an integral membrane protein expressed in all eukaryotic cells, where it functions as a key regulator of intracellular Na+ and K+ concentrations (1.Skou J.C. Esmann M. J. Bioenerg. Biomembr. 1992; 24: 249-261PubMed Google Scholar). Recent studies, however, point to an additional role for Na,K-ATPase as a signal transducer (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar, 3.Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 4.Lichtstein D. McGowan M.H. Russell P. Carper D.A. Hypertens. Res. 2000; 23: S51-S53Crossref PubMed Scopus (11) Google Scholar, 5.Peng M. Huang L. Xie Z. Huang W.H. Askari A. J. Biol. Chem. 1996; 271: 10372-10378Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Importantly, Na,K-ATPase has an endogenous ligand, ouabain, a steroid hormone that dose-dependently inhibits the activity of Na,K-ATPase. The biological role of ouabain is, despite extensive research, not well understood. Ouabain belongs to the family of cardiac glycosides, which have been used for centuries in the treatment of heart disease. Recently, several investigators have noted that cardiac glycosides may act as anticancer agents (6.Haux J. Med. Hypotheses. 1999; 53: 543-548Crossref PubMed Scopus (140) Google Scholar, 7.Repke K.R. Schon R. Megges R. Weiland J. Nissen E. Matthes E. Anti-Cancer Drug Des. 1995; 10: 177-187PubMed Google Scholar). We have described a new cell signaling pathway triggered by ouabain (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar). Using rat renal proximal tubule (RPT) 1The abbreviations used are: RPTrenal proximal tubuleInsP3inositol 1,4,5-trisphosphateInsP3Rinositol 1,4,5-trisphosphate receptorFRETfluorescent resonance energy transferPLCphospholipase CERendoplasmic reticulumSERCAsarco-endoplasmic reticulum Ca2+ ATPaseCPAcyclopiazonic acid2-APB2-aminoethoxydiphenyl borateCytDcytochalasin DAQP4aquaporin-4GSTglutathione S-transferaseGFPgreen fluorescent proteinEGFPenhanced GFP.1The abbreviations used are: RPTrenal proximal tubuleInsP3inositol 1,4,5-trisphosphateInsP3Rinositol 1,4,5-trisphosphate receptorFRETfluorescent resonance energy transferPLCphospholipase CERendoplasmic reticulumSERCAsarco-endoplasmic reticulum Ca2+ ATPaseCPAcyclopiazonic acid2-APB2-aminoethoxydiphenyl borateCytDcytochalasin DAQP4aquaporin-4GSTglutathione S-transferaseGFPgreen fluorescent proteinEGFPenhanced GFP. cells, we showed that exposure to concentrations of ouabain that cause only partial inhibition of Na,K-ATPase activity induces slow intracellular Ca2+ oscillations and subsequent activation of the transcription factors NF-κB and cAMP-response element-binding protein. Our results from that study indicated that Ca2+ oscillations occurred as an interplay between different Ca2+ transporters and that Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) was involved in this event. Na,K-ATPase does not possess the characteristics of a G-protein-coupled receptor. Given the generality of Na,K-ATPase expression and its significant role in cell homeostasis, it is important to identify the molecular mechanisms by which Na,K-ATPase can act as a signal transducer. Here we show that the generation of Ca2+ oscillations by ouabain is dependent on the physical association of Na,K-ATPase and InsP3R in a signaling microdomain. renal proximal tubule inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor fluorescent resonance energy transfer phospholipase C endoplasmic reticulum sarco-endoplasmic reticulum Ca2+ ATPase cyclopiazonic acid 2-aminoethoxydiphenyl borate cytochalasin D aquaporin-4 glutathione S-transferase green fluorescent protein enhanced GFP. renal proximal tubule inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor fluorescent resonance energy transfer phospholipase C endoplasmic reticulum sarco-endoplasmic reticulum Ca2+ ATPase cyclopiazonic acid 2-aminoethoxydiphenyl borate cytochalasin D aquaporin-4 glutathione S-transferase green fluorescent protein enhanced GFP. Expression Plasmids—A cDNA fragment encoding wild type rat Na,K-ATPase α1-subunit (NKAα1) was amplified by AmpliTaq Gold (Applied Biosystems). The PCR product was digested by ApaI/XbaI restriction enzymes and cloned into pEGFP-C2 (Clontech) to obtain pGFP-NKAα1. A mutant NKAα1 with truncation of the first 32 amino acids (NKAα1.M32) was generated using PCR. The region of truncation of the NH2 terminus was decided on the basis of structure/function analysis reported elsewhere (8.Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; 271: 23683-23689Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The sense primer sequence was 5′-AAAGGGCCCATGGAAGTGTCTATGGACGAC-3′, corresponding to nucleotide positions 349–366 of NKAα1 (GenBank™ accession number NM_012504) with an additional ApaI site and ATG codon on the 5′-end of the primer. The antisense primer was 5′-CTTGCCGTGGAGGAGGATAGAACT-3′, corresponding to nucleotide positions 1792–1815 of NKAα1. The PCR product and pGFP-NKAα1 were hydrolyzed by ApaI/AflII restriction enzymes and ligated for cloning pGFP-NKAα1.M32. A fusion protein with NH2-terminal glutathione S-transferase (GST) and 95 amino acids of NKAα1 (GST-NKAα1.N95) was constructed using Gateway Technology (Invitrogen). Briefly, a cDNA fragment encoding 95 amino acids of the Na,K-ATPase α1-subunit NH2 terminus was amplified by AmpliTaq Gold (Applied Biosystems). The PCR product was cloned in pENTR/D-TOPO vector using pENTR Directional TOPO cloning kit (Invitrogen) and subcloned into pDEST-15 using Gateway System (Invitrogen) to obtain pGST-NKAα1.N95. The nucleotide sequences of all constructs were confirmed by automated sequencing (KISEQ, Core Facilities of Karolinska Institutet, Stockholm, Sweden) and subsequent bioinformatics analysis using Lasergene software (DNASTAR). For FRET control experiments, the cytosolic NH2 terminus of aquaporin-4 (AQP4) was tagged with GFP to obtain pGFP-AQP4 (9.Zelenina M. Zelenin S. Bondar A.A. Brismar H. Aperia A. Am. J. Physiol. 2002; 283: F309-F318Crossref PubMed Scopus (173) Google Scholar). InsP3R type 1 ligand binding protein (226–604 amino acids) with point mutation (R441Q) encoding InsP3 sponge was cloned into a pEF-BOS-MCS vector (pEF-GSTm49-IRES-GFP) (10.Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). pEGFP-actin was from Clontech. Cell Culture and Transfections—Three types of renal cells were used: primary cultures of rat RPT cells prepared as described (11.Uhlen P. Laestadius A. Jahnukainen T. Soderblom T. Backhed F. Celsi G. Brismar H. Normark S. Aperia A. Richter-Dahlfors A. Nature. 2000; 405: 694-697Crossref PubMed Scopus (200) Google Scholar), COS-7 cells, a cell line derived from fetal monkey kidney, and LLC-PK1 cells, a cell line derived from pig kidney. GFP-NKAα1 was stably expressed in COS-7 cells (12.Belusa R. Wang Z.M. Matsubara T. Sahlgren B. Dulubova I. Nairn A.C. Ruoslahti E. Greengard P. Aperia A. J. Biol. Chem. 1997; 272: 20179-20184Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). pGFP-NKAα1, pGFP-NKAα1.M32, pEF-GSTm49-IRES-GFP, or pEGFP-actin was transiently transfected into RPT cells on culture day 2 using CLONfectin (Clontech). pGFP-AQP4 was transiently transfected into COS-7 cells on culture day 2 using CLONfectin (Clontech). Reagents—Reagents were used at the following concentrations: 20 μm cyclopiazonic acid (CPA), 5 μm cytochalasin D (CytD), 100 pm-250 μm ouabain, 5 μm 2-aminoethoxydiphenyl borate (2-APB), 0.5 μm bradykinin, and 5 μm U73122. All reagents were from Sigma. Intracellular Calcium and Sodium Measurements—Intracellular Ca2+ and Na+ measurements were performed using Fura-2/AM and SBFI/AM (Molecular Probes), respectively, as described previously (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar, 11.Uhlen P. Laestadius A. Jahnukainen T. Soderblom T. Backhed F. Celsi G. Brismar H. Normark S. Aperia A. Richter-Dahlfors A. Nature. 2000; 405: 694-697Crossref PubMed Scopus (200) Google Scholar). After baseline recording, cells were treated, and ratio images were recorded every 30 s for 45–90 min. In each dish, 20–30 individual cells from a single cluster of cells were analyzed. Results presented are representative single cell traces obtained from a minimum of 12 experiments. Immunocytochemistry and Confocal Microscopy—For co-immunolocalization and FRET studies, COS-7 cells stably expressing GFP-NKAα1 were fixed with acetone for 3 min at room temperature and then incubated with phosphate-buffered saline containing 5% (v/v) normal goat serum and 3% (w/v) bovine serum albumin for 1 h. InsP3Rs were probed with monoclonal mouse anti-rat InsP3R type 2 (KM1083) or type 3 (KM1082) antibodies (1 μg/ml) (13.Sugiyama T. Furuya A. Monkawa T. Yamamoto-Hino M. Satoh S. Ohmori K. Miyawaki A. Hanai N. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 354: 149-154Crossref PubMed Scopus (82) Google Scholar) overnight at 4 °C. Cy3-conjugated goat anti-mouse IgG antibody served as secondary antibody (1:1000, Jackson ImmunoResearch Laboratories). Cells were scanned with a Leica TCS SP inverted confocal scanning laser microscope. NF-κB activation was measured in RPT cells by immunocytochemical staining as described previously (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar). RPT cells transiently transfected with pGFP-NKAα1.M32 were treated with 250 μm ouabain for 30 min and then fixed using 3% paraformaldehyde (10 min). Following blocking as described above, cells were incubated with NF-κB p65 antibody (1:200, Santa Cruz Biotechnology) for 1 h and then with Alexa 546 fluorescent secondary antibody (1:500, Molecular Probes) for 30 min. Slides were scanned using a Leica TCS SP inverted confocal scanning laser microscope, and images of cells expressing the construct were identified by GFP signal; NF-κB immunostaining of cells was captured for the same field of view. NF-κB activation in individual cells was semiquantitatively estimated by measuring the ratio between the mean NF-κB immunosignal in a given comparable area in the nucleus and cytoplasm in cells expressing GFP-NKAα1.M32 or those adjacent cells not expressing the construct using ImageJ (Wayne Rasband, National Institutes of Health). FRET—Fluorescent resonance energy transfer (FRET) measurements were performed on a Leica TCS SP inverted confocal scanning laser microscope using a ×40/1.4 NA objective. A detailed description of the FRET technique can be found elsewhere (14.Kenworthy A.K. Methods (Orlando). 2001; 24: 289-296Google Scholar, 15.Wouters F.S. Verveer P.J. Bastiaens P.I. Trends Cell Biol. 2001; 11: 203-211Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). The Förster constant, R0, for the donor-acceptor pair, GFP and Cy3, used in this study was 6 nm (16.Ng T. Squire A. Hansra G. Bornancin F. Prevostel C. Hanby A. Harris W. Barnes D. Schmidt S. Mellor H. Bastiaens P.I. Parker P.J. Science. 1999; 283: 2085-2089Crossref PubMed Scopus (267) Google Scholar). FRET occurs when the fluorophores are separated by distances 0.5 R0 < r < 2 R0. Thus, it is possible to distinguish proteins that are spatially co-localized within a 12-nm radius. To determine FRET, we quantified the quenching of donor fluorescence by performing acceptor photobleaching (14.Kenworthy A.K. Methods (Orlando). 2001; 24: 289-296Google Scholar). COS-7 cells stably expressing GFP-NKAα1 and stained with Cy3-labeled secondary goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories) to detect mouse monoclonal antibody to InsP3R2 (KM1083) and InsP3R3 (KM1082) were excited with 488 and 543 nm and collected separately. The acceptor, Cy3, was then irreversibly photobleached in a selected adequate region by continuous excitation with 543 and 633 nm lasers for 30–90 s. Thereafter, the residual Cy3 and GFP image was obtained, and identical regions, at the plasma membrane on individual cells, were outlined in the photobleached area and processed using ImageJ (Wayne Rasband, National Institutes of Health). Ratios between GFP intensities of the plasma membrane region, after and before photobleaching, were calculated to quantify FRET. The FRET values presented are corrected for erroneous intensity changes in a selected region outside the bleached area. In a typical experiment, 10–15 cells were measured for each sample. Immunoprecipitation Studies—Cells were solubilized in lysis buffer (50 mm Tris/HCL (pH 7.4), 150 mm NaCl, 0.25% sodium deoxycholate, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, protease inhibitors (Roche Applied Science)) and left for 30 min on ice. Cell lysates were sonicated (3 × 2 s at setting 2 using a Branson Sonifier 250, Branson Ultrasonics) and centrifuged at 9,000 × g at 4 °C to obtain a crude cell extract. 500 μg of supernatant protein/reaction were precleared with protein G-Sepharose for 1 h to reduce background that may be caused by non-specific adsorption of cellular debris. After a low speed centrifugation, the resultant supernatant was incubated for 1 h at 4 °C with a mouse monoclonal anti-Na,K-ATPase α1-subunit antibody (1:250; Upstate Biotechnology), a mouse monoclonal anti-InsP3R2 antibody (1:50; Santa Cruz Biotechnology), or a mouse monoclonal antibody anti-InsP3R3 antibody (1:50; BD Biosciences). Immunocomplexes were incubated with protein G-Sepharose beads overnight at 4 °C. Beads were pelleted, washed, and incubated with 2× Laemmli buffer, and supernatants were subjected to SDS gel electrophoresis using 6% acrylamide gels. Membranes were incubated overnight with a mouse monoclonal anti-InsP3R3 antibody (1 μg/ml; KM1082) and then for 1 h using a horseradish peroxidase-conjugated secondary antibody (1:5000) prior to detection using ECL plus (Amersham Biosciences). The resultant protein bands were scanned digitally and densitometrically analyzed by Bio-Rad QuantitativeOne software. GST Pull-down Assay—GST-NKAα1.N95 was produced in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B beads (Amersham Biosciences). Non-recombinant GST was used as a control. Detergent-extracted RPT cell lysate (prepared as described above for the co-immunoprecipitation protocol) was added to the beads in a 5:1 ratio (v/v) and incubated overnight at 4 °C with gentle rotation. Beads were washed and resuspended in 2× Laemmli buffer prior to SDS gel electrophoresis (6% gel) and immunoblotting for InsP3R3. Data Presentation and Analysis—Data are presented as means ± S.E. of a minimum of 10 experiments, unless indicated otherwise. Student's t test was used, and significance was accepted at p < 0.05. In accordance with previous observations (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar), ouabain (250 μm) induced highly regular intracellular Ca2+ oscillations with a periodicity in the minute range in RPT cells (Fig. 1a). Typical Ca2+ oscillations were detected about 5–15 min after ouabain exposure in approximately one-third of the cells and were generally initiated in one cell at the periphery of a cell cluster. Quantitatively and qualitatively, COS-7 cells treated with ouabain showed a similar Ca2+ oscillatory response (Fig. 1b). Spontaneous oscillations in cytosolic Ca2+ were never observed in untreated cells. Na,K-ATPase activity is dose-dependently inhibited by ouabain, and 250 μm ouabain causes ∼50% inhibition of rat Na,K-ATPase activity (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar). Ouabain, 250 μm, exceeds circulating levels in rat, estimated to be in the pm-nm range (17.Hamlyn J.M. Blaustein M.P. Bova S. DuCharme D.W. Harris D.W. Mandel F. Mathews W.R. Ludens J.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6259-6263Crossref PubMed Scopus (704) Google Scholar). When cells were exposed to physiological ouabain doses (100 pm), Ca2+ oscillations were observed (Fig. 1c) in ∼1% of cells. For subsequent experiments designed to explore the mechanism by which Na,K-ATPase triggers Ca2+ oscillations, we used 250 μm ouabain. To elucidate the source of the Ca2+ oscillatory response, intracellular endoplasmic reticulum (ER) Ca2+ stores were depleted by preincubation with a sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, CPA (Fig. 1d). Ouabain did not induce Ca2+ oscillations in CPA-pretreated cells. Regulated Ca2+ release from intracellular ER Ca2+ stores occurs mainly via InsP3Rs or via ryanodine receptors. InsP3Rs are abundantly expressed in RPT cells, whereas ryanodine receptors do not have any functional importance for ouabain-triggered Ca2+ oscillations in these cells (2.Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (242) Google Scholar). The membrane-permeable substance, 2-APB, was initially introduced as a specific inhibitor of InsP3Rs (18.Bootman M.D. Collins T.J. Mackenzie L. Roderick H.L. Berridge M.J. Peppiatt C.M. FASEB J. 2002; 16: 1145-1150Crossref PubMed Scopus (597) Google Scholar). The IC50 for inhibition of InsP3R-evoked Ca2+ release was reported to be 1–20 μm. Since then, 2-APB has, in addition to its inhibitory effect on InsP3-induced Ca2+ release, been shown to block store-operated calcium-mediated cytosolic Ca2+ influx (19.Peppiatt C.M. Collins T.J. Mackenzie L. Conway S.J. Holmes A.B. Bootman M.D. Berridge M.J. Seo J.T. Roderick H.L. Cell Calcium. 2003; 34: 97-108Crossref PubMed Scopus (229) Google Scholar). Store-operated calcium is generally fully inhibited by 50–100 μm 2-APB. Exposure of cells to concentrations of 2-APB higher than 100 μm may also cause a pronounced increase of basal cytosolic Ca2+, consistent with inhibition of the SERCA pump (19.Peppiatt C.M. Collins T.J. Mackenzie L. Conway S.J. Holmes A.B. Bootman M.D. Berridge M.J. Seo J.T. Roderick H.L. Cell Calcium. 2003; 34: 97-108Crossref PubMed Scopus (229) Google Scholar). Since available data suggest that low concentrations (up to 20 μm) of 2-APB will preferentially inhibit InsP3Rs, we tested the effect of 5 μm 2-APB. Using this concentration, we found that ouabain-induced Ca2+ oscillations were abolished in the majority of cells treated with 2-APB (5 μm) (Fig. 1e). Collectively, the inhibitory effects of CPA and 2-APB demonstrate that release of Ca2+ via InsP3Ris an essential contributor to the Ca2+ oscillations triggered by the ouabain/Na,K-ATPase complex. Activation of InsP3Rs is critically dependent on activation of phospholipase C (PLC), phosphatidylinositol lipid hydrolysis, and liberation of InsP3. Notably, however, recent studies indicate that InsP3R function is also modulated by interaction with accessory proteins (20.Tang T.S. Tu H. Chan E.Y. Maximov A. Wang Z. Wellington C.L. Hayden M.R. Bezprozvanny I. Neuron. 2003; 39: 227-239Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 21.Yang J. McBride S. Mak D.O. Vardi N. Palczewski K. Haeseleer F. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7711-7716Crossref PubMed Scopus (168) Google Scholar, 22.Johenning F.W. Ehrlich B.E. Neuron. 2002; 34: 173-175Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To examine the role of InsP3 for the ouabain-induced Ca2+ oscillations, RPT cells were transfected with a construct encoding a hyper-affinity InsP3 absorbent, an InsP3 sponge. The InsP3 sponge, having more than 1000-fold higher affinity for InsP3 than InsP3R, traps InsP3 and abrogates InsP3-induced Ca2+ release (10.Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The construct also encoded GFP to facilitate identification of transfected cells. Ouabain triggered low frequency Ca2+ oscillations in one-third of the cells expressing the InsP3 sponge (Fig. 1f). The amplitude of the oscillatory response was attenuated in some, but not all, of the cells expressing the InsP3 sponge. To confirm the efficiency of the InsP3 sponge in quenching InsP3-mediated Ca2+ signaling in RPT cells, we treated cells with bradykinin, a well known activator of PLC and InsP3 production (23.Felder C.C. FASEB J. 1995; 9: 619-625Crossref PubMed Scopus (449) Google Scholar). Bradykinin induced single Ca2+ transients in virtually all non-transfected cells but was without effect in all cells expressing the InsP3 sponge (Fig. 1g). Cells expressing only GFP exhibited regular ouabain-induced Ca2+ oscillations (data not shown). It was further found that preincubation of RPT cells with a PLC inhibitor, U73122, abolished bradykinin-induced Ca2+ transients (data not shown) but did not influence ouabain-induced Ca2+ oscillations (Fig. 1h). These findings indicate that ouabain-induced Ca2+ oscillations do not require increased InsP3 levels to activate InsP3R in this model. Immunocytochemical studies, performed on COS-7 cells, revealed partial co-localization of Na,K-ATPase with InsP3R types 1, 2, and 3 (InsP3R1, InsP3R2, and InsP3R3), respectively. Only InsP3R2 (Fig. 2a) and InsP3R3 (Fig. 2b) were studied in subsequent experiments since these isoforms were more abundantly expressed than InsP3R1. To investigate the spatial relationship between Na,K-ATPase and InsP3R on a nanometer scale, FRET measurements were performed. In this protocol, we used COS-7 cells stably expressing GFP-tagged Na,K-ATPase α1-subunit. These cells express approximately the same level of Na,K-ATPase as wild type COS-7 cells (12.Belusa R. Wang Z.M. Matsubara T. Sahlgren B. Dulubova I. Nairn A.C. Ruoslahti E. Greengard P. Aperia A. J. Biol. Chem. 1997; 272: 20179-20184Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). GFP, which was fused to the cytosolic NH2 terminus of Na,K-ATPase, served as FRET donor (GFP-NKAα1). The primary antibodies against InsP3R2 or InsP3R3 were probed with a Cy3-conjugated IgG secondary antibody, which served as the FRET acceptor (InsP3R-Cy3). The epitopes recognized by the InsP3R2 and InsP3R3 antibodies are located in the cytoplasmic COOH terminus of the respective InsP3Rs (13.Sugiyama T. Furuya A. Monkawa T. Yamamoto-Hino M. Satoh S. Ohmori K. Miyawaki A. Hanai N. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 354: 149-154Crossref PubMed Scopus (82) Google Scholar). The GFP-NKAα1 fluorescence intensity was, following acceptor photobleaching, enhanced 12.5 ± 0.9% for InsP3R2 and 15.5 ± 2.0% for InsP3R3 (Fig. 3, a and b). These results imply that the donor and acceptor complexes, GFP-NKAα1 and anti-InsP3R-anti-mouse IgG-Cy3, were separated less than 12 nm, i.e. the maximal distance for FRET detection between GFP and Cy3 (16.Ng T. Squire A. Hansra G. Bornancin F. Prevostel C. Hanby A. Harris W. Barnes D. Schmidt S. Mellor H. Bastiaens P.I. Parker P.J. Science. 1999; 283: 2085-2089Crossref PubMed Scopus (267) Google Scholar). Ouabain treatment significantly increased FRET (from 15.5 ± 2.0% to 25.0 ± 1.6%) between Na,K-ATPase and InsP3R (Fig. 3, a and b).Fig. 3Studies of Na,K-ATPase and InsP3R signaling microdomain.a and b, FRET measurements between Na,K-ATPase and InsP3R3. a, GFP-NKAα1 images of COS-7 cells with and without ouabain treatment before and after acceptor photobleaching (bleached area indicated by square). b, quantitative changes in emission intensities after bleaching as compared with before bleaching, mean ± S.E., *, p < 0.05. FRET was enhanced by ouabain. c–e, co-immunoprecipitation (IP) studies followed by Western blotting (WB) for InsP3R3. c and d, representative Western blot (c) and densitometric analysis (d) of InsP3R3 content in Na,K-ATPase immunoprecipitates before and after 250 μm ouabain treatment for 30 min in COS-7 cells. Ouabain significantly increased the amount of InsP3R3 associated with Na,K-ATPase, mean ± S.E. (n = 3), *, p < 0.05. Molecular mass markers are indicated to the left of the blot. In e, InsP3R3 co-immunoprecipitated with Na,K-ATPase and InsP3R2 in COS-7, RPT and LLC-PK1 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that the observed FRET between Na,K-ATPase and InsP3R3 was a unique property of this pair of proteins and not merely the result of non-specific experimental artifacts, we designed control experiments using another integral plasma membrane protein, namely AQP4. For these negative control experiments, GFP-AQP4 was expressed in COS-7 cells. FRET analysis was performed using GFP-AQP4 (donor) and the same Cy3-labeled secondary antibody to detect the InsP3R3 antibody (InsP3R3-Cy3, acceptor). No change in donor emission ratio before and after acceptor photobleaching was found for this molecular pair (data not shown). This result indicates that FRET between Na,K-ATPase and InsP3R3 is not likely a result of non-specific effects of the fixation protocol on plasma membrane and ER membrane integrity and strengthens the conclusion that the physical association between Na,K-ATPase and InsP3R3 is specific. Co-immunoprecipitation studies added further support to the concept that Na,K-ATPase and InsP3R are linked together in a microdomain. As shown in Fig. 3c, InsP3R3 co-immunoprecipitated with Na,K-ATPase in COS-7 cells. The amount of InsP3R3 that co-immunoprecipitated with Na,K-ATPase represented only a fraction (<50%) of the total InsP3R3 present in the initial cell lysate. Non-immune IgG did not co-immunoprecipitate a detectable amount of InsP3R3 (data not shown). Incubation of COS-7 cells with ouabain significantly increased the amount of InsP3R3 associated with immunoprecipitated Na,K-ATPase (Fig. 3, c and d). The propensity of InsP3R3 to co-immunoprecipitate with Na,K-ATPase was also demonstrated in RPT and LLC-PK1 cells (Fig. 3e). InsP3R isoforms form heterotetrametric channels (24.Monkawa T. Miyawaki A. Sugiyama T. Yoneshima H. Yamamoto-Hino M. Furuichi T. Saruta T. Hasegawa M. Mikoshiba K. J. Biol. Chem. 1995; 270: 14700-14704Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), and as expected, InsP3R2 co-immunoprecipitated with InsP3R3 in all cell types (Fig. 3e). Both Na,K-ATPase and InsP3R bind to cytoskeleton proteins that are anchored by the actin network (25.Bourguignon L.Y. Jin H. Iida N. Brandt N.R. Zhang S.H. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar, 26.Devarajan P. Scaramuzzino D.A. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2965-2969Crossref PubMed Scopus (131) Google Scholar). To examine whether the signaling function of the Na,K-ATPase/InsP3R complex depends on an intact cytoskeleton, RPT cells were pretreated with CytD to depolymerize the
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