Sodium/Calcium Exchanger (NCX1) Macromolecular Complex
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m300754200
ISSN1083-351X
AutoresDan H. Schulze, Muqeem Muqhal, W. J. Lederer, A. Ruknudin,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings. The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings. The Na+/Ca2+ exchanger, NCX1, is an integral membrane protein that is expressed in many tissues and is involved in cellular Ca2+ homeostasis (1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1464) Google Scholar, 2Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Crossref PubMed Scopus (446) Google Scholar). The expression level of NCX1 is modulated during development (3Boerth S.R. Zimmer D.B. Artman M. Circ. Res. 1994; 74: 354-359Crossref PubMed Google Scholar, 4Boerth S.R. Coetzee W.A. Artman M. Ann. N. Y. Acad. Sci. 1996; 779: 536-538Crossref PubMed Scopus (4) Google Scholar) and under pathological conditions (5Studer R. Reinecke H. Bilger J. Eschenhagen T. Bohm M. Hasenfuss G. Just H. Drexler H. Circ. Res. 1994; 75: 443-453Crossref PubMed Scopus (518) Google Scholar, 6Studer R. Reinecke H. Vetter R. Holtz J. Drexler H. Basic. Res. Cardiol. 1997; 92: 53-58Crossref PubMed Google Scholar, 7O'Rourke B. Kass D.A. Tomaselli G.F. Kaab S. 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Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar), by protons (14Doering A.E. Lederer W.J. J. Physiol. (Lond.). 1993; 466: 481-499Google Scholar, 15Doering A.E. Lederer W.J. J. Physiol. (Lond.). 1994; 480: 9-20Crossref Scopus (52) Google Scholar), by phosphatidylinositol 4,5-bisphosphate in the membrane (16Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (563) Google Scholar), and by exogenous agents including intracellular application of an inhibitor peptide (XIP) (17Li Z. Nicoll D.A. Collins A. Hilgemann D.W. Filoteo A.G. Penniston J.T. Weiss J.N. Tomich J.M. Philipson K.D. J. Biol. Chem. 1991; 266: 1014-1020Abstract Full Text PDF PubMed Google Scholar). However, regulation of NCX1 by PKA 1The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; RyR, ryanodine receptors. mAKAP, muscle protein kinase A-anchoring protein; AKAP, A-kinase-anchoring proteins; TM, transmembrane; LZ, leucine/isoleucine-zipper. has remained controversial (18Collins A. Somlyo A.V. Hilgemann D.W. J. Physiol. (Lond.). 1992; 454: 27-57Crossref Scopus (125) Google Scholar, 19Condrescu M. Gardner J.P. Chernaya G. Aceto J.F. Kroupis C. Reeves J.P. J. Biol. Chem. 1995; 270: 9137-9146Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 20Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 21Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar, 22Linck B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar, 23DiPolo R. Berberian G. Delgado D. Rojas H. Beauge L. FEBS Lett. 1997; 401: 6-10Crossref PubMed Scopus (32) Google Scholar). Early studies suggested that ATP-dependent regulation of NCX1 occurred in squid axons (24DiPolo R. Beauge L. Am. J. Physiol. 1994; 266: C1382-C1391Crossref PubMed Google Scholar, 25DiPolo R. Beauge L. Biochim. Biophys. Acta. 1987; 897: 347-354Crossref PubMed Scopus (58) Google Scholar) and in cardiac sarcolemmal vesicles (26Caroni P. Carafoli E. Eur. J. Biochem. 1983; 132: 451-460Crossref PubMed Scopus (160) Google Scholar), but these studies did not distinguish between direct ATP binding and ATP-dependent phosphorylation. Several studies were designed to resolve this problem. Hilgemann and colleagues (11Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar, 12Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (240) Google Scholar, 27Hilgemann D.W. Nicoll D.A. Philipson K.D. Nature. 1991; 352: 715-718Crossref PubMed Scopus (178) Google Scholar, 28Matsuoka S. Hilgemann D.W. J. Physiol. (Lond.). 1994; 476: 443-458Crossref Scopus (68) Google Scholar) investigated the question by measuring NCX1 currents in giant excised patches. The work used two preparations, NCX1-expressing Xenopus oocytes (27Hilgemann D.W. Nicoll D.A. Philipson K.D. Nature. 1991; 352: 715-718Crossref PubMed Scopus (178) Google Scholar) or cardiac myocytes expressing native NCX1 (11Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar, 12Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (240) Google Scholar, 28Matsuoka S. Hilgemann D.W. J. Physiol. (Lond.). 1994; 476: 443-458Crossref Scopus (68) Google Scholar). These investigations found no functional change in cardiac NCX1 activity following application of PKA or protein kinase C (PKC) catalytic subunits to the intracellular side of the giant patch. Condrescu et al. (19Condrescu M. Gardner J.P. Chernaya G. Aceto J.F. Kroupis C. Reeves J.P. J. Biol. Chem. 1995; 270: 9137-9146Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) came to similar conclusions by examining the phosphorylation state of NCX1 in a heterologous expression system (19Condrescu M. Gardner J.P. Chernaya G. Aceto J.F. Kroupis C. Reeves J.P. J. Biol. Chem. 1995; 270: 9137-9146Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Less direct investigations, however, suggested that PKA affected NCX1 function in heart (29Han X. Ferrier G.R. Circ. Res. 1995; 76: 664-674Crossref PubMed Scopus (37) Google Scholar, 30Perchenet L. Hinde A.K. Patel K.C. Hancox J.C. Levi A.J. Pflugers Arch. Eur. J. Physiol. 2000; 439: 822-828Crossref PubMed Scopus (57) Google Scholar). The indirect nature of these studies in support of phosphorylation did not exclude the possibility that the measured changes were due to PKA phosphorylation of other proteins. The first unambiguous demonstration that phosphorylation affected NCX1 was provided by Iwamoto et al. (31Iwamoto T. Wakabayashi S. Shigekawa M. J. Biol. Chem. 1995; 270: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). They showed that NCX1 from rat aorta smooth muscle cells is phosphorylated by PKC and is activated in response to growth factors (32Shigekawa M. Iwamoto T. Wakabayashi S. Ann. N. Y. Acad. Sci. 1996; 779: 249-257Crossref PubMed Scopus (13) Google Scholar). Further studies demonstrated that cardiac NCX1 is regulated by PKC phosphorylation (20Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Additionally, Iwamoto et al. (20Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) provided evidence that the intracellular loop of NCX1 was phosphorylated by PKC and PKA. We were the first to show that PKA-dependent phosphorylation of NCX1 increased NCX1 activity both in Xenopus oocytes expressing cardiac NCX1 and in adult rat ventricular cardiomyocytes (33Ruknudin A. He S. Lederer W.J. Schulze D.H. J. Physiol. (Lond.). 2000; 529: 599-610Crossref Scopus (61) Google Scholar, 34Ruknudin A. Schulze D.H. Ann. N. Y. Acad. Sci. 2002; 976: 209-213Crossref PubMed Scopus (14) Google Scholar). To determine how such divergent conclusions could arise, we undertook an investigation of the molecular organization of NCX1 in heart. Regulation of NCX1 could arise through cytosolic enzymes bathing NCX1 or through local clustering of the regulatory proteins. The variable loss of cytosolic regulatory proteins during experiments on NCX1 phosphorylation could, in principle, account for the divergent findings. Alternatively, if NCX1 was part of a macromolecular complex that included other regulatory proteins, differences in experimental results may reflect the different levels of expression and activity of associated proteins under different experimental conditions. Recently, local signaling complexes have been shown to regulate ion channels similar the l-type Ca+ channel (35Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (444) Google Scholar) and specific K+ channels (36Marx S.O. Kurokawa J. Reiken S. Motoike H. D'Armiento J. Marks A.R. Kass R.S. Science. 2002; 295: 496-499Crossref PubMed Scopus (615) Google Scholar) and cardiac ryanodine receptors (RyR2) (37Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). These complexes are composed of kinases, phosphatases, and kinase-anchoring proteins (AKAPs) and regulate activation state, substrate specificity, and subcellular localization. Recently, it was shown that the RyR2 could be more rapidly de-phosphorylated by the phosphatases of the macromolecular cluster than the kinases phosphorylated RyR2 (38Reiken S.R. Gaburjakova M. Guatimosim S. Gomez A.M. D'Armiento J. Burkhoff D. Wang J. Vassort G. Lederer W.J. Marks A.R. J. Biol. Chem. 2002; 278: 444-453Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). This recent observation and the large number of Ca2+-regulatory proteins associated with macromolecular complexes that regulate function suggested that this hypothesis is particularly appealing for NCX1. The experiments that we present below examine the macromolecular complex hypothesis for NCX1. Recently, we have shown that phosphorylation of NCX1 is induced by β-adrenergic stimulation in pig heart resulting increased NCX1 exchanger current (INCX). More importantly, we found that NCX1 is hyperphosphorylated in pig model of heart failure but β-adrenergic response is attenuated in heart failure (39Wei S.K. Ruknudin A. Hanlon S.U. McCurley J.M. Schulze D.H. Haigney M.C. Circ. Res. 2003; 92: 897-903Crossref PubMed Scopus (79) Google Scholar). These findings underscore the significance of NCX1 phosphorylation in pathological heart, and we have investigated the regulation of NCX1 phosphorylation in this paper. Preliminary findings of this work were presented in abstract form previously (40Mughal M. Schulze D. Lederer J. Ruknudin A. Biophys. J. 2003; 84: 151aGoogle Scholar). F344 strain rats (NIA, National Institutes of Health) were sacrificed by injecting with pentobarbital, and the hearts were removed immediately. After rinsing in ice-cold phosphate buffered saline, the ventricles were frozen in liquid nitrogen. The ventricles were pulverized in liquid nitrogen using a mortar and pestle and then homogenized in glass homogenizer (Eberbach, Ann Arbor, MI) for 20 min over ice. During homogenization, the extraction buffer composed of 50 mm Hepes, 150 mm NaCl, 3 mm KCl, 25 mm sodium pyrophosphate, 10 mm ATP, 5 mm EDTA (pH 7.4), and protease inhibitors (50 μg/ml phenylmethylsulfonyl fluoride, 1 mm iodoacetamide, 1 μm pepstatin, 2 mg/ml leupeptin, 1000 units/ml aprotinin, and 1 mm 1,10-phenonthroline) was added gradually. Undissolved cell fragments were removed by centrifugation at 3000 rpm for 15 min at 4 °C. The turbid supernatant was homogenized again over ice and centrifuged at 5000 rpm and at 4 °C (Eppendorf centrifuge 5415C). The supernatant was concentrated 3-fold using Micron-50 (Millipore) at 4 °C, and the concentrate was used either for electrophoresis as cardiac lysate or for immunoprecipitation for NCX1 macromolecular complex. The cardiac lysate was incubated with NCX polyclonal antibody π11–13 (Swant, Bellinzona, Switzerland) overnight, and the antigen-antibody complex was precipitated by mixing with protein A-Sepharose beads (Sigma). To identify the full-length mAKAP, immunoprecipitation was done using monoclonal NCX1 antibody (R3F1, Swant). The complex was washed in buffer containing 150 mm NaCl, 6 mm EDTA, 50 mm Tris (pH 7.4), 0.1% Triton X-100, and 0.02% SDS. Phosphorylation was performed by incubation of the pellet with 1 μg of the catalytic subunit of PKA (PKA-CS reconstituted in 5 mm dithiothreitol) and 10 μCi of [32P]ATP (3000 Ci/mmol) in phosphorylation buffer (25 mm Hepes, 5 mm MgCl2,5mm EGTA, and 0.2% Triton X-100 (pH 7.4)) for 10 min at 37 °C. In experiments with inhibitor, PKA inhibitor (Sigma) was included along with PKA catalytic subunit. The reaction was stopped by washing with 1 ml of RIA buffer (50 mm sodium phosphate buffer (pH 7.4), 50 mm KF, 75 mm NaCl, 2.5 mm EDTA, 0.01% NaN3, and 25 mm Tris (pH 7.4)) (41Ivanina T. Perets T. Thornhill W.B. Levin G. Dascal N. Lotan I. Biochemistry. 1994; 33: 8786-8792Crossref PubMed Scopus (60) Google Scholar). Samples were heated to 75 °C for 3 min in gel loading buffer containing 100 mm dithiothreitol and analyzed using 8% polyacrylamide gels. The proteins from the gel were transferred to nitrocellulose membrane (Amersham Biosciences) and exposed to Kodak X-Omat AR at –80 °C. After the immunoprecipitation with NCX1 antibodies and protein A-Sepharose beads, the protein sample was treated as described above. After electrophoresis, the proteins were transferred to nitrocellulose membrane and the primary antibodies were added. The following antibodies were used for immunoblotting: rabbit polyclonal to NCX1 (π11–13, Swant); mouse monoclonals to PKA catalytic subunit (clone 5B), to PKA RI subunit (Clone 18), to PKC (Clone MC5), and to PP1 (clone 24) (BD Biosciences); rabbit polyclonal to mAKAP (Upstate Biotechnology); and mouse monoclonal to PP2A (Clone 6F9) (CRP Inc.). HRP-conjugated appropriate secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) were added, and the proteins in the nitrocellulose membranes were identified. The proteins were visualized using ECL kit (Amersham Biosciences), and the images were obtained using Kodak Biomax-MR film. Enzymatically isolated adult rat cardiomyocytes (33Ruknudin A. He S. Lederer W.J. Schulze D.H. J. Physiol. (Lond.). 2000; 529: 599-610Crossref Scopus (61) Google Scholar) were fixed in cold ethanol, permeabilized, and incubated with primary antibodies to NCX1 (mouse, Swant) and mAKAP (rabbit, Upstate Biotechnology). Another set of cardiomyocytes was incubated with antibodies to RI (Pan-antibody mouse, Clone 18, BD Biosciences) and NCX1 (rabbit, Swant) followed by appropriate secondary antisera (Alexa488 and Alexa633, Molecular Probes, Sunnyvale, CA) and imaged by confocal microscopy (Zeiss LSM 510). Images at each wavelength (488 and 633 nm) were collected separately to ensure that there was no fluorescent channel bleed-through. The fluorescence intensities in a chosen area of the cell were analyzed using Metamorph software (Universal Imaging Corporation, Downingtown, PA). Increased NCX1 activity in rat ventricular cardiomyocytes following PKA activation (30Perchenet L. Hinde A.K. Patel K.C. Hancox J.C. Levi A.J. Pflugers Arch. Eur. J. Physiol. 2000; 439: 822-828Crossref PubMed Scopus (57) Google Scholar, 33Ruknudin A. He S. Lederer W.J. Schulze D.H. J. Physiol. (Lond.). 2000; 529: 599-610Crossref Scopus (61) Google Scholar) could be attributed to the direct effect on NCX1 protein or could be due to PKA activation of another target protein. To investigate these alternatives, we examined the phosphorylation state of NCX1. Total cardiac protein was prepared, and NCX1 protein was immunoprecipitated with NCX1 antibody. A Western blot permitted identification of NCX1 protein as three bands at 120, 140, and 160 kDa (Fig. 1, left panel). These bands are characteristically observed for NCX1 protein (42Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). When the immunoprecipitated cardiac NCX1 was phosphorylated in vitro using catalytic subunit of PKA and [32P]ATP, the bands at 120 and 160 kDa were clearly phosphorylated (Fig. 1, middle panel). Inclusion of an inhibitor of PKA (PKI, the PKA inhibitory peptide) during the phosphorylation reaction prevented the labeling of these proteins, indicating the specificity of PKA on rat cardiac NCX1 (Fig. 1, right panel). This experiment demonstrates that cardiac NCX1 is phosphorylated by PKA in support of the findings of Iwamoto et al. (20Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). To examine the possibility that NCX1 associates with relevant kinases, NCX1 was immunoprecipitated and immunoblotting was performed. The PKA holoenzyme is a kinase composed of two identical catalytic subunits and two identical regulatory subunits (43Taylor S.S. Radzio-Andzelm E. Madhusudan Cheng X. Ten Eyck L. Narayana N. Pharmacol. Ther. 1999; 82: 133-141Crossref PubMed Scopus (45) Google Scholar). We show that the components of the PKA holoenzyme, both the catalytic and regulatory subunit RI, are recognized in the NCX1 immunoprecipitate by the antibodies specific for those proteins (Fig. 2AI). The other PKA regulatory subunit, RII, was not detected in the macromolecular complex. This indicates that only RI is associated with NCX1. All of the PKA components (including RII) were found in the cardiac lysate (Fig. 2, AII). The presence of both catalytic and regulatory subunits in the cardiac lysate shows that PKA components are also found in the soluble components of the cell as has been reported previously (44Corbin J.D. Sugden P.H. Lincoln T.M. Keely S.L. J. Biol. Chem. 1977; 252: 3854-3861Abstract Full Text PDF PubMed Google Scholar). Using the same immunoprecipitation strategy, we investigated whether or not PKC is associated with NCX1. As shown in Fig. 2B, a sharp single band is present on the immunoblot using a pan-PKC antibody. The absence of measured phosphorylation of NCX1 following PKA activation in some studies could be compatible with our results if there was a difference in the rate of dephosphorylation in the various studies. Conditions that may lead to such differences could occur if phosphatases colocalized with NCX1 as has been shown for RyR2 (38Reiken S.R. Gaburjakova M. Guatimosim S. Gomez A.M. D'Armiento J. Burkhoff D. Wang J. Vassort G. Lederer W.J. Marks A.R. J. Biol. Chem. 2002; 278: 444-453Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Fig. 3 examines this by immunoblot analysis with antibodies to PP1 and PP2A as shown in Fig. 3, A and B, respectively. We found that the serine/threonine phosphatases PP1 and PP2A were precipitated along with the NCX1. These phosphatases were also readily detected in the cardiac lysate. Targeting of kinases and phosphatases to specific proteins has been described extensively by Scott and co-workers (45Smith F.D. Scott J. Curr. Biol. 2001; 12: R32-R40Abstract Full Text Full Text PDF Scopus (97) Google Scholar, 46Bauman A.L. Scott J.D. Nat. Cell Biol. 2002; 4: E203-E206Crossref PubMed Scopus (117) Google Scholar). These studies have identified a family of "Akinase anchoring proteins" or AKAPs that serves as scaffolding proteins for this function. To explore the possibility that an AKAP may be associated with NCX1, we probed immunoblots with antibodies against six of the major AKAPs. Fig. 4A and B, shows that mAKAP coprecipitates with NCX1 while the other AKAPs tested (79, 95, 121, 149, and 220) did not. The size of mAKAP is 300 kDa and is identified by the specificity of the mAKAP antibody. 2J. Scott, personal communication. The cardiac lysate probed with the same antibody contained a 300-kDa band as well as the smaller bands (Fig. 4A). Others have also detected the proteolytic fragments of the 300-kDa mAKAP (47Kapiloff M.S. Schillace R.V. Westphal A.M. Scott J.D. J. Cell Sci. 1999; 112: 2725-2736Crossref PubMed Google Scholar). To identify the NCX1 and associated proteins of the macromolecular complex in situ, we used immunocytochemical techniques to localize these proteins in rat ventricular cardiomyocytes. NCX1 was identified in the sarcolemmal membrane and in the Z-lines of cardiomyocytes (Fig. 5) as already reported (48Kieval R.S. Bloch R.J. Lindenmayer G.E. Ambesi A. Lederer W.J. Am. J. Physiol. 1992; 263: C545-C550Crossref PubMed Google Scholar, 49Frank J.S. Mottino G. Reid D. Molday R.S. Philipson K.D. J. Cell Biol. 1992; 117: 337-345Crossref PubMed Scopus (164) Google Scholar). Interestingly, mAKAP is also localized in the Z-lines and the dual staining of NCX1 and mAKAP confirmed that both of these proteins are present in the same location in rat cardiomyocytes (Fig. 5A, overlay). The regulatory subunit of PKA, RI has been identified in the Z-lines and also in other regions of the cell (Fig. 5C). The overlay of the RI and NCX1 clearly shows that both of these proteins are colocalized in cardiomyocytes (Fig. 5C). Analysis of the fluorescence intensities in selected regions of the cell indicates that there is clear correspondence between NCX1 and mAKAP (Fig. 5B) and that there is even better support for the matching of PKA-RI and NCX1 (Fig. 5D). The mAKAP and PKA-RI are also expressed in places that do not contain NCX1 and that are also shown in immunofluorescence images. We have presented data demonstrating that the NCX1 protein in cardiac cells is part of a macromolecular complex (Fig. 6). The complex consists of two types of protein kinases (PKA and PKC), two types of phosphatases (PP1 and PP2A), and an anchoring protein (mAKAP). In addition, there is strong evidence that NCX1 also binds to the widely distributed adapter protein, ankyrin (50Li Z.P. Burke E.P. Frank J.S. Bennett V. Philipson K.D. J. Biol. Chem. 1993; 268: 11489-11491Abstract Full Text PDF PubMed Google Scholar, 51Chen F. Mottino G. Shin V.Y. Frank J.S. J. Mol. Cell Cardiol. 1997; 29: 2621-2629Abstract Full Text PDF PubMed Scopus (19) Google Scholar). The activity of NCX1 has been shown to be regulated by its interaction with a number of ions and molecules (Table I). How this regulation may be organized and controlled by the macromolecular complex is presented below.Table IInteractions of NCX1 macromolecular complexMajor itemInteractionCommentsReferencesNCX1AnkyrinSubcellular localization50Li Z.P. Burke E.P. Frank J.S. Bennett V. Philipson K.D. J. Biol. Chem. 1993; 268: 11489-11491Abstract Full Text PDF PubMed Google Scholar5′-TM segments with 3′-TM segmentsStudies reveal protein folding52Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 53Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. 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