Phospholemman Inhibition of the Cardiac Na+/Ca2+ Exchanger
2006; Elsevier BV; Volume: 281; Issue: 12 Linguagem: Inglês
10.1074/jbc.m512092200
ISSN1083-351X
AutoresXue-Qian Zhang, Belinda A. Ahlers, Amy L. Tucker, Jianliang Song, JuFang Wang, J. Randall Moorman, J. Paul Mounsey, Lois L. Carl, Lawrence I. Rothblum, Joseph Y. Cheung,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoWe have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (INaCa) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in INaCa, (ii) attenuation of the increase in INaCa by PMA, and (iii) additional reduction in INaCa in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of INaCa, whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on INaCa. Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of INaCa as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited INaCa. The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, INaCa was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in INaCa was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on INaCa in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart. We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (INaCa) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in INaCa, (ii) attenuation of the increase in INaCa by PMA, and (iii) additional reduction in INaCa in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of INaCa, whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on INaCa. Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of INaCa as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited INaCa. The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, INaCa was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in INaCa was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on INaCa in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart. Phospholemman (PLM), 2The abbreviations used are: PLM, phospholemman; 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; Cm, whole cell membrane capacitance; CMV, cytomegalovirus; Em, membrane potential; ENaCa, equilibrium potential for Na+/Ca2+ exchange current; HEK, human embryonic kidney; INaCa, Na+/Ca2+ exchange current; KO, knock-out; NCX1, Na+/Ca2+ exchanger; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SERCA2, sarco(endo)plasmic reticulum Ca2+-ATPase; WT, wild-type. 2The abbreviations used are: PLM, phospholemman; 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; Cm, whole cell membrane capacitance; CMV, cytomegalovirus; Em, membrane potential; ENaCa, equilibrium potential for Na+/Ca2+ exchange current; HEK, human embryonic kidney; INaCa, Na+/Ca2+ exchange current; KO, knock-out; NCX1, Na+/Ca2+ exchanger; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SERCA2, sarco(endo)plasmic reticulum Ca2+-ATPase; WT, wild-type. a 72-amino acid membrane phosphoprotein with a single transmembrane domain (1Palmer C.J. Scott B.T. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar), belongs to the FXYD gene family of small ion transport regulators (2Sweadner K.J. Rael E. Genomics. 2000; 68: 41-56Crossref PubMed Scopus (353) Google Scholar). With the exception of the γ-subunit of Na+-K+-ATPase (FXYD2), all other known members of the FXYD gene family have at least one serine or threonine within the cytoplasmic tail (2Sweadner K.J. Rael E. Genomics. 2000; 68: 41-56Crossref PubMed Scopus (353) Google Scholar), indicating potential phosphorylation sites. In particular, PLM (FXYD1) is the only FXYD family member to have a consensus sequence for phosphorylation by PKA (RRXS), PKC (RXXSXR), and NIMA (never in mitosis A) kinase (FRX(S/T)). Indeed PLM has been shown to be phosphorylated by PKA at serine 68 and PKC at both serine 63 and serine 68 (3Waalas S.I. Czernik A.J. Olstad O.K. Sletten K. Walaas O. Biochem. J. 1994; 304: 635-640Crossref PubMed Scopus (84) Google Scholar). To date, PLM has been demonstrated to modulate ion fluxes through both the Na+-K+-ATPase (4Crambert G. Fuzesi M. Garty H. Karlish S. Geering K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11476-11481Crossref PubMed Scopus (223) Google Scholar, 5Fuller W. Eaton P. Bell J.R. Shattock M.J. FASEB J. 2004; 18: 197-199Crossref PubMed Scopus (96) Google Scholar, 6Silverman B.Z. Fuller W. Eaton P. Deng J. Moorman J.R. Cheung J.Y. James A.F. Shattock M.J. Cardiovasc. Res. 2005; 65: 93-103Crossref PubMed Scopus (104) Google Scholar, 7Bossuyt J. Ai X. Moorman J.R. Pogwizd S.M. Bers D.M. Circ. Res. 2005; 97: 558-565Crossref PubMed Scopus (85) Google Scholar, 8Despa S. Bossuyt J. Han F. Ginsburg K.S. Jia L.G. Kutchai H. Tucker A.L. Bers D.M. Circ. Res. 2005; 97: 252-259Crossref PubMed Scopus (151) Google Scholar) and the cardiac Na+/Ca2+ exchanger (NCX1) (9Zhang X.Q. Qureshi A. Song J. Carl L.L. Tian Q. Stahl R.C. Carey D.J. Rothblum L.I. Cheung J.Y. Am. J. Physiol. 2003; 284: H225-H233Crossref PubMed Google Scholar, 10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 11Mirza M.A. Zhang X.Q. Ahlers B.A. Qureshi A. Carl L.L. Song J. Tucker A.L. Mounsey J.P. Moorman J.R. Rothblum L.I. Zhang T.S. Cheung J.Y. Am. J. Physiol. 2004; 286: H1322-H1330Crossref PubMed Scopus (40) Google Scholar). Based on analogy of phospholamban inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) (12Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (464) Google Scholar) and experimental observation on the effects of PLMS (a 15-kDa homologue of PLM isolated from shark rectal glands) on shark Na+-K+-ATPase (13Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 275: 35969-35977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Mahmmoud Y.A. Cramb G. Maunsbach A.B. Cutler C.P. Meischke L. Cornelius F. J. Biol. Chem. 2003; 278: 37427-37438Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), the current working hypothesis is that the Na+ pump is inhibited by unphosphorylated PLM. On phosphorylation of PLM, inhibition of Na+-K+-ATPase is relieved. This hypothesis has been given strong support by the observation that the Vmax of sarcolemmal Na+-K+-ATPase is increased 3-fold after acute cardiac ischemia in association with increased PLM phosphorylation by >300% (5Fuller W. Eaton P. Bell J.R. Shattock M.J. FASEB J. 2004; 18: 197-199Crossref PubMed Scopus (96) Google Scholar). In addition, Na+ pump current has been demonstrated to directly increase in association with PLM phosphorylation in response to forskolin (6Silverman B.Z. Fuller W. Eaton P. Deng J. Moorman J.R. Cheung J.Y. James A.F. Shattock M.J. Cardiovasc. Res. 2005; 65: 93-103Crossref PubMed Scopus (104) Google Scholar). More recently, comparison of β-adrenergic effects on Na+ pump function between wild-type and PLM-knock-out (KO) myocytes supports the notion that the inhibitory effects of PLM on Na+-K+-ATPase are relieved by phosphorylation (8Despa S. Bossuyt J. Han F. Ginsburg K.S. Jia L.G. Kutchai H. Tucker A.L. Bers D.M. Circ. Res. 2005; 97: 252-259Crossref PubMed Scopus (151) Google Scholar). It is at present not clear whether dissociation of the phosphorylated PLM from Na+-K+-ATPase is required to relieve its inhibition on the Na+ pump (5Fuller W. Eaton P. Bell J.R. Shattock M.J. FASEB J. 2004; 18: 197-199Crossref PubMed Scopus (96) Google Scholar, 6Silverman B.Z. Fuller W. Eaton P. Deng J. Moorman J.R. Cheung J.Y. James A.F. Shattock M.J. Cardiovasc. Res. 2005; 65: 93-103Crossref PubMed Scopus (104) Google Scholar, 8Despa S. Bossuyt J. Han F. Ginsburg K.S. Jia L.G. Kutchai H. Tucker A.L. Bers D.M. Circ. Res. 2005; 97: 252-259Crossref PubMed Scopus (151) Google Scholar, 13Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 275: 35969-35977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Mahmmoud Y.A. Cramb G. Maunsbach A.B. Cutler C.P. Meischke L. Cornelius F. J. Biol. Chem. 2003; 278: 37427-37438Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). With respect to the cardiac Na+/Ca2+ exchanger, previous studies demonstrated that overexpression of PLM inhibits Na+/Ca2+ exchange activity (9Zhang X.Q. Qureshi A. Song J. Carl L.L. Tian Q. Stahl R.C. Carey D.J. Rothblum L.I. Cheung J.Y. Am. J. Physiol. 2003; 284: H225-H233Crossref PubMed Google Scholar, 10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), whereas down-regulation of PLM enhances NCX1 current (INaCa) (11Mirza M.A. Zhang X.Q. Ahlers B.A. Qureshi A. Carl L.L. Song J. Tucker A.L. Mounsey J.P. Moorman J.R. Rothblum L.I. Zhang T.S. Cheung J.Y. Am. J. Physiol. 2004; 286: H1322-H1330Crossref PubMed Scopus (40) Google Scholar). The importance of PLM phosphorylation in mediating its modulatory effects on NCX1 was not addressed in these early studies except that serine 68 in PLM was found to be important (15Song J. Zhang X.Q. Ahlers B.A. Carl L.L. Wang J. Rothblum L.I. Stahl R.C. Mounsey J.P. Tucker A.L. Moorman J.R. Cheung J.Y. Am. J. Physiol. 2005; 288: H2342-H2354Crossref PubMed Scopus (47) Google Scholar). Here we demonstrated that PKC but not PKA activation enhanced INaCa when NCX1 was expressed alone in HEK293 cells. Co-expression of PLM with NCX1 resulted in decreased INaCa in the basal state, an additional decrease in INaCa when stimulated with forskolin, and attenuation of the magnitude of increase in INaCa by PKC activation. Mutating serine 68 to glutamic acid (S68E) enhanced whereas substituting serine 68 with alanine (S68A) abolished the inhibitory effect of PLM on INaCa. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of additional inhibition of INaCa. Using a fundamentally different model system of murine cardiac myocytes, we first showed that endogenous INaCa was larger in PLM-KO myocytes when compared with wild-type (WT) myocytes despite similar NCX1 protein levels. PKC but not PKA activation increased INaCa in WT myocytes. PLM-KO myocytes exhibited significantly larger increases in INaCa when stimulated with phorbol 12-myristate 13-acetate (PMA) as compared with WT myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits cardiac Na+/Ca2+ exchanger. Construction of PLM Mutants and NCX1 Clones—PLM serine mutants (S63A, S68A, and S68E) were constructed with PLM in pAlter-1 using Altered Sites II in vitro mutagenesis system (Promega, Madison, WI) as described previously (15Song J. Zhang X.Q. Ahlers B.A. Carl L.L. Wang J. Rothblum L.I. Stahl R.C. Mounsey J.P. Tucker A.L. Moorman J.R. Cheung J.Y. Am. J. Physiol. 2005; 288: H2342-H2354Crossref PubMed Scopus (47) Google Scholar). PLM and its serine mutants were authenticated by DNA sequencing and subcloned into the mammalian expression vector pAdTrack-CMV (16He T. Zhou S. da Costa L. Yu L. Kinzler K. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3236) Google Scholar). Rat cardiac NCX1 clone in pcDNA3.1(+) was a generous gift from Dr. J. Lytton and subcloned into pAdTrack-CMV as previously described (17Zhang X.Q. Song J. Rothblum L.I. Lun M. Wang X. Ding F. Dunn J. Lytton J. McDermott P.J. Cheung J.Y. Am. J. Physiol. 2001; 281: H2079-H2088Crossref PubMed Google Scholar). We chose the pAdTrack shuttle vector because it allowed us to identify successfully transfected HEK293 cells through a separate cytomegalovirus (CMV) promoter present on the vector backbone that drives the expression of green fluorescent protein. Transfection of HEK293 Cells—HEK293 cells (American Type Culture Collection (ATCC), Manassas, VA) were cultured and transfected with various combinations of NCX1 and PLM or its mutant clones as described previously (10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Briefly cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 containing 10% heat-inactivated fetal bovine serum at a density of 1.2 × 106 cells/100-mm dish. After 24 h, medium was changed, and cells were transfected with 25 μl of Lipofectamine and a total of 3 μg of plasmid DNA/dish: either pAdTrack-CMV alone (3μg), pAdTrack-CMV-NCX1 (1 μg) + pAdTrack-CMV (2 μg), pAdTrack-CMV-NCX1 (1 μg) + pAdTrack-CMV-PLM (1 μg) + pAdTrack-CMV (1 μg), or pAdTrack-CMV-NCX1 (1 μg) + pAdTrack-CMV-PLM serine mutant (1 μg) + pAdTrack-CMV (1 μg). The lipid-DNA complex was left on cells for 5 h at 37 °C in 5% CO2. Medium was then replaced with Dulbecco's modified Eagle's medium/Ham's F-12 + 10% fetal bovine serum, and cells were cultured for an additional 48 h before experiments. For patch-clamp applications, cells were trypsinized at 24 h post-transfection using trypsin-EDTA, transferred to 35-mm dishes containing sterile glass coverslips, and incubated a further 24 h prior to experiments. Transfection according to this protocol routinely yielded 30–50% transfection efficiency. For brevity, HEK293 cells expressing NCX1 alone are referred in the text as NCX1 cells, whereas cells co-expressing NCX1 and PLM or its serine mutants are referred as PLM cells or SnnX cells (where nn is either 63 or 68, and X is either Ala or Glu). Na+/Ca2+ Exchange Current (INaCa) Measurements—Whole cell patch-clamp recordings were performed at 30 °C as described previously (10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 11Mirza M.A. Zhang X.Q. Ahlers B.A. Qureshi A. Carl L.L. Song J. Tucker A.L. Mounsey J.P. Moorman J.R. Rothblum L.I. Zhang T.S. Cheung J.Y. Am. J. Physiol. 2004; 286: H1322-H1330Crossref PubMed Scopus (40) Google Scholar, 18Tadros G.M. Zhang X.Q. Song J. Carl L.L. Rothblum L.I. Tian Q. Dunn J. Lytton J. Cheung J.Y. Am. J. Physiol. 2002; 283: H1616-H1626Crossref PubMed Google Scholar, 19Zhang X. Tillotson D. Moore R. Zelis R. Cheung J. Am. J. Physiol. 1996; 271: C1800-C1807Crossref PubMed Google Scholar). Briefly fire-polished pipettes (tip diameter, 2–3 μm) were filled with a buffered Ca2+ solution containing 100 mm Cs+-glutamate, 7.25 mm Na+-HEPES, 1 mm MgCl2, 12.75 mm HEPES, 2.5 mm Na2ATP, 10 mm EGTA, and 6 mm CaCl2, pH 7.2. Free Ca2+ in the pipette solution was 205 nm, measured fluorometrically with fura 2. Cells were bathed in an external solution containing 130 mm NaCl, 5 mm CsCl, 1.2 mm MgSO4, 1.2 mm NaH2PO4, 5 mm CaCl2, 10 mm HEPES, 10 mm Na+-HEPES, and 10 mm glucose, pH 7.4. Verapamil (1 μm), ouabain (1 mm), and niflumic acid (30 μm) were used to block Ca2+, Na+-K+-ATPase, and Cl– currents, respectively. K+ currents were minimized by Cs+ substitution for K+ in both pipette and external solutions. Only cells that fluoresced green (excitation, 380 nm; emission, 510 nm), indicating successful pAdTrack transfection, were selected for current measurements. Membrane potential (Em) was held at the calculated reversal potential of INaCa (–73 mV) for 5 min before stimulation. A descending voltage ramp (from +100 to –120 mV; 500 mV/s) was immediately followed by an ascending voltage ramp (from –120 to +100 mV; 500 mV/s) (Fig. 1A). Membrane currents were measured both before and after addition of 1 mm CdCl2 to the external solution (Fig. 1B). INaCa was defined as the difference current measured during the descending voltage ramp in the absence and presence of Cd2+ (Fig. 1C). To facilitate comparison of NCX1 currents, INaCa of each cell was divided by its whole cell membrane capacitance (Cm) to account for variations in cell sizes. Except as otherwise stated, all results were obtained using these standard solutions. When indicated, PMA (0.1 μm) or forskolin (1 μm) (both dissolved in Me2SO) was added to cells after base-line INaCa was obtained. Repeat INaCa was measured ∼3–5 min after drug addition. In a second series of experiments, the effects of PMA on INaCa were measured under Cl–-free conditions. Pipette solutions consisted of 100 mm Cs+-glutamate, 7.25 mm Na+-HEPES, 1 mm MgSO4, 12.75 mm HEPES, 2.5 mm Na2ATP, 10 mm EGTA, and 6 mm Ca(OH)2, pH 7.2. External solutions contained 130 mm Na+-aspartate, 5 mm Cs+-glutamate, 1.2 mm MgSO4, 1.2 mm NaH2PO4, 5 mm Ca(OH)2, 10 mm HEPES, 10 mm Na+-HEPES, and 10 mm glucose, pH 7.4. Verapamil, ouabain, and niflumic acid were added to the bath as before. Holding potential was –73 mV. INaCa was defined as the difference current measured during the descending voltage ramp in the absence and presence of Cd2+ (1 mm) or Ni2+ (5 mm). In a third series of experiments, the effects of PMA on INaCa were measured under high [Na+]i conditions. Pipette solutions contained 60 mm Cs+-glutamate, 40 mm Na+-glutamate, 7.25 mm Na+-HEPES, 1 mm MgCl2, 12.75 mm HEPES, 2.5 mm Na2ATP, 10 mm EGTA, and 6 mm CaCl2, pH 7.2. External solution consisted of 130 mm NaCl, 5 mm CsCl, 1.2 mm MgSO4, 1.2 mm NaH2PO4, 0.2 mm CaCl2, 10 mm HEPES, 10 mm Na+-HEPES, and 10 mm glucose, pH 7.4, and the usual inhibitors. [Ca2+]o was deliberately lowered to 0.2 mm so that the calculated reversal potential of INaCa (–103 mV), and thus the holding potential, was closer to the holding potential of –73 mV used in other experiments. Keeping [Ca2+]o at 5 mm would have resulted in a very negative holding potential of –188 mV. INaCa was defined as the difference current measured during the descending voltage ramp in the absence and presence of Ni2+ (5 mm). Generation of PLM-KO Mice—A mouse line deficient in PLM was generated by replacing exons 3–5 of the PLM gene with lacZ and neomycin resistance genes as described in detail previously (20Jia L.G. Donnet C. Bogaev R.C. Blatt R.J. McKinney C.E. Day K.H. Berr S.S. Jones L.R. Moorman J.R. Sweadner K.J. Tucker A.L. Am. J. Physiol. 2005; 288: H1982-H1988Crossref PubMed Scopus (72) Google Scholar). These mice grow to adulthood and are fertile. Studies were performed using mice backcrossed to a pure congenic C57BL/6 background. Homozygous adult littermates that were 3–6 months old were used in the experiments. Mice were housed in ventilated racks in a barrier facility supervised by the Department of Comparative Medicine at the Pennsylvania State University College of Medicine. Standard care was provided to all mice used for experiments. PLM, NCX1, and Calsequestrin Immunoblotting—Mouse left ventricles were excised, rinsed in ice-cold phosphate-buffered saline, and cut into small pieces. Approximately 60 mg of tissue were suspended in 700 μl of ice-cold lysis buffer containing 50 mm Tris (pH 8.0), 150 mm NaCl, 1 mm Na+-orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 100 mm NaF, 1 mm EGTA, and 0.5% Nonidet P-40. A Complete Mini protease inhibitor mixture tablet (Roche Applied Science) was also added to 10 ml of lysis buffer. The tissue was homogenized with a glass Dounce homogenizer (15–20 strokes) and placed on ice for 15 min before centrifugation at 20,800 × g for 10 min at 4 °C. The supernatant was snap frozen with dry ice-ethanol and stored at –80 °C. Proteins in heart homogenates were subjected to 7.5% (NCX1 and calsequestrin) or 12% (PLM) SDS-PAGE under either non-reducing (10 mm N-ethylmaleimide for NCX1 and calsequestrin) or reducing (5% β-mercaptoethanol for PLM) conditions. The fractionated proteins were transferred to ImmunBlot polyvinylidene difluoride membranes. Primary antibodies used were polyclonal antibody C2Ab (1:10,000) for PLM (21Song J. Zhang X. Carl L. Qureshi A. Rothblum L. Cheung J. Am. J. Physiol. 2002; 283: H576-H583Crossref PubMed Scopus (36) Google Scholar), polyclonal antibody π 11-13 (1:500; Swant, Bellinzona, Switzerland) for NCX1, and rabbit anti-calsequestrin antibody (1:5,000; Swant). The secondary antibodies used were donkey anti-rabbit IgG (Amersham Biosciences). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting system. Protein band signal intensities were quantitated by scanning autoradiograms of the blots with a PhosphorImager (Amersham Biosciences). Because calsequestrin expression has been shown to be unchanged during ontogenic development, aging, cardiac hypertrophy, and failing human myocardium (22Hasenfuss G. Cardiovasc. Res. 1998; 37: 279-289Crossref PubMed Google Scholar), we used calsequestrin as an internal control for protein loading. Isolation of Murine Myocytes and Measurement of INaCa—Cardiac myocytes were isolated from the septum and left ventricular free wall of WT and PLM-KO mice (25–37 g) according to the protocol of Zhou et al. (23Zhou Y.Y. Wang S.Q. Zhu W.Z. Chruscinski A. Kobilka B.K. Ziman B. Wang S. Lakatta E.G. Cheng H. Xiao R.P. Am. J. Physiol. 2000; 279: H429-H436Crossref PubMed Google Scholar). Briefly mice were heparinized (1500 units/kg intraperitoneally) and anesthetized (pentobarbital sodium, 50 mg/kg intraperitoneally). The heart was excised, mounted on a steel cannula, and retrograde perfused (100 cm of H2O at 37 °C) with Ca2+-free bicarbonate buffer followed by enzymatic digestion (collagenases B and D and protease XIV) as described previously (23Zhou Y.Y. Wang S.Q. Zhu W.Z. Chruscinski A. Kobilka B.K. Ziman B. Wang S. Lakatta E.G. Cheng H. Xiao R.P. Am. J. Physiol. 2000; 279: H429-H436Crossref PubMed Google Scholar). Isolated myocytes were plated on laminin-coated glass coverslips in a Petri dish, and the Ca2+ concentration of the buffer was progressively increased from 0.05 to 0.125 to 0.25 to 0.5 mm in three steps (10-min interval for each). The 0.5 mm Ca2+ buffer was then aspirated and replaced with minimal essential medium (Sigma catalogue number M1018) containing 1.2 mm Ca2+, 2.5% fetal bovine serum, and antibiotics (1% penicillin/streptomycin). After 1 h (in 5% CO2 at 37 °C), medium was replaced with fetal bovine serum-free minimal essential medium. Myocytes were used within 2–8 h of isolation. The protocol for heart excision for myocyte isolation was approved by the Institutional Animal Care and Usage Committee. INaCa was measured in isolated murine myocytes with the same protocol and standard solutions used for transfected HEK293 cells except that pipette tip diameter was increased to 4–6 μm, and niflumic acid was decreased to 10 μm. Statistical Analysis—All results are expressed as means ± S.E. For analysis of a parameter (e.g. INaCa) as a function of group (e.g. NCX1 versus PLM) and voltage, two-way analysis of variance was used to determine statistical significance. For analysis of Cm, Student's t test was used. A commercial software package (JMP, version 4.0.5; SAS Institute, Cary, NC) was used. In all analyses, p < 0.05 was taken to be statistically significant. Effects of PMA or Forskolin on INaCa in HEK293 Cells Expressing NCX1 Alone—We have shown previously that HEK293 cells do not express NCX1 or demonstrate measurable INaCa or Na+-dependent Ca2+ uptake (10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). When transfected with rat cardiac NCX1, HEK293 cells exhibited characteristic INaCa demonstrating both forward (inward current, 3 Na+ in:1 Ca2+ out) and reverse (outward current, 3 Na+ out:1 Ca2+ in) Na+/Ca2+ exchange (Fig. 2A). In addition, the reversal potential of INaCa was between –70 and –60 mV, close to its theoretical equilibrium potential of –73 mV under our experimental conditions (Fig. 2A). There were no significant (p < 0.76) differences in base-line INaCa measured with either Cd2+ or Ni2+ (data not shown). Treatment with PMA, which activates PKC, resulted in a large increase in INaCa in NCX1 cells (Fig. 2A; p < 0.0001). For example, at +100 mV, PKC stimulation resulted in an ∼120% increase in INaCa. Control experiments performed in Cl–-free solutions demonstrated that the PMA-induced current increase was not due to an increase in Cl– currents (Fig. 2B). In addition, PMA induced large increases in currents whether Cd2+ (∼122% at +100 mV) (Fig. 2B) or Ni2+ (∼81% at +100 mV) (data not shown) was used to define INaCa under Cl–-free conditions. To control for the possibility that the observed PMA-induced INaCa increase was due to small changes in [Na+]i rather than enhancing intrinsic NCX1 activity, experiments were performed in high [Na+]i conditions such that INaCa would not be so sensitive to small changes in [Na+]i. Fig. 2C shows that base-line INaCa was significantly (p < 0.0001) smaller in high [Na+]i and low [Ca2+]o (0.2 mm) when compared with normal [Na+]i and high [Ca2+]o (5 mm) conditions (Fig. 2A), likely due to the 25-fold reduction of [Ca2+]o. However, addition of PMA increased INaCa (∼84% at +100 mV) under high [Na+]i conditions, similar to the observations obtained under lower but more physiological [Na+]i conditions. In contrast to results obtained with PMA stimulation, forskolin treatment did not affect INaCa in NCX1-expressing cells (Fig. 3B; p < 0.64).FIGURE 3Effects of PMA and forskolin on INaCa in transfected HEK293 cells. A, HEK293 cells were transfected with either NCX1 alone (open circles, n = 14) or PLM + NCX1 (open diamonds, n = 15). At 48 h post-transfection, INaCa was measured at 5 mm [Ca2+]o and 30 °C as described in Fig. 1. After base-line INaCa was obtained, PMA (0.1 μm) was added to NCX1 (open squares, n = 8) and PLM + NCX1 (filled triangles, n = 9) cells. Measurement of INaCa was repeated ∼3–5 min after drug addition. B, HEK293 cells were transfected with either NCX1 alone (open circles, n = 14) or PLM + NCX1 (open diamonds, n = 15). At 48 h post-transfection, base-line INaCa was obtained. Forskolin (1 μm) was then added to NCX1 (open squares, n = 4) and PLM + NCX1 (open triangles, n = 7) cells. Measurement of INaCa was repeated ∼3–5 min after drug addition. Error bars are not shown if they fall within boundaries of the symbols. pF, picofarad.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effects of PMA or Forskolin on INaCa in Cells Expressing Both NCX1 and PLM—Co-expression of PLM with NCX1 in HEK293 cells resulted in a significant decrease in INaCa compared with cells expressing NCX1 alone (Fig. 3, A and B; p < 0.0005), consistent with our previous observations (10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). At +100 mV, PLM inhibited INaCa by ∼26%. PMA treatment of PLM cells resulted in a significant increase in INaCa when compared with unstimulated NCX1 or PLM cells (Fig. 3A; p < 0.0001). However, the magnitude of INaCa increase by PMA was much smaller in PLM cells when compared with NCX1 cells (39 versus 120% at +100 mV). Despite the absence of an effect of forskolin on INaCa in cells expressing NCX1 alone, PKA stimulation in PLM cells resulted in a significant decrease in INaCa compared with unstimulated PLM cells (Fig. 3B; p < 0.0001). For example, at +100 mV, forskolin effected an ∼49% decrease in INaCa in PLM cells (Fig. 3B). Effects of PLM Serine 68 Mutants on INaCa in Transfected HEK293 Cells—Because serine 68 in PLM is the common phosphorylation target for both PKA and PKC, we next investigated the effects of serine 68 mutants on INaCa in cells co-expressing NCX1 and PLM serine 68 mutants. Mutating serine 68 to alanine (S68A) resulted in abolition of the effect of WT PLM on INaCa (Fig. 4A; p < 0.08), consistent with our previous observations (10Ahlers B.A. Zhang X.Q. Moorman J.R. Rothblum L.I. Carl L.L. Song J. Wang J. Geddis L.M. Tucker A.L. Mounsey J.P. Cheung J.Y. J. Biol. Chem. 2005; 280: 19875-19882Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Treating S68A cells with PMA, instead of increasing INaCa as observed in PLM cells (Fig. 3A), resulted in a modest but significant suppression of INaCa when compared with unstimulated NCX1 cells (Fig. 4A; p < 0.0004). Mutating serine 68 to glutamic acid (S68E) resulted in greater suppression of INaCa
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