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• W1997503239 abstract "The N-methyl-d-aspartate (NMDA) receptor is a glutamate gated cation channel prevalent in the postsynaptic membranes of central nervous system neurons. The neurotransmitter receptor complex is thought to represent a tetramer where variable NR2 or NR3 polypeptides form heteromeric assemblies with an obligatory NR1 subunit. Recently, we showed that cardiac myocytes from perinatal rats transiently express the NMDA receptor subunit NR2B, the function of which in heart is unknown. To characterize the cardiac NR2B protein, we determined its subcellular distribution and specific molecular interaction partners. By immunostaining of rat heart tissue slices and acutely dissociated cardiac myocytes, the NR2B antigen was localized at the sarcomeric Z-bands. Using immunoprecipitation of detergent-solubilized NR2B protein and subsequent analysis employing matrix-assisted laser desorption/ionization time of flight mass spectrometry, ryanodine receptor 2 was identified as a molecular interaction partner of the cardiac NR2B polypeptide. Differences in antibody recognition indicate that the cardiac NR2B polypeptide carries a structurally altered C terminus as compared with the NR2B variant prevalent in central nervous system. Based on its localization and protein interaction, the function of cardiac NR2B protein may relate to mechanosensitivity or play a role in the regulation of the contractile apparatus of neonatal heart. The N-methyl-d-aspartate (NMDA) receptor is a glutamate gated cation channel prevalent in the postsynaptic membranes of central nervous system neurons. The neurotransmitter receptor complex is thought to represent a tetramer where variable NR2 or NR3 polypeptides form heteromeric assemblies with an obligatory NR1 subunit. Recently, we showed that cardiac myocytes from perinatal rats transiently express the NMDA receptor subunit NR2B, the function of which in heart is unknown. To characterize the cardiac NR2B protein, we determined its subcellular distribution and specific molecular interaction partners. By immunostaining of rat heart tissue slices and acutely dissociated cardiac myocytes, the NR2B antigen was localized at the sarcomeric Z-bands. Using immunoprecipitation of detergent-solubilized NR2B protein and subsequent analysis employing matrix-assisted laser desorption/ionization time of flight mass spectrometry, ryanodine receptor 2 was identified as a molecular interaction partner of the cardiac NR2B polypeptide. Differences in antibody recognition indicate that the cardiac NR2B polypeptide carries a structurally altered C terminus as compared with the NR2B variant prevalent in central nervous system. Based on its localization and protein interaction, the function of cardiac NR2B protein may relate to mechanosensitivity or play a role in the regulation of the contractile apparatus of neonatal heart. The NMDA 1The abbreviations used are: NMDA, N-methyl-d-aspartate; CM, cardiac myocytes; BSA, bovine serum albumin; DABCO™, 1,4-diacabicyclo[2.2.2]octane; EDAC, N-(3-methylaminopropyl)-N′-ethylcarbodiimide; IP3, inositol triphosphate; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; P 4, P 5, P 12, P 74, postnatal days 4, 5, 12, and 74, respectively; PBS, phosphate-buffered saline; RT, reverse transcription; TRITC, 5,6-tetramethylrhodamine-5,6-isothiocyanate; sulfo-SMCC, sulfosuccinimidyl-4-(N-maleinidomethyl)cyclohexane-1-carboxalate; TM, transmembrane. 1The abbreviations used are: NMDA, N-methyl-d-aspartate; CM, cardiac myocytes; BSA, bovine serum albumin; DABCO™, 1,4-diacabicyclo[2.2.2]octane; EDAC, N-(3-methylaminopropyl)-N′-ethylcarbodiimide; IP3, inositol triphosphate; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; P 4, P 5, P 12, P 74, postnatal days 4, 5, 12, and 74, respectively; PBS, phosphate-buffered saline; RT, reverse transcription; TRITC, 5,6-tetramethylrhodamine-5,6-isothiocyanate; sulfo-SMCC, sulfosuccinimidyl-4-(N-maleinidomethyl)cyclohexane-1-carboxalate; TM, transmembrane. receptor is a subtype of the excitatory glutamate receptor family prevalent in mammalian central nervous system. After activation by glutamate binding, the functional NMDA receptor complex forms an ion channel which permits the influx of Na+ and Ca2+ into the cell. The NMDA receptor complex is thought to represent a tetrameric assembly of the subunits NR1, NR2A-D, and NR3A-B (1Hollmann M. Boulter J. Maron C. Heinemann S. Ren. Physiol. Biochem. 1994; 17: 182-183PubMed Google Scholar, 2Nishi M. Hinds H. Lu H.P. Kawata M. Hayashi Y. J. Neurosci. 2001; 21: 1-6Google Scholar, 3Matsuda K. Kamiya Y. Matsuda S. Yuzaki M. Brain Res. Mol. Brain Res. 2002; 100: 43-52Crossref PubMed Scopus (161) Google Scholar). Members of the NR2A-D subunits as well as some NR1 splice variants use their cytoplasmic C-terminal domains to interact with a variety of intracellular proteins including the cytoskletal protein α-actinine (4Dunah A.W. Wyszynski M. Martin D.M. Sheng M. Standaert D.G. Brain Res. Mol. Brain Res. 2000; 79: 77-87Crossref PubMed Scopus (50) Google Scholar) and, via their PDZ domains, the postsynaptic density proteins PSD-95 and SAP-102 (5Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1611) Google Scholar, 6Niethammer M. Kim E. Sheng M. J. Neurosci. 1996; 16: 2157-2163Crossref PubMed Google Scholar, 7Rutter A.R. Freeman F.M. Stephenson F.A. J. Neurochem. 2002; 81: 1298-1307Crossref PubMed Scopus (20) Google Scholar, 8Sans N. Prybylowski K. Petralia R.S. Chang K. Wang Y.X. Racca C. Vicini S. Wenthold R.J. Nat. Cell Biol. 2003; 5: 520-530Crossref PubMed Scopus (256) Google Scholar). In the central nervous system, formation of functional NMDA receptors requires the presence of at least one NR1 subunit in complex with at least one of the NR2 subunits (9Ishii T. Moriyoshi K. Sugihara H. Sakurada K. Kadotani H. Yokoi M. Akazawa C. Shigemoto R. Mizuno N. Masu M. J. Biol. Chem. 1993; 268: 2836-2843Abstract Full Text PDF PubMed Google Scholar, 10Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2819) Google Scholar). In contrast, NR3 subunits act as regulators that decrease channel currents in NR1/NR2 heteromeric receptors but are not able to form ion channel complexes (2Nishi M. Hinds H. Lu H.P. Kawata M. Hayashi Y. J. Neurosci. 2001; 21: 1-6Google Scholar, 3Matsuda K. Kamiya Y. Matsuda S. Yuzaki M. Brain Res. Mol. Brain Res. 2002; 100: 43-52Crossref PubMed Scopus (161) Google Scholar, 11Das S. Sasaki Y.F. Rothe T. Premkumar L.S. Takasu M. Crandall J.E. Dikkes P. Conner D.A. Rayudu P.V. Cheung W. Chen H.S. Lipton S.A. Nakanishi N. Nature. 1998; 393: 377-381Crossref PubMed Scopus (503) Google Scholar). In the mammalian central nervous system, developmental and regional variations in NMDA receptor subunit composition result in altered channel properties, including differences in drug sensitivity and channel conductance (12Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3638) Google Scholar). As evident from developmental distribution studies, the NR1 subunit appears to be uniformly expressed throughout the brain starting early in development. In contrast, the different NR2 and NR3 subunits are strictly regulated, both developmentally and spatially (11Das S. Sasaki Y.F. Rothe T. Premkumar L.S. Takasu M. Crandall J.E. Dikkes P. Conner D.A. Rayudu P.V. Cheung W. Chen H.S. Lipton S.A. Nakanishi N. Nature. 1998; 393: 377-381Crossref PubMed Scopus (503) Google Scholar, 13Takai H. Katayama K. Uetsuka K. Nakayama H. Doi K. Exp. Mol. Pathol. 2003; 75: 89-94Crossref PubMed Scopus (55) Google Scholar). NMDA receptors have been implicated in learning and memory by pattern formation and long term potentiation (14Kullmann D.M. Asztely F. Walker M.C. Cell. Mol. Life Sci. 2000; 57: 1551-1561Crossref PubMed Scopus (58) Google Scholar) but are also associated with excitotoxic cell death of neurons during neurodegeneration and stroke (15Faden A.I. Demediuk P. Panter S.S. Vink R. Science. 1989; 244: 798-800Crossref PubMed Scopus (1304) Google Scholar, 16Cohen R.A. Hasegawa Y. Fisher M. Neurol. Res. 1994; 16: 443-448Crossref PubMed Scopus (12) Google Scholar, 17Crair M.C. Malenka R.C. Nature. 1995; 375: 325-328Crossref PubMed Scopus (590) Google Scholar). While the developmental and regional distribution of NMDA receptor subunits in the central nervous system has been extensively investigated, little is known about the expression of NMDA receptor subunits in non-neuronal tissues. Recently, evidence has accumulated that glutamate receptors may also possess important functions other than synaptic transmission of neuronal impulses in extraneuronal tissues such as lung (18Said S.I. Berisha H.I. Pakbaz H. Neuroscience. 1995; 65: 943-946Crossref PubMed Scopus (46) Google Scholar) or pancreas (19Inagaki N. Kuromi H. Gonoi T. Okamoto Y. Ishida H. Seino Y. Kaneko T. Iwanaga T. Seino S. FASEB J. 1995; 9: 686-691Crossref PubMed Scopus (172) Google Scholar). In a previous study, we found NR2B protein to be transiently expressed in rat heart during perinatal development. Here, the NR2B polypeptide was located to sarcomeric Z-bands in both the neonatal heart ventricle and acutely dissociated cardiac myocytes. In contrast to the aorticopulmonary septum and conotruncal cushions (20Jiang X. Rowitch D.H. Soriano P. McMahon A.P. Sucov H.M. Development (Camb.). 2000; 127: 1607-1616PubMed Google Scholar), cells forming the NR2B expressing ventricle are not of neural crest origin. While the NR2B subunit is known to functionally depend on co-expression with the NR1 subunit, we previously failed to detect either NR1 antigen or transcripts as analyzed by Western blot and RT-PCR, respectively (21Seeber S. Becker K. Rau T. Eschenhagen T. Becker C.M. Herkert M. J. Neurochem. 2000; 75: 2472-2477Crossref PubMed Scopus (33) Google Scholar). Thus, an identification of potential interaction partners of the NR2B polypeptide was attempted in neonatal rat heart. By immunoprecipitation and matrix-assisted laser desorption/ionization time of flight mass spectrometry, we identified the ryanodine receptor 2 as a molecular interaction partner of the cardiac NR2B protein. Generation and Use of NR2B Antibodies—Antibodies were raised in rabbits (Chinchilla Bastard) against a synthetic peptide representing the N-terminal region of subunit NR2B (439RIISENKTDEEPGYCIC454) as described before (21Seeber S. Becker K. Rau T. Eschenhagen T. Becker C.M. Herkert M. J. Neurochem. 2000; 75: 2472-2477Crossref PubMed Scopus (33) Google Scholar, 22Herkert M. Rottger S. Becker C.M. Eur. J. Neurosci. 1998; 10: 1553-1562Crossref PubMed Scopus (45) Google Scholar). Numbers indicate the position of the first and last amino acid in the mature subunit sequence. The underlined cysteine residue was added to the C terminus for coupling to keyhole limpet hemocyanine (Calbiochem, Bad Soden, Germany) or EAH-Sepharose (Amersham Biosciences, Freiburg, Germany) with the heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleinidomethyl)cyclohexane-1-carboxalate (sulfo-SMCC; Pierce). Peptide-specific antibodies were purified by affinity chromatography on immobilized peptides to sulfo-SMCC activated EAH-Sepharose. A polyclonal anti-NR2B antibody from rabbit recognizing a C-terminal epitope consisting of 19 amino acids (positions 1436–1457) was purchased from Upstate Biotechnology (Lake Placid, NY). An N-terminal polyclonal anti-NR2B antibody directed against an epitope covering 54 amino acids (amino acids 27–76) was obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). Reverse Transcriptase PCR—Total RNA from heart was reverse transcribed into cDNA using the Superscript II reverse transcriptase (Invitrogen, Eggenstein, Germany) and random hexameric primers. Aliquots of the cDNA obtained were amplified by PCR performed in 15 mm Tris (pH 8.3), 2 mm MgCl2, 50 mm KCl, 125 μm concentration of each deoxynucleoside triphosphate, and 10 pmol of each oligonucleotide given in Table I (all purchased from MWG, München, Germany). PCR conditions were as follows: heating to 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min. The cycles were performed 30 times. The amplificats were subjected to electrophoresis on 1% agarose gels, stained with propidium iodide, and visualized under UV light.Table IOligonucleotides used for RT-PCR amplification of the entire coding region of NMDA receptor subunit NR2B Sequences of the corresponding forward and reverse primers and positions of the first and last nucleotide of the resulting amplimer in the mature NR2B transcript from rat are given. Right column shows the predicted amplimer size. nt, nucleotide.SequencePositionSizeNR2B I forward:5′-CGTACCCCAAGAGAGCCGACTAGCTG-3′nt 324-16241300 bpNR2B I reverse:5′-CGATTACCACGCTTTCCACAATGACAA-3′NR2B II forward:5′-TGTCCAGAGACTGAAGAACAGGAAG-3′nt 1532-2187655 bpNR2B II reverse:5′-GACCCCAGAGTAACCAAATCGC-3′NR2B III forward:5′-TGTGTCACCTTCTGCCTTCTTAGAG-3′nt 1982-2633651 bpNR2B III reverse:5′-TGGATAGCAATGCCATAGCCCG-3′NR2B IV forward:5′-CGGTACCGCATTGCTCTCCCTGAAAAC-3′nt 2498-35541056 bpNR2B IV reverse:5′-CGATTACGACAGTGTGCGTGGAGATGT-3′NR2B V forward:5′-CGGTACCAGCGACCTGTATGGCAAGTT-3′nt 3456-48341378 bpNR2B V reverse:5′-CGATTACCCCGTACCCACCTTAACCTC-3′ Open table in a new tab Preparation of Tissue Slices and Cardiac Myocytes Culture—Tissue slices from rat cardiac ventricels were prepared following the method of Koulen et al. (23Koulen P. Fletcher E.L. Craven S.E. Bredt D.S. Wassle H. J. Neurosci. 1998; 18: 10136-10149Crossref PubMed Google Scholar). On postnatal day 4 (P 5) and at the adult stage (P 74), heart tissue was dissected from Wistar rats and fixed by diffusion with 4% EDAC in 0.1 m sodium phosphate buffer (pH 7.4) for 30 min. After fixation, hearts were washed three times with PBS (pH 7.4) and cryopreserved in 25% sucrose in PBS (pH 7.4). Slices (7 μm) were dissected with a cryotome (MICROM, Walldorf, Germany) at -30 °C and immunostained immediately. Cardiac myocytes (CM) were cultivated as described elsewhere (24Webster K.A. Bishopric N.H. J. Mol. Cell. Cardiol. 1992; 24: 741-751Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Briefly, heart tissue from P 5 rats was dissociated by trypsinization and trituration cycles, centrifuged (400 × g, 1 min), and resuspended in non-CM medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum). Non-CM cells were allowed to attach to the bottom of a 10-cm culture plate for 90 min. The CMs in the supernatant were then plated on 3-cm culture plates containing four coverslips each (coated with 50 mg/ml poly-l-lysine, 10% silane) at a density of 200,000 cells per plate. Cells were grown at 5% CO2 and 37 °C in CM medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum, 10 mm bromodesoxyuridine) for 4 days. CMs were washed three times with PBS (pH 7.4) and fixed using 4% formaldehyde in PBS (pH 7.4) for 10 min. After removal of formaldehyde, reactive carbonyl groups were blocked by 0.1 m ammoniumchloride for 10 min followed by another three washing steps with PBS (pH 7.4). For staining procedures, coverslips with fixed CMs were transferred into 24-well plates. Immunohistochemistry—Before addition of primary antibody, fixed cells and tissue slices were incubated in blocking buffer containing 10% normal goat serum in PBS for 1 h. Samples were incubated with affinity-purified polyclonal NR2B antiserum (protein concentration: ∼20 μg/ml) in addition to a monoclonal antibody directed against tubuline, ryanodine receptor 2, or actinine (Dianova, Hamburg, Germany; Alexis, Cologne, Germany) in 1% normal goat serum in PBS for 1 h. Following three washing steps with PBS, samples were incubated with Alexa-conjugated goat anti-rabbit and goat anti-mouse antibodies (Molecular Probes, Leiden, Netherlands) or with TRITC-conjugated phalloidin (Sigma, Deisenhofen, Germany) for 30 min. After an additional washing step, samples were mounted in Mowiol containing the antifading reagent DABCO™ (Sigma). Stained cells and tissue slices were analyzed using a confocal laser scanning microscope (Bio-Rad, München, Germany) and a fluorescence microscope (Zeiss, Jena, Germany), respectively, and photographs were taken with a CCD camera (Hamamatsu, Shizouka, Japan). Preparation of Membrane Proteins and Immunprecipitation—For preparation of membrane proteins, heart and cerebral cortex tissue from Wistar rats (P 5 or P 74) was dissected and homogenized for 3 min in buffer HP1 (0.32 m sucrose, 20 mm Tris-HCl (pH 7.4), 5 mm o-phenantroline, 2 μg/ml E64, 10 μg/ml pepstatinA, 10 μg/ml leupeptin, 30 μg/ml Pefabloc, 2.5 mm EDTA, 2.5 mm EGTA; Sigma) using a glass homogenizer. The homogenate was centrifuged (1000 x g; 5 min), and the low speed supernatant was pelleted at 20,000 x g for 20 min. The resulting crude membrane fraction was resuspended in hypotonic buffer HP2 (HP1 without sucrose), homogenized for 30 s, and recentrifuged at 20,000 x g for 20 min. The final pellet was resuspended in HP2 at a final protein concentration of 5–10 mg/ml. Immunoprecipitations were performed as described previously (22Herkert M. Rottger S. Becker C.M. Eur. J. Neurosci. 1998; 10: 1553-1562Crossref PubMed Scopus (45) Google Scholar). In short, membrane proteins were solubilized from crude membrane fractions by incubation with 1% Triton, 0.5% sodium desoxycholate in 50 mm Tris-HCl (pH 7.0) for 1 h at 30 °C followed by centrifugation at 100,000 x g for 30 min. Solubilized proteins were reacted with affinity-purified antiserum against NR2B or NR1 or a monoclonal antibody against ryanodine receptor 2 (Alexis, Cologne, Germany) (antibody concentration: ∼20 μg/ml). Antibody-coupled protein complexes were precipitated using protein A-conjugated Sepharose beads. Protein complexes were precipitated (3000 × g; 5 min), washed three times with 50 mm Tris-HCl containing 1.5 m NaCl, and resuspended in Laemmli buffer containing 2 m urea. All steps were carried out at 4 °C. SDS Electrophoresis and Western Blotting—Protein concentrations were estimated by the method of Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using BSA as a standard. Membrane preparations or immunoprecipitates were incubated (56 °C; 20 min) in Laemmli sample buffer containing 2 m urea. Proteins (50 μg of protein per lane) were separated by SDS-polyacrylamide gelelectrophoresis on 6% polyacrylamide slab gels under reducing conditions. Proteins were visualized with SyproRuby™ protein stain or transferred onto nitrocellulose for subsequent in-gel digestion or Western blot analysis, respectively. Following SDS gelelectrophoresis, proteins were transferred on nitrocellulose using a semidry blotting apparatus (Biometra, Göttingen, Germany). Protein transfer was controlled by reversible staining with Ponceau S. Blots were blocked with 5% Blotto (PBS containing 5% nonfat dry milk powder and 0.1% Triton X-100) for 1 h. Incubation with affinity-purified polyclonal antiserum K39 (10 μg/ml) or antibodies against the ryanodine receptor 2, NR1, and NR2B proteins (dilution 1:500) was carried out overnight in 0.5% Blotto at 4 °C, followed by three washing steps with PBS. Antibodies were detected by fluorescence using Cy5-conjugated goat anti-rabbit IgG (1:200; Dianova, Hamburg, Germany) for 30 min, followed by extensive washing with PBS. Finally, fluorescence was visualized using the Storm Fluorescense Imager (Amersham Biosciences). Peptide Mass Fingerprinting—After SDS-gel electrophoresis and SyproRuby™ staining, protein gels were visualized using UV light and bands of interest excised. Gel bands were crushed into 1 mm2 pieces and destained using 50% acetonitrile, 25 mm ammoniumhydrogencarbonate. Following complete drying in a vacuum centrifuge, gel pieces were resuspended in 50 μl of 25 mm ammonium hydrogen carbonate containing 0.2 μg/μl sequencing grade trypsin (Promega, München, Germany). After enzymatic digest at 37 °C overnight, the resulting peptide fragments were eluted from the gel by adding 5% acetic acid, 50% actonitrile and sonification (20 min; room temperature). Supernatants were collected, and elution was repeated four times. Finally, supernatants were dried completely in a vacuum centrifuge, and peptides were resuspended in 10 μl of H2O containing 0.1% trifluoroacetic acid. For MALDI-TOF-MS, digests were diluted with H2O (1:10 to 1:50). The sample (0.5 μl) was mixed with 0.5 μl of 3-hydroxy-α-cyanocinnamic acid matrix (saturated solution in 30% acetonitrile, 0.1% trifluoroacetic acid), dotted onto a steel target, and air-dried. MALDI-TOF analysis was performed on a Biflex III (Bruker Daltonics, Bremen, Germany) in the reflector mode. Desorption of the samples was carried out using a nitrogen laser (337 nm), and the probes were accelerated with 19 kV after a delay of 3500 ns. For one sample spectrum, 25–50 individual spectra of the respective probes were performed and averaged. A peptide standard mix served as an external calibration. The masses obtained by MALDI-TOF-MS were used for a peptide mass fingerprint search. Experimentally obtained masses were aligned to theoretical peptide masses for trypsin digestion of all proteins available in the NCBI data base. For this purpose, we employed the search program MS-Fit, available at prospector.ucsf.edu (26Clauser K.R. Baker P. Burlingame A.L. Anal. Chem. 1999; 71: 2871-2882Crossref PubMed Scopus (972) Google Scholar). Data base search settings were as follows: protein mass (apparent molecular mass of the protein in SDS-PAGE according to the molecular mass of the standard proteins), ±50 kDa of the respective cut out protein band; species, rodentia; mass tolerance, 0.05%; maximum number of missed cleavages, 1; standard modifications, none; minimum number of peptides required for match, 4; data base, NCBI Protein. Immunological Variations of NR2B Antigen in Rat Heart and Central Nervous System—During perinatal development, the NMDA receptor subunit NR2B is detectable in rat heart (21Seeber S. Becker K. Rau T. Eschenhagen T. Becker C.M. Herkert M. J. Neurochem. 2000; 75: 2472-2477Crossref PubMed Scopus (33) Google Scholar) using an affinity-purified rabbit antiserum (K39) directed against an extracellular epitope formed by amino acids 439–454 that is adjacent to transmembrane region 1 (TM1) (Fig. 1A). In contrast, studies using antibodies directed against the very C-terminal part of NR2B failed to detect the corresponding antigen in rat heart at any age analyzed (27Gill S.S. Pulido O.M. Mueller R.W. McGuire P.F. Brain Res. Bull. 1998; 46: 429-434Crossref PubMed Scopus (89) Google Scholar, 28Gill S.S. Pulido O.M. Toxicol. Pathol. 2001; 29: 208-223Crossref PubMed Scopus (149) Google Scholar, 29Leung J.C. Travis B.R. Verlander J.W. Sandhu S.K. Yang S.G. Zea A.H. Weiner I.D. Silverstein D.M. Am. J. Physiol. 2002; 283: R964-R971Crossref PubMed Scopus (64) Google Scholar). To elucidate these apparently contradictory reports, we employed three antibodies defining distinct epitopes of NR2B. In Western blot analysis, affinity-purified antiserum K39 stained a 180-kDa protein present in both heart and cerebral cortex tissue. In the immunostains from central nervous tissue, a microheterogeneity of the NR2B antigen was observed. An identical result was reproducibly obtained in with an antibody directed against an epitope formed by amino acids 27–76 that is situated within the N-terminal extracellular domain of NR2B. In contrast, an antibody against the C-terminal part of NR2B detected this antigen in cerebral cortex but not in heart (Fig. 1B). To corroborate these results, we performed peptide mass fingerprinting of a K39 immunoprecipitate of the 180-kDa antigen from rat heart. Following SDS electrophoresis and in-gel digest, an informative peptide fragment pattern was obtained and subjected to a data bank search. Indeed, the NR2B polypeptide turned out to be the most probable candidate (compare Table II). Thus, by showing immunoreactivity with two anti-NR2B antibodies with different specificities, directed against distinct epitopes situated at the very N terminus and an extracellular pre-TM1 segment of NR2B (Fig. 1A), and the peptide mass fingerprint of the 180-kDa immunoband from heart, the expression of NR2B in juvenile rat heart could be proven unambiguously. To exclude alternative splicing to underlie the differences in antibody binding, RT-PCRs of overlapping fragments comprising the entire coding region of the NR2B transcript were performed. Sizes of the DNA fragments obtained were as predicted by gene bank data (see Fig. 1C and Table I), and partial sequencing of these fragments revealed no obvious sequence heterogeneity, regardless of their origin from either heart or central nervous system (data not shown). Thus, no evidence was obtained for transcript variability affecting the C terminus of the NR2B subunit.Table IIPeptide mass fingerprints of proteins precipitated from neonatal heart by anti NR2B antiserum K39 Left column lists the protein band excised from SDS-PAGE (Fig. 4B). Middle column indicates the masses obtained after trypsin digest. In right column, high scoring candidate proteins for the respective samples are listed.Sample bandMasses (m/z)Candidate proteinsBSA927.570BSA1001.6401249.7701305.8801402.4701464.8901479.9701505.0201946.080No. 11010.2318NMDA receptor subunit 2B, rat RNA polymerase 1-4 (194-kDa subunit), nitricoxidase synthase, rat voltage-gated sodium channel, type VIII (α-polypeptide)1038.95521094.17541104.21621155.36281180.08971210.98871261.28131284.71121309.96391337.98561346.98641486.25671507.45091656.88341804.94012391.73362700.39533336.0561No. 6957.4432Spectrin, rat ryanodine receptor cardiac, rodent sacsin, rodent myosin actin cross-linking family protein 7, mouse IP3 receptor, mouse dynein.971.5962993.10341002.06511034.48061045.55121055.64781146.58671187.30131291.64291307.63001494.36201526.66191650.83322062.98632212.83852327.9869 Open table in a new tab Accumulation of NR2B Protein at Cardiac Z-bands—To elucidate the subcellular localization of NR2B antigen in native cardiac tissue, rat hearts were dissected at postnatal days P 5 and P 74, fixed, and cryoslices were immunostained using the affinity-purified anti-NR2B antiserum K39. As a marker of filamentous actin, TRITC-conjugated phalloidin was used to determine the type of cells stained. In agreement with our previous results, tissue slices of P 5 rat yielded intense NR2B signals (Fig. 2, B and D). The striated pattern generated by phalloidin staining clearly identified the anti-NR2B reactive cells as cardiac myocytes (Fig. 2A). After preincubation of the antiserum with antigenic peptide, the NR2B signals were completely abolished (Fig. 2C). In contrast to slices from P 5 rat heart, adult heart did not produce any signal above background levels (Fig. 2F). The NR2B signal obtained with P 5 slices followed a well defined intracellular striated pattern (Fig. 2D). The subcellular distribution of NR2B antigen was investigated more closely in cultured cardiac myocytes. Single cells were prepared from ventricles of P 5 rats, fixed after 36 h in culture, and double-stained with affinity-purified anti-NR2B antiserum and monoclonal antibodies against cytoskeletal proteins. As analyzed by confocal microscopy, all myocytes showed significant immunosignals for NR2B. In addition to a weak intracellular reticular staining pattern, an intense accumulation of antigen was observed at distinct sarcomeric bands (Fig. 3, A, D, and G). By immunological co-localization with α-actinin, a protein forming the Z-bands, these structures could be confirmed to represent the Z-bands (Fig. 3C). We also found a less defined co-distribution of NR2B antigen with filamentous actin (Fig. 3F). In contrast, the reticular staining pattern of tubulin and the striated pattern of NR2B antigen was clearly distinct and did not overlap (Fig. 3I).Fig. 3Subcellular localization of NR2B antigen in acutely dissociated cardiac myocytes. Cardiac myocytes were double-stained with anti-NR2B antiserum and antibodies against typical structural proteins of cardiac myocytes. NR2B antigen appeared intracellularly, accumulating at distinct bands within the cells (A, D, G; arrows). A–C, double staining of NR2B (A) with actinin (B) antibody revealed clear co-localization, thereby indicating an accumulation of NR2B antigen at the sarcomeric Z-bands (C; arrows). D–F, double staining of NR2B (D) with TRITC-conjugated phalloidin (E). Staining patterns for both proteins, NR2B and actin, showed related distributions accumulating at the sarcomeric Z-band (F; arrows). G–I, double staining NR2B (G) with tubulin (H). Tubulin showed a reticular distribution throughout the cell (H); NR2B and tubulin staining patterns were mutually exclusive (I).View Large Image Figure ViewerDownload (PPT) Different Molecular Interaction Partners of NR2B in Cerebral Cortex and Heart—To identify proteins interacting with the cardiac NR2B polypeptide, detergent extracts from membranes from heart and cerebral cortex were subjected to immunoprecipitation using affinity-purified anti-NR2B antiserum. The protein complexes precipitated were separated on SDS gels, stained with SyproRuby™, and visualized under UV light. In particular in the higher molecular mass range, we found significant differences in the composition of protein complexes precipitated from either from heart or from central nervous system preparations. In samples from cerebral cortex, signals were prominent in molecular mass ranges of 180 kDa (Fig. 4A: band 1) and 120 kDa (Fig. 4A, band 5), most l" @default.
• W1997503239 created "2016-06-24" @default.
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• W1997503239 creator A5035957608 @default.
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• W1997503239 date "2004-05-01" @default.
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• W1997503239 title "Formation of Molecular Complexes by N-Methyl-d-aspartate Receptor Subunit NR2B and Ryanodine Receptor 2 in Neonatal Rat Myocard" @default.
• W1997503239 cites W1508050508 @default.
• W1997503239 cites W1548457940 @default.
• W1997503239 cites W1594111906 @default.
• W1997503239 cites W1608934724 @default.
• W1997503239 cites W1653383134 @default.
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• W1997503239 cites W1996809674 @default.
• W1997503239 cites W2000250462 @default.
• W1997503239 cites W2014739886 @default.
• W1997503239 cites W2017213222 @default.
• W1997503239 cites W2019121351 @default.
• W1997503239 cites W2029400956 @default.
• W1997503239 cites W2031923353 @default.
• W1997503239 cites W2046485926 @default.
• W1997503239 cites W2050824263 @default.
• W1997503239 cites W2053980736 @default.
• W1997503239 cites W2058779467 @default.
• W1997503239 cites W2065169750 @default.
• W1997503239 cites W2073041310 @default.
• W1997503239 cites W2073799314 @default.
• W1997503239 cites W2085475269 @default.
• W1997503239 cites W2087916050 @default.
• W1997503239 cites W2093573816 @default.
• W1997503239 cites W2097424141 @default.
• W1997503239 cites W2105752803 @default.
• W1997503239 cites W2120699640 @default.
• W1997503239 cites W2129108032 @default.
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• W1997503239 cites W2148037744 @default.
• W1997503239 cites W2152541148 @default.
• W1997503239 cites W4293247451 @default.
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