Functional N-methyl-D-aspartate receptors in clonal rat phaeochromocytoma cells.
1. To characterize from a molecular and functional point of view the endogenous NMDA receptors expressed by phaeochromocytoma (PC12) cells, experiments involving polymerase chain reaction (PCR) amplification, Western blotting and patch-clamp analysis of undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells were performed. 2. Analysis of PC12 mRNA demonstrated the presence of NMDAR1 and NMDAR2C transcripts. The NMDAR1 subunits lack the amino terminal insert of twenty-one amino acid residues, where as transcripts with and without deletions I and II at the 3' end of the coding region were detected. Thus, NMDA receptors of the PC12 cells might include NMDAR1A, NMDAR1E, NMDAR1C and NMDAR1D subunits. 3. Differentiation by NGF treatment of PC12 cells did not alter mRNA expression for NMDA receptor subunits significantly but induced an increase in both the NMDAR1 protein and the total amount of functional receptors that correlated well with a parallel increase in membrane area. 4. NMDA receptors in differentiated PC12 cells had a high affinity for both glutamate and glycine. These were estimated kinetically as 0.59 microM and 74 nM, respectively. Responses to glutamate or NMDA were non-desensitizing in the presence of saturating glycine, but slowly desensitized with low concentrations of glycine. Currents were completely blocked by D-aminophosphonovalerate (APV), 7-Cl-kynurenate and phencyclidine, and showed a voltage-dependent magnesium blockade. Spermine did not potentiate but inhibited NMDA receptor-mediated responses in a voltage-independent manner. 5. With 0.5 mM Ca2+, single-channel analysis revealed very brief openings (mean open time (t(o)) = 0.42 ms), with at least two conductive states, 55 and 33 pS, both having markedly low open probability. At 2 mM Ca2+, conductances were reduced to 39 and 19 pS, without an effect in open probability or mean open time. 6. The functional properties of NMDA receptors in PC12 cells were very similar to those described for NMDAR1A-NMDAR2C heteromers recombinantly expressed. The PC12 cell line provides a simple and reproducible system to analyse some specific NMDA receptor properties.
Casado
M
M
Department of Neural Plasticity, Instituto Cajal, Madrid, Spain.
López-Guajardo
A
A
Mellström
B
B
Naranjo
J R
JR
Lerma
J
J
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002460:Cell Line; D002999:Clone Cells; D004229:Dithiothreitol / Q000494:pharmacology; D004594:Electrophysiology; D005728:Ganglia, Sympathetic / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D010673:Pheochromocytoma; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology
D015220:Calcium Channels; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D013096:Spermine; D018698:Glutamic Acid; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol; D005998:Glycine