FUNCTIONAL N-METHYL-D-ASPARTATE RECEPTORS IN CLONAL RAT PHEOCHROMOCYTOMA CELLS

1. To characterize from a molecular and functional point of view the e ndogenous NMDA receptors expressed by phaeochromocytoma (PC12) cells, experiments involving polymerase chain reaction (PCR) amplification, W estern blotting and patch-clamp analysis of undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells were performed. 2. Anal ysis of PC12 mRNA demonstrated the presence of NMDAR1 and NMDAR2C tran scripts. The NMDAR1 subunits lack the amino terminal insert of twenty- one amino acid residues, whereas transcripts with and without deletion s I and II at the 3' end of the coding region were detected. Thus, NMD A receptors of the PC12 cells might include NMDAR1A, NMDAR1E, NMDAR1C and NMDAR1D subunits. 3. Differentiation by NGF treatment of PC12 cell s did not alter mRNA expression for NMDA receptor subunits significant ly but induced an increase in both the NMDAR1 protein and the total am ount of functional receptor s that correlated well with a parallel inc rease in membrane area. 4. NMDA receptors in differentiated PC12 cells had a high affinity for both glutamate and glycine. These were estima ted kinetically as 0.59 mu M and 74 nM, respectively. Responses to glu tamate or NMDA were non-desensitizing in the presence of saturating gl ycine, but slowly desensitized with low concentrations of glycine. Cur rents 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-med iated responses in a voltage-independent manner. 5. With 0.5 mM Ca2+, single-channel analysis revealed very brief openings (mean open time ( (t) over bar o)= 0.42 ms), with at least two conductiue states, 55 and 33 pS, both having markedly low open probability. At 2 mM Ca2+, condu ctances were reduced to 39 and 19 pS, without an effect in open probab ility or mean open time. 6. The functional properties of NMDA receptor s in PC12 cells were very similar to those described for NMDAR1A-NMDAR 2C heteromers recombinantly expressed. The PC12 cell line provides a s imple and reproducible system to analyse some specific NMDA receptor p roperties.