Functional and Pharmacological Differences Between Recombinant N-Methyl-D- Aspartate Receptors. Stefano Vicini, Jian Feng Wang, Jin Hong Li, Wei Jian Zhu, Yue Hua Wang, Jian Hong Luo, Barry B. Wolfe1 and Dennis R.Grayson. Dept. of Physiology and Biophysics and 1Dept. of Pharmacology, Georgetown University School of Medicine, 3900 Reservoir Road, N.W., Washington D.C. 20007, and Neurosciences Research Center, Allegheny Singer Research Institute, Depts. of Psychiatry and Anatomy and Neurobiology, Allegheny University of the Health Sciences, 320 East North Avenue, Pittsburgh, PA 15212.
APStracts 4:289N, 1997.
ABSTRACT
N-methyl-D-aspartic acid receptors transiently transfected into mammalian HEK- 293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Deactivation time course recorded in response to fast L-glutamate pulses were studied in isolated and lifted cells, as well as in outside-out membrane patches excised from cells expressing recombinant NR1 subunits in combination with the NR2A, NR2B, NR2C or NR2D NMDA receptor subunits. Transfected cells were pre-identified by the fluorescence emitted from the co-expressed Aequorea victoria jellyfish Green Lantern protein. Currents generated by NR1/NR2A channels displayed double exponential deactivation time course being faster than that in NR1/NR2B or NR1/NR2C channels. However, a large decay variability was observed within each co- transfection, suggesting that mechanisms additional to subunit composition may also regulate deactivation time course. NR1/NR2D channels displayed slowly deactivating currents. Channel deactivation was fast and comparable amongst receptor obtained by cotransfecting five distinct spliced variants of the NR1 subunit, each with the NR2A subunit. Additionally, recovery from desensitization was slower for NR1/NR2B than for NR1/NR2A channels. The average deactivation time course of responses to brief L-glutamate applications in cells where NR1/NR2A/NR2B cDNAs was cotransfected at variable ratio were intermediate between those of the NR1/NR2A and NR1/NR2B channels. Although immunocytochemical evidence indicates that the majority of cells are cotransfected by all plasmids in triple transfection, our experimental condition did not allow for a tight control of the expression of NMDA receptor subunits. This produced the result that many cells were characterized by deactivation time course and haloperidol sensitivities of separate NR1/NR2A and NR1/NR2B subunit heteromers. We also speculate on the possible formation of channels resulting from the coassembly in the same receptor of NR1/NR2A/NR2B subunits from a minority of cells that gave responses to brief application of L-glutamate characterized by slow deactivation time course and decreased haloperidol sensitivity. 

Received 6 June 1997; accepted in final form 13 Ocotber 1997.
APS Manuscript Number J465-7.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1997 The American Physiological Society.
Published in APStracts on 29 October 1997