Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors.

N-methyl-D-aspartic acid (NMDA) 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-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 preidentified by the fluorescence emitted from the coexpressed 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 cotransfection, 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 among receptors 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 were cotransfected at variable ratio was 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.

[1]  D. Laurie,et al.  Ligand affinities at recombinant N-methyl-D-aspartate receptors depend on subunit composition. , 1994, European journal of pharmacology.

[2]  D. Feldmeyer,et al.  Functional Correlation of NMDA Receptor ε Subunits Expression with the Properties of Single-Channel and Synaptic Currents in the Developing Cerebellum , 1996, The Journal of Neuroscience.

[3]  P. Stern,et al.  Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors , 1992, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[4]  J. Rossier,et al.  Neuronal activity differentially regulates NMDA receptor subunit expression in cerebellar granule cells , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[5]  CE Jahr,et al.  NMDA channel behavior depends on agonist affinity , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[6]  W. Zieglgänsberger,et al.  The distribution of splice variants of the NMDAR1 subunit mRNA in adult rat brain. , 1995, Brain research. Molecular brain research.

[7]  CE Jahr,et al.  Interactions between the glycine and glutamate binding sites of the NMDA receptor , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[8]  H. Monyer,et al.  NMDA receptor channels: Subunit-specific potentiation by reducing agents , 1994, Neuron.

[9]  B. Sakmann,et al.  Developmental and regional expression in the rat brain and functional properties of four NMDA receptors , 1994, Neuron.

[10]  S. Tonegawa,et al.  Modification of NMDA Receptor Channels and Synaptic Transmission by Targeted Disruption of the NR2C Gene , 1996, The Journal of Neuroscience.

[11]  J Kerby,et al.  Pharmacological properties of recombinant human N-methyl-D-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fibroblast cells. , 1995, Molecular pharmacology.

[12]  E. R. Whittemore,et al.  Subtype-selective inhibition of N-methyl-D-aspartate receptors by haloperidol. , 1996, Molecular pharmacology.

[13]  K. Sakimura,et al.  Developmental changes in distribution of NMDA receptor channel subunit mRNAs. , 1992, Neuroreport.

[14]  Shaul Hestrin,et al.  Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse , 1992, Nature.

[15]  F. Stephenson,et al.  Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule. , 1994, The Journal of biological chemistry.

[16]  P. Bregestovski,et al.  Calcium‐dependent inactivation of heteromeric NMDA receptor‐channels expressed in human embryonic kidney cells. , 1995, The Journal of physiology.

[17]  R. Huganir,et al.  Differential Tyrosine Phosphorylation of N-Methyl-D-aspartate Receptor Subunits (*) , 1995, The Journal of Biological Chemistry.

[18]  P. Seeburg,et al.  Subtype‐specific regulation of recombinant NMDA receptor‐channels by protein tyrosine kinases of the src family. , 1996, The Journal of physiology.

[19]  P. Seeburg,et al.  Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[20]  K Williams,et al.  Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. , 1993, Molecular pharmacology.

[21]  G. Westbrook,et al.  Desensitized states prolong GABAA channel responses to brief agonist pulses , 1995, Neuron.

[22]  M. Bennett,et al.  Alternatively spliced isoforms of the NMDARI receptor subunit , 1995, Trends in Neurosciences.

[23]  G. Carmignoto,et al.  Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. , 1992, Science.

[24]  Jianhong Luo,et al.  The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). , 1997, Molecular pharmacology.

[25]  T. Kanno,et al.  Photodynamic triggering of calcium oscillation in the isolated rat pancreatic acini , 1997, The Journal of physiology.

[26]  N. Spruston,et al.  Dendritic glutamate receptor channels in rat hippocampal CA3 and CA1 pyramidal neurons. , 1995, The Journal of physiology.

[27]  G. Westbrook,et al.  Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents , 1990, Nature.

[28]  R. Huganir,et al.  Inactivation of NMDA Receptors by Direct Interaction of Calmodulin with the NR1 Subunit , 1996, Cell.

[29]  A. J. Scheetz,et al.  Modulation of NMDA receptor function: implications for vertebrate neural development , 1994, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  G. Westbrook,et al.  Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. , 1996, Molecular pharmacology.

[31]  C. McBain,et al.  N-methyl-D-aspartic acid receptor structure and function. , 1994, Physiological reviews.

[32]  Y. Jan,et al.  Changing subunit composition of heteromeric NMDA receptors during development of rat cortex , 1994, Nature.

[33]  S. Vicini,et al.  Characterization of NMDA Receptor Subunit‐Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum , 1995, Journal of neurochemistry.

[34]  S. Heinemann,et al.  Zinc potentiates agonist-lnduced currents at certain splice variants of the NMDA receptor , 1993, Neuron.

[35]  Y. Yaari,et al.  Synaptic NMDA receptors in developing mouse hippocampal neurones: functional properties and sensitivity to ifenprodil. , 1996, The Journal of physiology.

[36]  C. Portera-Cailliau,et al.  N‐Methyl‐d‐Aspartate Receptor Proteins NR2A and NR2B Are Differentially Distributed in the Developing Rat Central Nervous System as Revealed by Subunit‐Specific Antibodies , 1996, Journal of neurochemistry.

[37]  H. Bayley,et al.  Combinatorial RNA splicing alters the surface charge on the NMDA receptor , 1992, FEBS letters.

[38]  S. Nakanishi,et al.  Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing. , 1992, Biochemical and biophysical research communications.

[39]  M. Kennedy,et al.  The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[40]  H. Monyer,et al.  NR2A Subunit Expression Shortens NMDA Receptor Synaptic Currents in Developing Neocortex , 1997, The Journal of Neuroscience.

[41]  O. Garaschuk,et al.  Molecular determinants of NMDA receptor function in GABAergic neurones of rat forebrain. , 1997, The Journal of physiology.