L-glutamate binding site on N18-RE-105 neuroblastoma hybrid cells is not coupled to an ion channel.
We studied the properties of the N18-RE-105 neuronal cell line to determine if its glutamate binding site represents a neurotransmitter receptor. In immunocytochemical experiments, these cells stained strongly for neurofilament, but not for glial fibrillary acidic protein. In whole-cell patch clamp experiments, cells exhibited voltage-dependent Na+, Ca2+, and K+ currents characteristic of neurons. However, perfusion with L-glutamate or other excitatory amino acids did not evoke the inward current expected of a receptor/channel complex. In binding studies, the maximum accumulation of L-[3H]glutamate by washed membrane vesicles at 37 degrees C was 69 pmol/mg protein, and half-maximal accumulation occurred at 0.64 microM. This accumulation was blocked completely by quisqualate, partially by DL-2-amino-4-phosphonobutyric acid and L-cystine, but not at all by 1 mM kainate or N-methylaspartate. L-[3H]Glutamate accumulation was stimulated by Cl-, but reduced by Na+, 0.01% digitonin, or hyperosmotic (400 mM glucose) assay medium. The release of L-[3H]glutamate from vesicles was much faster in the presence of 100 microM unlabelled glutamate than 100 microM unlabelled quisqualate or DL-2-amino-4-phosphonobutyric acid. Thus, although N18-RE-105 cells possess many neuronal properties, the results obtained are not those expected from reversible binding of L-glutamate to a receptor/channel complex, but are consistent with a Cl- -stimulated sequestration or exchange process.
Berry
B W
BW
Department of Pharmacology and Neurobiology Curriculum, University of North Carolina, Chapel Hill 27599-7365.
Boland
L M
LM
Hoch
D B
DB
Dingledine
R
R
D000613:Aminobutyrates / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D003553:Cystine / Q000378:metabolism / Q000494:pharmacology; D004553:Electric Conductivity; D005455:Fluorescent Antibody Technique; D005971:Glutamates / Q000378:metabolism* / Q000494:pharmacology; D018698:Glutamic Acid; D006822:Hybrid Cells / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000032:analysis; D007473:Ion Channels / Q000378:metabolism*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D016900:Neurofilament Proteins; D009474:Neurons / Q000378:metabolism; D010069:Oxadiazoles / Q000378:metabolism / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D016318:Quisqualic Acid; D051381:Rats; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000613:Aminobutyrates; D002712:Chlorides; D005971:Glutamates; D007381:Intermediate Filament Proteins; D007473:Ion Channels; D016900:Neurofilament Proteins; D010069:Oxadiazoles; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D018698:Glutamic Acid; D003553:Cystine; D016318:Quisqualic Acid; D012964:Sodium; C012729:2-amino-4-phosphonobutyric acid; D011188:Potassium; D002118:Calcium