Identification of an intracellular pool of gamma-aminobutyric acidA/benzodiazepine receptors en route to the cell surface of brain neurons in culture.
We have previously shown that approximately 20% of the gamma-aminobutyric acid (GABA)A/benzodiazepine receptors in intact neurons are intracellular [J. Neurosci. 6:2857-2863 (1986); Mol. Pharmacol., 35: 75-84], but the nature of this pool remained unknown. In this report, we describe the synthesis, appearance in the plasma membrane, and degradation of the GABAA/benzodiazepine receptor complex in nerve cells derived from embryonic chick brain and grown in primary monolayer cell culture. Irreversible photoaffinity labeling of the benzodiazepine-sensitive modulator site on the GABAA/benzodiazepine receptor using [3H]flunitrazepam as a permeable probe was used in conjunction with exhaustive trypsinization of intact cells and competition binding using Ro7-0213 (a benzodiazepine bearing a charged quarternary ammonium moiety). Newly synthesized intracellular receptors that are transported to the surface membrane comprise at most only 4% of total receptors and up to one fifth of the steady state intracellular pool. The kinetics of receptor recovery after photoaffinity blockade are consistent with a model in which newly synthesized receptors first appear within the cell and then undergo intracellular transit before appearing on the cell surface. This suggests a "precursor-product" relationship between newly synthesized intracellular receptors and surface receptors. However, the majority of intracellular receptors are not transported to the cell surface and may represent a "nonprecursor" pool. The kinetics of degradation for intracellular receptors are consistent with the overall kinetics for total (surface plus intracellular) receptors. Intracellular and surface receptors are pharmacologically similar with regard to their sensitivity to methyl-beta-carboline-3-carboxylate binding and their affinity for flunitrazepam. Degradation of the individual receptor subunits (with apparent molecular weights of 51,000 and 48,000) was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both photoaffinity-labeled subunits degrade with indistinguishable biphasic kinetics. From these results, and the results of our previous studies, we propose a minimal model describing the dynamic cellular pathway for GABAA/benzodiazepine receptor metabolism in central nervous system neurons.
Czajkowski
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Department of Anatomy and Cell Biology, State University of New York, Brooklyn 11203.
Farb
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D000818:Animals; D001923:Brain Chemistry; D002478:Cells, Cultured; D002642:Chick Embryo; D007700:Kinetics; D008970:Molecular Weight; D009474:Neurons / Q000032:analysis*; D011963:Receptors, GABA-A / Q000032:analysis* / Q000378:metabolism
D011963:Receptors, GABA-A