GAMMA-AMINOBUTYRIC ACID-A [GABA(A)] AGONISTS DOWN-REGULATE GABA(A) BENZODIAZEPINE RECEPTOR POLYPEPTIDES FROM THE SURFACE OF CHICK CORTICAL-NEURONS

The impermeant SH-cleavable reagent, I-125-labeled 3,3'-dithiopropiony l 1-sulfosuccinimidyl 1'-glycyltyrosine (DPSgt) and specific immunopre cipitation were used to quantitate gamma-aminobutyric acid-A (GABA(A)) receptor polypeptides with domains exposed on the surface of chick co rtical neurons. The I-125 label incorporated into 50- and 53-kDa recep tor subunits was completely removed by washing the cells with glutathi one (GSH) buffer. A single dose of 100 muM GABA or GABA agonist was ad ded to the neurons, and after 5 days in culture the cells were washed and labeled with I-125-DPSgt. This treatment with GABA or isoguvacine reduced the level of the receptor I-125-labeled subunits by 50-60%, wh ile the reduction by 4,5,6,7-tetrahydroisoazolo[5,4-c]pyridin-3-ol was less than 17%. The subunit down-regulation by agonist was prevented b y the GABA(A) antagonist pha-hydroxy-16-imino-5beta-17-aza-androstan-1 1-one (R5135). Direct iodination of membranes isolated from treated ce lls revealed a similar loss of subunits, indicating that the down-regu lated polypeptides are not retained intracellularly. Furthermore, the level of intracellular [H-3]flunitrazepam binding (per mg of protein) did not change significantly during chronic GABA treatment, while the fraction of intracellular binding rose from 7% to 15% of the total, ow ing to a decline in surface binding. The fate of the surface subunits during acute agonist exposure was examined by labeling intact neurons with I-125-DPSgt at 0-degrees-C, incubating with GABA for 2-4 h at 37- degrees-C and then washing with GSH buffer. Of the 50-53-kDa receptor peptides originally on the surface, 16 +/- 2% became protected from GS H during the 2-h GABA treatment. This sequestration was not found in c ells incubated without GABA, with GABA + R5135, or with GABA at 0-degr ees-C. A consistently lower level of sequestered subunits was recovere d after a 4- versus 2-h GABA treatment at 37-degrees-C, suggesting pol ypeptide degradation. Overall, the results indicate that GABA(A) recep tor sequestration and subsequent degradation play an important role in agonist-dependent down-regulation.