REPRESSION OF GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR ALPHA-1-POLYPEPTIDE BIOSYNTHESIS REQUIRES CHRONIC AGONIST EXPOSURE

Although it is well established that the number of gamma-aminobutyric acid type A (GABA(A)) receptors declines in cortical neurons exposed t o GABA(A) receptor agonists, the mechanisms responsible for this use-d ependent down-regulation remain unclear. Two hypotheses have been prop osed: (i) agonist-evoked sequestration and degradation of surface GABA (A) receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [S-35]Met/Cys pulse-chase labeling of chick cortical newtons in culture and immunoprecipitation and immun oblotting with an antibody (RP4) directed against a GABA(A) receptor a lpha(1)-(331-381) fusion protein. Exposure of the cells to GABA or iso guvacine for 2 h to 4 days had no effect on the initial rate of S-35 i ncorporation into the GABA(A) receptor 51-kDa alpha 1 polypeptide, but this rate declined by 33% after a 7-day treatment, This is consistent with a previous report (Baumgartner, B. J., Harvey, R. J., Darlison, M. G., and Barnes, E. M. (1994) Mol. Brain Res. 26, 9-17) that a 7-day GABA treatment of this preparation produced a 45% reduction in the al pha 1 subunit mRNA level, while a 4-day exposure had no detectable eff ect. On the other hand, after a 4-day exposure to these agonists, a 30 % reduction in the level of the alpha 1 polypeptide was observed on im munoblots, similar to that found previously for down-regulation of GAB A(A) receptor ligand-binding sites, Thus, the de nova synthesis of GAB A(A) receptor alpha 1 subunits is subject to a delayed use-dependent r epression that was observed after, rather than before, the decline in neuronal levels of the polypeptide. Pulse-chase experiments showed a m onophasic degradation of the GABA(A) receptor S-35-alpha 1 subunit Wit h a t(1/2) = 7.7 h, a process that was unaffected by the addition of G ABA to neurons during the chase period. These nascent S-35-labeled pol ypeptides are presumably diluted into the neuronal pool of unlabeled u nassembled al subunits, which was found to exceed by a 4:1 molar ratio the amount assembled into [H-3]flunitrazepam binding sites. Thus, the data reveal an alternative scheme for degradation of GABA(A) receptor polypeptides: a pathway that may participate in the agonist-independe nt degradation of unassembled receptor subunits. This differs from ano ther pathway for the agonist-dependent degradation of mature GABA(A) r eceptors derived from the neuronal surface (Calkin, P. A. and Barnes, E. M., Jr. (1994) J. Biol. Chem. 269, 1548-1553).