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Volume 272, Number 26, Issue of June 27, 1997 pp. 16288-16294
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Repression of -Aminobutyric Acid Type A Receptor 1 Polypeptide Biosynthesis Requires Chronic Agonist Exposure

(Received for publication, September 4, 1996, and in revised form, April 8, 1997)

Jorge D. Miranda and Eugene M. Barnes Jr.^¶

From the  Neuroscience Division and ^¶ Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030

Although it is well established that the number of -aminobutyric acid type A (GABA_A) receptors declines in cortical neurons exposed to GABA_A receptor agonists, the mechanisms responsible for this use-dependent down-regulation remain unclear. Two hypotheses have been proposed: (i) agonist-evoked sequestration and degradation of surface GABA_A receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [³⁵S]Met/Cys pulse-chase labeling of chick cortical neurons in culture and immunoprecipitation and immunoblotting with an antibody (RP4) directed against a GABA_A receptor 1-(331-381) fusion protein. Exposure of the cells to GABA or isoguvacine for 2 h to 4 days had no effect on the initial rate of ³⁵S incorporation into the GABA_A receptor 51-kDa 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 1 subunit mRNA level, while a 4-day exposure had no detectable effect. On the other hand, after a 4-day exposure to these agonists, a 30% reduction in the level of the 1 polypeptide was observed on immunoblots, similar to that found previously for down-regulation of GABA_A receptor ligand-binding sites. Thus, the de novo synthesis of GABA_A receptor 1 subunits is subject to a delayed use-dependent repression that was observed after, rather than before, the decline in neuronal levels of the polypeptide.

Pulse-chase experiments showed a monophasic degradation of the GABA_A receptor ³⁵S-1 subunit with a t1/2 = 7.7 h, a process that was unaffected by the addition of GABA to neurons during the chase period. These nascent ³⁵S-labeled polypeptides are presumably diluted into the neuronal pool of unlabeled unassembled 1 subunits, which was found to exceed by a 4:1 molar ratio the amount assembled into [³H]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-independent degradation of unassembled receptor subunits. This differs from another pathway for the agonist-dependent degradation of mature GABA_A receptors derived from the neuronal surface (Calkin, P. A., and Barnes, E. M., Jr. (1994) J. Biol. Chem. 269, 1548-1553).

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