The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.
1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes.
Platt
K P
KP
Neuroscience Division, School of Biological Sciences, University of Manchester.
Zwartjes
R E
RE
Bristow
D R
DR
D000591:Amino Acid Isomerases / Q000096:biosynthesis; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D011963:Receptors, GABA-A / Q000096:biosynthesis*; D013268:Stimulation, Chemical; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase