Cyclic AMP-dependent protein kinase decreases gamma-aminobutyric acidA receptor-mediated 36Cl- uptake by brain microsacs.
The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP-dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-alpha 1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.
Leidenheimer
N J
NJ
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Machu
T K
TK
Endo
S
S
Olsen
R W
RW
Harris
R A
RA
Browning
M D
MD
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008813:Mice, Inbred ICR; D009118:Muscimol / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000187:drug effects / Q000502:physiology*
D002712:Chlorides; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D009118:Muscimol; D011494:Protein Kinases