Reduced function of gamma-aminobutyric acidA receptors in tottering mouse brain: role of cAMP-dependent protein kinase.
The single-locus mutant mouse tottering (tg) displays spontaneous seizures that resemble those in human petit-mal epilepsy. In order to examine alterations in GABAA receptor function which could arise as a result of this mutation, the influx of 36Cl- was determined using microsacs (membrane vesicles) isolated from the brain of tg/tg and coisogenic C57BL/6J (+/+) control mice. In microsacs from both tg/tg and +/+ strains, the maximum level of 36Cl- uptake induced by 50 microM GABA was observed during five seconds of incubation at 28 degrees C. Compared to +/+, the GABA-dependent 36Cl- uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The levels of gated 36Cl- uptake in tg/tg microsacs were 45 +/- 6.3%, 65 +/- 9.9%, and 33 +/- 6.1% of control (+/+) values for 3-, 5-, and 10-s incubations, respectively. GABAA receptor-specific agonists (30 microM), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced 36Cl- influx in the order muscimol > GABA > isoguvacine > THIP. This order was similar for both strains, but the agonist-dependent influx was always significantly lower in tg/tg compared to +/+. Treatment of the microsacs with 10 microM H-89, a membrane-permeant inhibitor of the cAMP-dependent protein kinase (protein kinase A, PKA), was without effect on GABA-gated 36Cl- uptake in +/+, but increased the gated uptake in tg/tg microsacs by 44 +/- 16%. PKA was assayed using [gamma-32]ATP and kemptide as the substrate. Triton X-100 (0.1%) increased both the basal and 8-Br-cAMP dependent PKA activity in microsacs by 3-4 four fold, showing that most of the enzyme was intravesicular. In the presence of Triton, the basal activity of PKA in the tg/tg preparations was twice that of +/+, while the strain difference was no longer apparent in assays containing 8-Br-cAMP. The data suggest that an abnormal elevation of protein kinase A activity in tottering mouse brain contributes to an impairment of GABAA receptor function. It is suggested that the resulting loss of inhibition could play a role in induction of the seizures which characterize the mutant phenotype.
Tehrani
M H
MH
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA.
Barnes
E M
EM
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D002713:Chlorine; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018755:GABA Agonists / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D007555:Isoxazoles / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008818:Mice, Neurologic Mutants; D047428:Protein Kinase Inhibitors; D011868:Radioisotopes; D011963:Receptors, GABA-A / Q000378:metabolism*; D013449:Sulfonamides; D005680:gamma-Aminobutyric Acid / Q000502:physiology
D018118:Chloride Channels; D004791:Enzyme Inhibitors; D018755:GABA Agonists; D007546:Isoquinolines; D007555:Isoxazoles; D047428:Protein Kinase Inhibitors; D011868:Radioisotopes; D011963:Receptors, GABA-A; D013449:Sulfonamides; D002713:Chlorine; D005680:gamma-Aminobutyric Acid; D017868:Cyclic AMP-Dependent Protein Kinases; C015542:gaboxadol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide