REDUCED FUNCTION OF GAMMA-AMINOBUTYRIC-ACID(A) 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 GABA(A) receptor function which could arise as a result of this mutation, the influx of Cl-36(-) 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 Cl-36(-) uptake induced by 50 mu M GABA was observed during five seconds of incubation at 28 degrees C. Compared to +/+, the GABA-dependent Cl-36(-) uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The levels of gated Cl-36(-) 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. GABA(A) receptor-specific agonists (30 mu M), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced Cl-36(-) 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 mu M H-89, a membrane-permeant inhibitor of the cAMP-dependent protein kinase (protein kinase A,PKA), was without effect on GABA-gated Cl-36(-) 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 GABA(A) 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.