REDUCED FUNCTION OF GAMMA-AMINOBUTYRIC-ACID(A) RECEPTORS IN TOTTERINGMOUSE-BRAIN - ROLE OF CAMP-DEPENDENT PROTEIN-KINASE

The single-locus mutant mouse tottering (tg) displays spontaneous seiz ures that resemble those in human petit-mal epilepsy. In order to exam ine alterations in GABA(A) receptor function which could arise as a re sult of this mutation, the influx of Cl-36(-) was determined using mic rosacs (membrane vesicles) isolated from the brain of tg/tg and coisog enic 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. Compa red to +/+, the GABA-dependent Cl-36(-) uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The l evels 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 ag onist-dependent influx was always significantly lower in tg/tg compare d to +/+. Treatment of the microsacs with 10 mu M H-89, a membrane-per meant inhibitor of the cAMP-dependent protein kinase (protein kinase A ,PKA), was without effect on GABA-gated Cl-36(-) uptake in +/+, but in creased the gated uptake in tg/tg microsacs by 44 +/- 16%. PKA. was as sayed using [gamma-(32)]ATP and kemptide as the substrate. Triton X-10 0 (0.1%) increased both the basal and 8-Br-cAMP dependent PKA. activit y in microsacs by 3-4 four fold, showing that most of the enzyme was i ntravesicular. In the presence of Triton, the basal activity of PKA in the tg/tg preparations was twice that of +/+, while the strain differ ence was no longer apparent in assays containing 8-Br-cAMP. The data s uggest that an abnormal elevation of protein kinase A activity in tott ering mouse brain contributes to an impairment of GABA(A) receptor fun ction. It is suggested that the resulting loss of inhibition could pla y a role in induction of the seizures which characterize the mutant ph enotype.