Reduced function of γ-aminobutyric acidA receptors in tottering mouse brain: Role of cAMP-dependent protein kinase

Jalilian Tehrani, Mohammad H.; Barnes, Eugene M.

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Volume 22, Issue 1 , Pages 13-21, September 1995

Reduced function of γ-aminobutyric acid_A receptors in tottering mouse brain: Role of cAMP-dependent protein kinase

Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

Received 28 November 1994; accepted 19 April 1995.

Abstract

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 ³⁶C1⁻ 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 μM GABA was observed during five seconds of incubation at 28°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 ³⁶C1⁻ 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 μM), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced ³⁶C1⁻ 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 μ M 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 [γ-³²]ATP and kemptide as the substrate. Triton ×-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.