Dual character of GABA action on Cl–-transport by the reconstituted Cl–/-ATPase from rat brain

Abstract

The Cl–/-ATPase from the plasma membranes of animal brains is an ATP-consuming Cl–-transporting ATPase P-type that is structurally coupled to GABAA receptors. The aim of work was to study the GABA effect on Cl– transport across liposomal membranes by the reconstituted ATPase under various conditions (i.e. in the absence and in the presence of ATP in the incubation medium). We reconstituted the affinity-purified enzyme in the liposomes with a fluorescent dye for Cl–. The Cl–-transport in proteoliposomes was evaluated from variations in fluorescence. Native-PAGE of the purified enzyme preparation, as well as western blot analysis with an antibody against the GABAAR β3 subunit, showed one band with a molecular mass of 300 kDa. The addition of GABA (100 μM) quickly resulted (1–3 s) in an increase in the Cl– influx into proteoliposomes. The application of ATP (3 mM) resulted in a gradual increase in the Cl– inflow into the proteoliposomes at 0.5–4 min. Vanadate (10 μM) did not change the GABA-induced Cl– inflow into the liposomes, but completely inhibited the ATP-dependent Cl– input. Under conditions where Cl– was previously loaded into proteoliposomes by ATP-dependent Cl– input, the addition of GABA to the incubation medium caused a short-lasting Cl– efflux from the proteoliposomes. However, an additional long-time incubation of these proteoliposomes resulted in the restoration of Cl– accumulation. Pentobarbital, propofol, or diazepam elevate and picrotoxin or furosemide eliminate the effect of GABA on the Cl–-transport processes. These findings indicate a dual nature of GABA action on the Cl–/-ATPase.