Dual character of GABA action on Cl--transport by the reconstituted Cl-/HCO3--ATPase from rat brain

摘要

The Cl-/HCO3--ATPase from the plasma membranes of animal brains is an ATP-consuming Cl(-)transporting ATPase P-type that is structurally coupled to GABA(A) 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 GABA(A)R beta 3 subunit, showed one band with a molecular mass of 300 kDa. The addition of GABA (100 mu M) quickly resulted (1-3s) 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 mu M) did not change the GABA-induced Cl- inflow into the liposomes, but completely inhibited the ATP-dependent Cl- input. Under conditions where a - 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-/HCO3--ATPase.