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 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_AR 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.

Keywords:

• GABA
• Cl^–/-ATPase
• Cl^–-transport
• vanadate
• proteoliposomes
• furosemide

Ethical approval

This study follows the principles of the Declaration of Helsinki.

Disclosure statement

The author reports no conflict of interest.