Effect of phenol on the GABAAR-coupled Cl−/HCO3−-ATPase from fish brain: An in vitro approach on the enzyme function

Phenol (C6H5OH) has a toxic effect on the central nervous system of animals and humans. The Cl−/HCO3−-ATPase from the plasma membranes of animal brains is the primary active P-type Cl−-transporting system that is coupled to GABAA receptor (GABAAR). In this paper, we used an in vitro approach to assess the effects of phenol (1-500 μM) on the functional parameters of the Cl−/HCO3−-ATPase isolated from the fish brain. The enzyme is insensitive to phenol in the presence of Cl− or HCO3− in the incubation medium. By contrast, in the presence of Cl−/HCO3−, phenol inhibits (I50 = 27 μM) both the enzyme activity and its participation in ATP-dependent Cl− transport through the membranes of artificial liposomes. Enriched plasma membranes and purified enzyme preparations were separated using hrCNE-PAGE. The ATPase activity in native gels was detected in the presence of phenol (100 μM). Detection of ATPase activity in a purified preparation, showed a native protein of 300 kDa, in agreement with western blot analysis with antibodies against GABAAR β3 subunits. SDS-PAGE showed that one subunit with a molecular weight of 56 kDa was directly phosphorylated by γ-32P-ATP and dephosphorylated in the presence of phenol. The in vitro approach described in this work allowed the first demonstration that GABAAR-coupled Cl−/HCO3−-ATPase can be a protein marker for assessment of the toxicity of phenolics on the central nervous system.