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

Abstract

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 (1–500μM) 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=27μM) 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 (100μM). Detection of ATPase activity in a purified preparation, showed a native protein of 300kDa, in agreement with western blot analysis with antibodies against GABAAR β3 subunits. SDS-PAGE showed that one subunit with a molecular weight of 56kDa 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.