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 GABA_A receptor (GABA_AR). 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 GABA_AR 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 GABA_AR-coupled Cl^-/HCO₃^--ATPase can be a protein marker for assessment of the toxicity of phenolics on the central nervous system.

Keywords: Cl(−)-transport; Cl(−)/HCO(3)(−)-ATPase; Fish brain; Phenol; Phosphorylation.