Biochemical properties of the sensitivity to GABA_Aergic ligands, Cl⁻/HCO₃ ⁻-ATPase isolated from fish (Cyprinus carpio) olfactory mucosa and brain

Abstract

This paper presents a comparative study of the roles of Cl⁻ and HCO₃ ⁻ in the functioning of the GABA_AR-associated Cl⁻/HCO₃ ⁻-ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl⁻(15–30 mmol l⁻¹), whereas the enzyme from MBNs was not significantly affected by Cl⁻. By contrast, HCO₃ ⁻(15–30 mmol l⁻¹) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl⁻(15 mmol l⁻¹)/HCO₃ ⁻(27 mmol l⁻¹) and these activities were inhibited in the presence of picrotoxin (100 μmol l⁻¹), bumetanide (150 μmol l⁻¹), and DIDS (1000 μmol l⁻¹). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~ 56 kDa that binds [³H]muscimol and [³H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-³²P and Mg²⁺, as well as Cl⁻/HCO₃ ⁻ sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABA_AR β3 subunit. The use of a fluorescent dye for Cl⁻ demonstrated that HCO₃ ⁻(27 mmol l⁻¹) causes a twofold increase in Cl⁻ influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO₃ ⁻ had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl⁻ and HCO₃ ⁻ in the regulation of the Cl⁻/HCO₃ ⁻-ATPases functioning in neurons with different specializations.