TITLE:
GABAA-Coupled Cl-/ HCO3--ATPase from Plasma Membrane of the Rat Brain: Role of HCO3- in the Enzyme Activation
AUTHORS:
Sergey A. Menzikov, Margarita N. Karpova, Lada V. Kuznetsova, Natalya Y. Klishina
KEYWORDS:
Rat Brain, Plasma Membrane, Mg2+-ATPase, Chloride, Bicarbonate, Mg2+-ATP, Picrotoxin, Bicuculline, Autoradiography, Molecular Weight, Subunits
JOURNAL NAME:
Advances in Enzyme Research,
Vol.3 No.1,
March
18,
2015
ABSTRACT: This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl-/25mM HCO3- and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl--transport through the plasma membrane by utilizing neuronal Cl-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.