GABAA-Coupled Cl-/ HCO3--ATPase from Plasma Membrane of the Rat Brain: Role of HCO3- in the Enzyme Activation

Natalya Y.  Klishina; Lada V.  Kuznetsova; Margarita N.  Karpova; Sergey A.  Menzikov

doi:10.4236/aer.2015.31002

Sergey A. Menzikov^*, Margarita N. Karpova, Lada V. Kuznetsova, Natalya Y. Klishina

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.

Keywords: Rat Brain, Plasma Membrane, Mg2+-ATPase, Chloride, Bicarbonate, Mg2+-ATP, Picrotoxin, Bicuculline, Autoradiography, Molecular Weight, Subunits

Cite this paper: Menzikov, S. , Karpova, M. , Kuznetsova, L. and Klishina, N. (2015) GABA_A-Coupled Cl-/ HCO₃^--ATPase from Plasma Membrane of the Rat Brain: Role of HCO₃^- in the Enzyme Activation. Advances in Enzyme Research, 3, 9-18. doi: 10.4236/aer.2015.31002.

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