Background: The Cl-/HCO3--ATPase from animal brain is a new primary active Cl-- transport system that is coupled to the GABAA receptor (GABAAR). The structural coupling of the enzyme with GABAAR, however, has not been confirmed by the presence of GABAAR subunits within the purified protein complex.
Objective/Design: Identification of the enzyme using methodological approaches that would allow the demonstration of GABAAR peptide subunits within a purified enzyme preparation.
Main outcome measures: A plasma membrane-enriched preparation (PMP) and a purified enzyme preparation (PEP) from rat brain were loaded onto high resolution clear native-PAGE (hrCNE-PAGE) gels and analyzed on a denaturing gel system. The Cl-/HCO3--ATPase activity was detected in gel strips in the presence of Mg2+-ATP and 5 mM Cl-/25 mM HCO3-, and was inhibited by ethanol (500 mM). The enzyme in the gel strips was detected by western blotting with antibodies against GABAAR 1-3 subunit.
Results: The PEP gave one band with a molecular weight of 300 kDa. HrCNEPAGE/ SDS and western blotting results confirmed the presence of the GABAAR β 1-3 subunits in the PMP. The PEP showed two subunits with molecular masses of 52 kDa and 57 kDa that were immuno-reactive only with GABAAR 3 antibodies. These peptide sub-units were directly phosphorylated by γ-32 P-ATP and dephosphorylated in the presence of Cl-/HCO3-, hydroxylamine and ethanol.
Conclusion: This proteomic approach and western blot analysis allowed the detection of the Cl-/HCO3--ÃÂTPase along with GABAAR 3 subunits isolated from rat brain.
Sergey A. Menzikov