Irreversible site-directed labeling of the 4-aminobutyrate binding site bytritiated mefa-sulfonate benzene diazonium - Contribution of a nucleophilic amino acid residue of the alpha 1 subunit

Tritiated meta-sulfonate benzene diazonium ([H-3]MSBD), a molecule structur ally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA b inding site on the GABA(A) receptor. Irreversible labeling reactions using [H-3]MSBD were performed on purified GABA(A) receptors isolated from cow br ain membranes and labeled receptors were analyzed by SDS/PAGE. [H-3]MSBD wa s found to be specifically incorporated into proteins in the 45-60 kDa mole cular mass range which were identified as al subunits and beta 2/beta 3 sub units by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [H -3]MSBD occurs at the boundary of these subunits. These labeling results co nfirm the involvement of nucleophilic residues from the beta subunit but re veal also the contribution of yet unidentified nucleophilic residues on the cu subunit for the GABA binding site.