An heterobifunctional probe binds an allosteric site on the nicotínic acetylcholine receptor.

PAVáN, CARLOS; DEL CANTO, SERGIO GABRIEL; BISCOGLIO DE JIMéNEZ BONINO, MIRTHA.

Huerta Grande, Cordoba

Congreso; XXVII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias; 2012

The probe was developed as a tool for the study of cholinergic receptor binding sites. Acetylcholine was derivatized at its alkyl end and, through a short spacer, with a photoactivatable aryl-azide group susceptible to radioiodination. The probe was able to interact specifically with the muscle nicotinic receptor  and has a considerable selectivity for its á/ä binding site. This ligand showed the capability of modyfing the affininty of (-)-[3H]-nicotine for the muscle-type nicotinic receptor. Competition experiments between AC4-ASA and (-)-[3H]-nicotine revealed that the ligand could perform its modulating activity through, at least, one new allosteric binding site, different from the typical orthosteric binding sites. With the aim of delineating this site, the Torpedo californica receptor was modified with the ligand and submitted to SDS-PAGE. All subunits were digested with trypsin and peptide mixtures analyzed by MALDI-TOF-TOF mass spectrometry. Spectra from the alpha and delta subunits allowed us to find several m/z signals, absent in the non-modified receptor subunits. After a detailed analysis of such signals we were able to postulate that they correspond to determinants of ligand binding sites. Moreover, most of them are not involved in the orthosteric sites thus suggesting that could be participating in the allosteric one.