AC4-ASA, a novel heterobifunctional probe, binds an allosteric site on the muscle nicotinic acetylcholine receptor.

PAVÁN CARLOS HUMBERTO; BISCOGLIO DE JIMÉNEZ BONINO MIRTHA JOSEFA; DEL CANTO SEGIO GABRIEL

Potrero de los Funes

Congreso; XLVII Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

Socieada Argentina de Investigación en Bioquímica y Biología Molecular

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 alfa/delta binding site.The ligand showed the capability of modyfing the affininty of (-)-[3H]-nicotine by 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 spectrometryAnalysis of the spectrum from the alpha subunit allowed us to find a 1600.8 Da molecular mass - absent in the non-modified receptor subunit - and corresponding to the mass of the tryptic peptide WNPADYGGIKK plus that of the ligand. Moreover, such peptide is not involved in the orthosteric receptor binding sites. New photolabelling experiments are in progress to define the location of other ligand binding determinants at the receptor.