A novel heterobifunctional probe binds an allosteric site on the nicotinic acetylcholine receptor

PAVáN C.; BISCOGLIO DE JIMéNEZ BONINO M.J. ; DEL CANTO S.

San Luis

Congreso; 47th Annual Meeting SAIB; 2011

SAIB

The probe (AC4-ASA) 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. 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 spectrometry. Analysis 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.