Mapping of exposed surfaces of the nicotinic acetylcholine receptor by identification of iodinated tyrosine residues.

摘要: Here we report on the use of iodination membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions molecule. Membrane-bound nAChR was 125I-iodinated using oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. resulting peptides reverse-phase HPLC radioactive identified mass spectrometry protein sequencing. For delta-subunit, five containing tyrosine residues deltaTyr17, deltaTyr74, deltaTyr365, deltaTyr372, deltaTyr428. surface exposition these amino acids is agreement four-transmembrane-segment model (4TM model) nAChR, but assignment intra- or extracellular doubtful. According this model, N-terminal portion including deltaTyr17 deltaTyr74 deltaTyr372 as a site phosphorylation located cytoplasmic side. But since latter residue among first be an immobilized agent, its true position respect membrane bilayer not clear.