Localization of regions of the Torpedo californica nicotinic acetylcholine receptor labeled with an aryl azide derivative of phosphatidylserine.
A photoactivatable analog of phosphatidylserine, 125I-labeled 4-azidosalicylic acid-phosphatidylserine (125I-ASA-PS) (Blanton, M. and Wang, H.H. (1990) Biochemistry 29, 1186-1194) was used to label the nicotinic acetylcholine receptor. The photoactivatable group of 125I-ASA-PS is attached directly to the phospholipid head group making it an excellent probe of regions of the AchR structure in contact with the negatively-charged head group of phosphatidylserine. The 'binding domains' were localized by chemically cleaving the labeled receptor with cyanogen bromide (CNBr), separating the generated peptides by reverse-phase HPLC, and N-terminal sequence analysis of radiolabeled material. CNBr fragments containing flanking regions of the transmembrane spanning region M4 as well as within M3 were identified within HPLC separated radiolabeled material. The results suggest a topological arrangement of the transmembrane helices in which the hydrophobic faces of M3 and M4 form the boundary of the receptor complex in contact with the lipid bilayer.
Blanton
M P
MP
Department of Biology, University of California, Santa Cruz 95064.
Wang
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010718:Phosphatidylserines; D011978:Receptors, Nicotinic / Q000032:analysis*; D014101:Torpedo / Q000378:metabolism*
D001386:Azides; D010446:Peptide Fragments; D010718:Phosphatidylserines; D011978:Receptors, Nicotinic; C070385:4-azidosalicylic acid-phosphatidylserine; D003488:Cyanogen Bromide