Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site.
The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.
Dennis
M
M
Laboratoire Associé au CNRS (UA 041149), Institut Pasteur, Paris, France.
Giraudat
J
J
Kotzyba-Hibert
F
F
Goeldner
M
M
Hirth
C
C
Chang
J Y
JY
Lazure
C
C
Chrétien
M
M
Changeux
J P
JP
D000109:Acetylcholine / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003488:Cyanogen Bromide; D003979:Diazonium Compounds / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo
D000345:Affinity Labels; D000596:Amino Acids; D003979:Diazonium Compounds; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; C028214:4-dimethylaminobenzenediazonium fluoroborate; D000109:Acetylcholine; D003488:Cyanogen Bromide