The noncompetitive blocker [(3)H]chlorpromazine labels segment M2 but not segment M1 of the nicotinic acetylcholine receptor alpha-subunit.
The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker ]3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity side for noncompetitive blockers. The alpha-subunit was purified and digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the alpha-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta-chain [1] and Ser-254 and Leu-257 in the beta-chain [2]. Extended sequence analysis of the hydrophobic segment M1 further showed that no labeling-occurred in this region.
Giraudat
J
J
URA CNRS 0210, Départment des Biotechnologies, Institut Pasteur, Paris, France.
Gali
J
J
Revah
F
F
Changeux
J
J
Haumont
P
P
Lederer
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002746:Chlorpromazine / Q000378:metabolism*; D003488:Cyanogen Bromide; D066298:In Vitro Techniques; D007473:Ion Channels / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011978:Receptors, Nicotinic / Q000378:metabolism* / Q000648:ultrastructure; D014101:Torpedo
D007473:Ion Channels; D011978:Receptors, Nicotinic; D003488:Cyanogen Bromide; D002746:Chlorpromazine