Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [3H]DCTA: identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine.
Torpedo marmorata acetylcholine binding sites were photolabeled using 360 nm light, at equilibrium in the desensitized state, with the agonist [3H]DCTA utilizing the CeIV/glutathione procedure described previously (Grutter, et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [3H]DCTA was concentration-dependent with a maximum of 7.5% specific labeling on the alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation constants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and 3.6 +/- 2.8 microM, respectively. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or in the presence of carbamylcholine were cleaved with CNBr using an efficient "in gel" procedure. The resulting peptide fragments were purified by HPLC and further submitted to trypsinolysis. The digest was analyzed by HPLC leading to a single radioactive peak which, by microsequencing, revealed two sequences extending from alpha Lys-179 and from alpha His-186, respectively. Radioactive signals could be unambiguously attributed to positions corresponding to residues alpha Tyr-190, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [3H]DCTA-labeled residues, which have been also labeled with other affinity and photoaffinity probes including the agonist [3H]nicotine, belong to loop C of the ACh binding site. The chemical structure of [3H]DCTA, together with its well-defined and powerful photochemical reactivity, provides convincing evidence that loop C-labeled residues are primarily involved in the interaction with the ester moiety of acetylcholine.
Grutter
T
T
Laboratoire de Chimie Bio-Organique, UMR 7514 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, France. grutter@bioorga.u-strasbg.fr
Ehret-Sabatier
L
L
Kotzyba-Hibert
F
F
Goeldner
M
M
D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003488:Cyanogen Bromide; D003979:Diazonium Compounds / Q000378:metabolism*; D004952:Esters; D006868:Hydrolysis; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D003979:Diazonium Compounds; D004952:Esters; D018722:Nicotinic Agonists; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; C119727:diazocyclohexadienoylpropyltrimethylammonium; D014316:Tritium; D014357:Trypsin; D000109:Acetylcholine; D003488:Cyanogen Bromide