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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

Grutter, T.; Ehret-Sabatier, L.; Kotzyba-Hibert, F.; Goeldner, M.

Biochemistry 39(11): 3034-3043

2000

ISSN/ISBN: 0006-2960
PMID: 10715124
Accession: 009938493
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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 a-subunit and 1.2% on the c-subunit. The apparent dissociation constants for labeling of the a- and c-subunits were 2.2 [plus or minus] 1.1 and 3.6 [plus or minus] 2.8 mM, respectively. The a-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 aLys-179 and from aHis-186, respectively. Radioactive signals could be unambiguously attributed to positions corresponding to residues aTyr-190, aCys-192, aCys-193, and aTyr-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. Copyright 2000, American Chemical Society.

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Related References

Grutter, T.; Ehret-Sabatier, L.; Kotzyba-Hibert, F.; Goeldner, M. 2000: Photoaffinity Labeling of Torpedo Nicotinic Receptor with the Agonist [ 3 H]Dcta: Identification of Amino Acid Residues Which Contribute to the Binding of the Ester Moiety of Acetylcholine Biochemistry 39(11): 3034-3043
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