Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [H-3]DCTA: Identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine
T. Grutter et al., Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [H-3]DCTA: Identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine, BIOCHEM, 39(11), 2000, pp. 3034-3043
Torpedo marmorata acetylcholine binding sites were photolabeled using 360 n
m light, at equilibrium in the desensitized state, with the agonist [H-3]DC
TA utilizing the Ce-IV/glutathione procedure described previously (Grutter,
et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [H-3]DCTA
was concentration-dependent with a maximum of 7.5% specific labeling on th
e alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation co
nstants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and
3.6 +/- 2.8 mu M, respectively. The alpha-chains isolated from receptor-ric
h membranes photolabeled in the absence or in the presence of carbamylcholi
ne were cleaved with CNBr using an efficient "in gel" procedure. The result
ing peptide fragments were purified by HPLC and further submitted to trypsi
nolysis. The digest was analyzed by HPLC leading to a single radioactive pe
ak which, by microsequencing, revealed two sequences extending from alpha L
ys-179 and from alpha His-186, respectively. Radioactive signals could be u
nambiguously attributed to positions corresponding to residues alpha Tyr-19
0, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [
H-3]DCTA-labeled residues, which have been also labeled with other affinity
and photoaffinity probes including the agonist [H-3]nicotine, belong to lo
op C of the ACh binding site. The chemical structure of [H-3]DCTA, together
with its well-defined and powerful photochemical reactivity, provides conv
incing evidence that loop C-labeled residues are primarily involved in the
interaction with the ester moiety of acetylcholine.