Abstract
Analogs of d-tubocurarine were used to determine the individual effects of methylation, stereoisomerization, and halogenation of d-tubocurarine on the affinity for each of the two acetylcholine (ACh) binding sites of the Torpedo nicotinic acetylcholine receptor (AChR) and for the noncompetitive antagonist site. Eight analogs were synthesized, including three new compounds: 7'-O-methyl-chondocurarine, 12'-O-methyl-chondocurarine, and 13'-bromo-d-tubocurarine. The two ACh sites differ in their affinities for d-tubocurarine by 400-fold, as shown by inhibition of [3H]ACh binding, whereas the affinity ratio for metocurine, the trimethylated derivative of d-tubocurarine, is reduced to 30 due to a decreased affinity for the high affinity site. Binding analysis of five d-tubocurarine analogs demonstrates that methylation of the phenols alone is responsible for the observed changes in affinity. Substitution with bromine or iodine at the 13'-position affected affinity at both sites with a net increase in site selectivity. Stereoisomers of d-tubocurare had decreased affinity for only the high affinity ACh site. Thus, the ring systems, including the 12'- and 13'-positions and the 1-position stereocenter, appear to be important in discriminating between the two ACh binding sites. Desensitization of the AChR was measured by increased affinity for [3H]phencyclidine. Binding to only the single, high affinity acetylcholine binding site, comprised by the alpha gamma-subunits, was required for partial desensitization of the AChR by d-tubocurarine and its analogs. Stronger desensitization, to the same extent observed in the presence of the agonist carbamylcholine, occurred upon binding by iodonated or brominated d-tubocurarine. Interaction of the analogs at the noncompetitive antagonist site of the AChR was also measured by [3H]phencyclidine binding. The bis-tertiary ammonium analogs of either the d- or l-stereoisomers bound to the noncompetitive antagonist binding site of the AChR with 100-fold higher affinity than the corresponding quaternary ammonium analogs.
Highlights
The nicotinic acetylcholine receptor (AChR)1 from Torpedo californica electric organ is a ligand gated cation channel com
Because the difference in structure lay in the methylation of the tertiary ammonium to a quaternary ammonium and methylation of the two phenols, we examined whether the difference could be ascribed to a particular site of methylation or resulted from smaller, additive effects on binding
The work presented in this article correlates the structure of d-tubocurarine with its site selectivity for the nicotinic acetylcholine binding sites
Summary
The nicotinic acetylcholine receptor (AChR) from Torpedo californica electric organ is a ligand gated cation channel com-. Several residues of the AChR that interact with d-tubocurarine have been identified by affinity labeling or by site directed mutagenesis. Further complications of d-tubocurarine structurefunction analysis was the correction of the structure by Everett et al (1970) from a bis-quaternary ammonium to a monoquaternary, mono-tertiary ammonium as well as the appreciation by Naghaway and Soine (1978a, 1978b) that some previous synthetic procedures did not yield the expected derivatives (e.g. Marshall et al (1967)). Interpretations of such experiments often emphasized the importance of bis-onium structure for antagonism (Sobell et al, 1972).
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