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Flapping loops: roles for hinges in a ligand-binding domain of the nicotinic receptor.

ABSTRACT: One of the goals of molecular pharmacology is to understand the machinery that converts information about the presence of a chemical (binding) to a functional consequence. Agonists are drugs that bind to their molecular targets and cause conformational changes underlying activation of the target. Inevitably, therefore, it can be difficult to disentangle the affinity of the agonist for the target from its efficacy in producing the ensuing conformational change. Efficacy depends on two factors: the intrinsic equilibrium between active and inactive states in the absence of agonist, and the energy contributed by the agonist as a result of the relative affinities of agonist for the active and inactive states. These difficulties are particularly frustrating when the goal is to determine the role(s) that particular residues in a target protein have in shaping the overall efficacy of a drug: how is it possible to unambiguously distinguish a role in determining intrinsic efficacy from one in determining relative affinity? This perspective highlights a research article in this issue (p. 351) that demonstrates a quantitative approach to the resolution of this problem in the case of activation of the muscle nicotinic receptor. This elegant (if demanding) approach involves determining, separately, the consequences of specific mutations on gating in the unliganded and liganded states.

SUBMITTER: Steinbach JH

PROVIDER: S-EPMC3061362 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Flapping loops: roles for hinges in a ligand-binding domain of the nicotinic receptor.

Steinbach Joe Henry JH

Molecular pharmacology 20101220 3

One of the goals of molecular pharmacology is to understand the machinery that converts information about the presence of a chemical (binding) to a functional consequence. Agonists are drugs that bind to their molecular targets and cause conformational changes underlying activation of the target. Inevitably, therefore, it can be difficult to disentangle the affinity of the agonist for the target from its efficacy in producing the ensuing conformational change. Efficacy depends on two factors: th ...[more]

PMID: 21173041

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Project description:A large number of structurally diverse ligands have been produced to selectively target ?7 nicotinic acetylcholine receptors (nAChRs). We applied the method of scanning cysteine accessibility mutations (SCAM) to the ligand-binding domain of the ?7 nAChR to identify subdomains of particular importance to the binding and subsequent activation by select agonists. We evaluated the activity of four structurally distinct ?7 agonists on wild-type human ?7 and 44 targeted mutants expressed in Xenopus oocytes. Responses were measured prior and subsequent to the application of the sulfhydryl reagent methanethiosulfonate ethylammonium (MTSEA). One mutant (C116S) served as a Cys-null control, and the additional mutants were made in the C116S background. In many cases, the insertion of free cysteines into the agonist-binding site had a negative effect on function, with 12 of 44 mutants showing no detectable responses to ACh, and with only 19 of the 44 mutants showing sufficiently large responses to permit further study. Several of the cysteine mutations, including W55C, showed selectively reduced responses to the largest agonist tested, 2-methoxy,4-hydroxy-benzylidene anabaseine. Interestingly, although homology models suggest that most of the introduced cysteine mutations should have had good solvent accessibility, application of MTSEA had no effect or produced only modest changes in the agonist response profile of most mutants. Consistent with previous studies implicating W55 to play important roles in agonist activation, MTSEA treatment further decreased the functional responses of W55C to all the test agonists. While the cysteine mutation at L119 itself had relatively little effect on receptor function, treatment of L119C receptors with MTSEA or alternative cationic sulfhydryl reagents profoundly decreased activation by all agonists tested, suggesting a general block of gating. The homologous mutation in heteromeric nAChRs produced similar results, provided that the mutation was placed in the beta subunit complementary surface of the ligand-binding domain. Structural models locate the L119 residue directly across the subunit interface from the C-loop of the primary face of the binding domain. Our data suggest that a covalent modification of L119C by MTSEA or other cationic reagents might block the binding of even small agonists such as TMA through electrostatic interactions. Reaction of L119C with small non-polar reagents increases activation by small agonists but can block the access of large ligands such as benzylidene anabaseines to the ligand-binding domain.

Dataset Information

Flapping loops: roles for hinges in a ligand-binding domain of the nicotinic receptor.

Publications

Flapping loops: roles for hinges in a ligand-binding domain of the nicotinic receptor.

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