ABSTRACT
BACKGROUND AND PURPOSE: Many local anaesthetics are non-competitive inhibitors of nicotinic receptors (acetylcholine receptor, AChR). Proadifen induces a high-affinity state of the receptor, but its mechanism of action and that of an analogue, adiphenine, is unknown. EXPERIMENTAL APPROACH: We measured the effects of proadifen and adiphenine on single-channel and macroscopic currents of adult mouse muscle AChR (wild-type and mutant). We assessed the results in terms of mechanisms and sites of action. KEY RESULTS: Both proadifen and adiphenine decreased the frequency of ACh-induced single-channel currents. Proadifen did not change cluster properties, but adiphenine decreased cluster duration (36-fold at 100 micromolxL(-1)). Preincubation with proadifen decreased the amplitude (IC(50)= 19 micromolxL(-1)) without changing the decay rate of macroscopic currents. In contrast, adiphenine did not change amplitude but increased the decay rate (IC(50)= 15 micromolxL(-1)). Kinetic measurements demonstrate that proadifen acts on the resting state to induce a desensitized state whose kinetics of recovery resemble those of ACh-induced desensitization. Adiphenine accelerates desensitization from the open state, but previous application of the drug to resting receptors is required. Both drugs stabilize desensitized states, as evidenced by the decrease in the number of clusters elicited by high ACh concentrations. The inhibition by adiphenine is not affected by proadifen, and the mutation alphaE262K decreases the sensitivity of the AChR only for adiphenine, indicating that these drugs act at different sites. CONCLUSIONS AND IMPLICATIONS: Two analogous local anaesthetics bind to different sites and inhibit AChR activity via different mechanisms and conformational states. These results provide new information on drug modulation of AChR.
Subject(s)
Anesthetics, Local/pharmacology , Diphenylacetic Acids/pharmacology , Ion Channel Gating/drug effects , Nicotinic Antagonists/pharmacology , Proadifen/pharmacology , Receptors, Nicotinic/drug effects , Acetylcholine/metabolism , Anesthetics, Local/metabolism , Animals , Binding Sites , Cell Line , Diphenylacetic Acids/metabolism , Dose-Response Relationship, Drug , Humans , Kinetics , Membrane Potentials , Mice , Nicotinic Antagonists/metabolism , Proadifen/metabolism , Protein Conformation , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , TransfectionABSTRACT
Ion-channel blockers are molecules that obstruct the path used by ions to cross the membrane through a protein channel. Many of these are local anesthetics, toxins or drugs of abuse, and the knowledge of their mechanism of action at the atomic level is an important step towards the development of new compounds on a structural basis. A molecular model of the transmembrane region of the nicotinic acetylcholine receptor, an important brain and muscle fast signaling protein, was used as a target for docking several channel blockers by means of an automatic docking method. The combination of the independent docking method and molecular models (of the receptor and blockers) reproduced or explained quite accurately experimental data (photoaffinity labeling, site-directed mutagenesis, binding assays). This represents a strong support for the validity of the predictions made for those molecules for which no experimental data is available and also for the models and methods on which are based.