Abstract
We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.
Highlights
From the $.Division of Biomedical Sciences, University of California, Riverside, California 92521, the §Department of Pharmacology, University ofCalifornia, San Diego, La Jolla, California 92093, and the TDepartment ofBiochemical Pharmacology, State University of New York atBuffalo, Buffalo, New York 14260
We have examined the interaction of the nicotinic The nicotinic acetylcholine receptor (AcChR)' represents a acetylcholine receptor with decidium diiodide, a bis- prototypic transmembraneligand-regulated ionchannel
A ligand which emits at wavelengths greater than 600 nm would be useful for assessing intersite distances throughexcitation energy transfer.Tothisend, we have characterized the interaction of decidium diiodide with the AcChR
Summary
Decidium Inhibition of Initial Rate of lZ51-Labeledcy-Toxin Bindingand Agonist-stimulated "Nu+ Permeability-Decidium inhibition of theinitialrate of lZ5I-labeleda-toxin bindingtoboth Torpedo AcChR-enriched membrane fragments and intact BC3H-1cells was examined after instantaneous and prolonged (30 min) exposure to decidium. The decidium protection constantsdecreased 2-4-fold when decidium was added 30 min prior to insteaodf simultaneously with radiolabeled cy-toxin addition (Fig. 1, TableI).This observation suggests that decidium possessesa limited capacitytoconvert AcChR to a state which displaysincreased affinity for decidium.Such a conversion in AcChR state might occur by decidium acting at the agonist site as a metaphilic antagonist (Rang and Ritte1r,970a, 1970b) or by acting at an allosteric site as a noncompetitive, heterotropic inhibitor. In both cases, a slow conversion to a state possessing a higher affinity for decidium is predicted. Values are eiven fS.D. and thenumber of determinations are in Darentheses
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