STUDIES OF THE INTERACTION OF AGONIST, ANTAGONIST AND ION CONDUCTANCE CHANNEL BINDING SITES IN THE FUNCTIONAL ACETYLCHOLINE RECEPTOR COMPLEX FROM FROG NEUROMUSCULAR JUNCTION.
Item
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Title
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STUDIES OF THE INTERACTION OF AGONIST, ANTAGONIST AND ION CONDUCTANCE CHANNEL BINDING SITES IN THE FUNCTIONAL ACETYLCHOLINE RECEPTOR COMPLEX FROM FROG NEUROMUSCULAR JUNCTION.
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Identifier
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AAI8302514
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identifier
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8302514
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Creator
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GROSS, STEVEN S.
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Contributor
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Tom Mittag
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Date
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1982
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Language
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English
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Publisher
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City University of New York.
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Subject
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Health Sciences, Pharmacology
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Abstract
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This study attempts to determine allosteric changes in the acetylcholine receptor ion channel complex (nAChR) of frog neuromuscular junction that may be induced by the binding of small ligands prototypic of each of three functional classes, i.e., agonists (carbachol), antagonists (d-tubocurarine), and ion channel blockers (phencyclidine). The study characterizes and makes use of the compound N,N,dimethyl phenylaziridinium (DPA) which is shown to behave as an irreversible agonist in dithiothreitol (DTT) reduced frog muscle. Changes in the binding characteristics of {lcub}('125)I{rcub} (alpha)-bungarotoxin ((alpha)-BTX) were employed as a specific probe to detect for the conformational changes of the receptor. The major findings of this study are summarized below.;(1) The actions of DPA following DTT reduction of rectus abdominis muscle are consistent with that of an irreversible (alkylating) nicotinic agonist. (A) The DPA response is prevented by pretreatment with BTX (2 x 10('-7) M) or with dTC (10('-4) M). (B) DPA induced contracture of reduced muscle is protected by carbachol (10('-3)M) and dTC (10('-4)M). (C) DPA induced contracture is reverted by the channel blocking ligands PCP (25(mu)M) and adiphenine (25(mu)M). (D) In common with carbachol the initial tension response to DPA is diminished by muscle reduction with DTT.;(2) A "ternary" complex is formed with nAChR, (alpha)-BTX and DPA. (A) DPA alkylation results in no reduction in the number of binding sites for (alpha)-BTX, however, it greatly reduces the affinity of (alpha)-BTX for the receptor. (B) (alpha)-BTX (2 x 10('-7)M) reverts the contracture in DPA alkylated muscle indicating simultaneous occupancy.;(3) The agonist and antagonist binding sites are distinct in intact muscle nAChR. The ability of carbachol but not dTC to compete for {lcub}('125)I{rcub} (alpha)-BTX binding to whole sartorius muscle is greatly diminished by DPA alkylation.;(4) The binding of a channel binding ligand may influence the conformation of the ACh recognition site. PCP (25(mu)M) protects against DPA alkylation, however, at this concentration PCP has no effect on (alpha)-BTX binding.;(5) The binding of agonist influences the conformation of the binding site for PCP on the ion conductance channel. PCP binds with higher affinity to the agonist activated than to the resting (non-conducting) conformation of the channel.;(6) Extraction of the nAChR from its membrane environment results in drastic changes in ligand binding properties and/or the ability of a ligand to induce conformational changes.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
