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Title:Single-Channel Studies of Agonist Interactions With the Neurotransmitter-Binding and Pore Domains of the Muscle Nicotinic Acetylcholine Receptor
Author(s):Purohit, Yamini
Doctoral Committee Chair(s):Claudio Grosman
Department / Program:Molecular and Integrative Physiology
Discipline:Molecular and Integrative Physiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Health Sciences, Pharmacology
Abstract:The nicotinic acetylcholine receptor, a ligand-gated ion channel, is activated by a variety of naturally occurring and synthetic ligands. The overall 'response' elicited by any ligand of the nicotinic receptor is not only a consequence of its binding to the extracellular neurotransmitter-binding domain, but also reflects its interaction with the transmembrane pore domain (a phenomenon, typically, referred to as 'channel block'). The focus of studies, documented in this dissertation, was to apply single-channel methodologies to investigate quantitative aspects of ligand interactions with the extracellular and transmembrane domains of the muscle nicotinic receptor. First, details of a novel single-channel based methodology, which we termed the 'activation competition assay', are presented. This assay was developed to obtain, previously unavailable, conformation-specific estimates of the nicotinic receptor's neurotransmitter-binding-site affinity (in terms of a dissociation equilibrium constant, KD) for its low-efficacy agonists. Specifically, the assay was applied to obtain affinity estimates of the closed conformation of the nicotinic receptor for its low-efficacy agonist, choline. The dissociation equilibrium constant for the binding of the closed wild-type nicotinic receptor to choline (KD, Choline) was estimated to be (4.1 +/- 0.5) mM. Further, we illustrate that low-efficacy agonists of the acetylcholine receptor, and estimates of the wild-type's affinities for these agonists, can serve as essential tools for assessing the effect of gain-of-function mutations on the binding-site affinity of the receptor. Next, the experimental and analytical procedures, and the results from the characterization of fast-channel block by four nicotinic agonists (trimethylammonium (TriMA), tetramethylammonium (TMA), ethyltrimethylammonium (ETMA) and choline), are presented. Our key findings from this study were as follows: although the four quaternary ammonium compounds bind the nicotinic-receptor pore with comparable affinities, they produce distinct effects on the channel gating kinetics when bound to the pore. Also, our results indicate that upon binding to the pore domain, agonists that permeated the nicotinic receptor pore at immeasurably slow rates (TMA, ETMA and choline), slow down shutting of diliganded receptors and, thereby, prolong the mean duration of diliganded openings. Analysis of the agonist concentration-dependence of the total open-time within clusters revealed that the slower shutting of diliganded receptors, blocked by TMA, ETMA or choline, could be ascribed to their slower closing rate constants (and not to a change in the entry-into-desensitization rate constants). This finding led us to conclude that the activation and desensitization gates of mouse muscle nicotinic receptors act as distinct molecular entities. Finally, since channel-block by TMA, ETMA and choline modifies kinetics of receptors diliganded with these agonists, we propose a method to correct for this artifact. This correction is a prerequisite to obtaining accurate estimates of channel activation parameters (such as, K D: the dissociation equilibrium constant for ligand-binding to the neurotransmitter-binding sites of the closed receptor, and theta2: the diliganded gating-equilibrium constant) extracted from concentration-response relationships.
Issue Date:2006
Type:Text
Language:English
Description:101 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.
URI:http://hdl.handle.net/2142/87243
Other Identifier(s):(MiAaPQ)AAI3250310
Date Available in IDEALS:2015-09-28
Date Deposited:2006


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