Single-Channel Studies of Agonist Interactions With the Neurotransmitter-Binding and Pore Domains of the Muscle Nicotinic Acetylcholine Receptor
type: CreativeWork
description: 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 nic…
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 nic…
author: Purohit, Yamini
language: eng
keywords: Health Sciences, Pharmacology
date published: 2015-09-28T15:50:12Z
date modified: 2026-07-22T22:26:28Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/87243