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Title:Molecular determinants of conductance and charge-selectivity in pentameric ligand-gated ion channels
Author(s):Harpole, Tyler
Director of Research:Grosman, Claudio
Doctoral Committee Chair(s):Grosman, Claudio
Doctoral Committee Member(s):Gennis, Robert; Gruebele, Martin; Tajkhorshid, Emad
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Ion Channels
Pentameric ligand-gated ion channels (pLGIC)
molecular simulations
Abstract:Pentameric ligand-gated ion channels (pLGICs) are a superfamily of neurotransmitter-gated ion channels, that includes (in vertebrates) the nicotinic Acetylcholine Receptor (AChR), Serotonin Receptor type 3 (5-HT3R), γ-Aminobutyric acid Receptor type A (GABAAR), and the Glycine Receptor (GlyR). All of these channels are made up of five subunits. Some pLGICs are homomeric and others are heteromeric. For example, the adult muscle-type AChR contains two α1, one β1, one δ, and one ε subunit, and each of these individual subunits is an AChR subtype within the superfamily. pLGICs are involved in processes as diverse as muscle contraction, cognition, nicotine addiction and inflammation. Beyond their particular biological roles, the pLGIC superfamily has been highly studied because the channels are well suited for single molecule electrophysiology experiments. Therefore, it is possible to robustly measure both the selectivity and conductance of these channels experimentally, but inferences about the molecular mechanisms responsible for these biophysical properties are often indirect because the technique lacks the resolution to see structural features responsible for these phenomena. In order to link electrophysiological data to molecular mechanisms, computational modeling is a highly suitable technique. Simulations can both verify inferences made using electrophysiology while simultaneously providing the resolution needed to extend the mechanism to an atomic scale. Here, two biophysical properties of pLGICs, charge selectivity and conductance, are studied using computational techniques such as molecular dynamics, Brownian dynamics, continuum electrostatics, and bioinformatics, which are then compared to experimental data. In the first part, the molecular mechanism of conductance through the cation-selective nicotinic AChR from muscle is studied. In the second part, the molecular determinants of charge-selectivity in anion-selective pLGICs is studied. In cation-selective members of the superfamily, rings of glutamates (and, more rarely, aspartates) that line the ion-permeation pathway are responsible for increasing the single-channel conductance. Among these rings, one in particular, at the intracellular mouth of the pore, catalyzes the movement of cations more so than the other rings. This ring is made up of four glutamates and one glutamine in the muscle-type AChR, but only two of the four glutamates are needed for a wild-type conductance. A channel with two glutamates also has two different current amplitudes within a single opening. It has been shown experimentally that the current fluctuations were not because one of the two glutamates became protonated. Rather, it was proposed that these fluctuations were due to a change in the side chain rotamers of these glutamates. This hypothesis was tested using a variety of computational techniques and showed great agreement with electrophysiology experiments. Simulations were also able to extend the experimentally generated hypothesis. Simulations showed that the rotamer distribution of these glutamates in the protein is asymmetric in that two of the glutamates tended to adopt one particular rotamer that was different from the rotamer adopted by the other two glutamates. These different classes of rotamers had a significantly different impact on conductance, and only two of the four glutamates were needed for a wild-type conductance. Furthermore, simulations showed that the rotamers that catalyze cation conduction most effectively are the mm and tp rotamers (using rotamer nomenclature as in the Penultimate Rotamer Library), and that these glutamates exert their effects by positioning the carboxylic acid up and into the pore. The speed at which ions go through the channel is of fundamental importance, but it is also essential to understand how these proteins are able to selectively transport some ions and not others. pLGICs are unique in that the superfamily contains highly cation-selective channels, such as the AChR and 5-HT3R, and highly anion-selective channels such as the GABAAR and GlyR. Therefore, the superfamily mediates both fast synaptic excitation and inhibition and is the sole source of fast synaptic inhibition in the nervous system of animals. This phenomenon of charge selectivity has been studied in pLGICs for many years, but the molecular details of how these channels discriminate based on the formal charge have been rather elusive. This is because some important residues for charge selectivity are experimentally difficult to mutate and because some results were not consistent among members of the superfamily. To study this problem, we have used multiscale simulations and made comparisons to experimental data. We find that charge-selectivity is determined by side-chain electrostatics, and more specifically, that the side-chain rotamers of a conserved ring of basic residues, even when not pointing directly towards the pore, plays an essential role in the charge selectivity of the anion-selective pLGICs receptors. Pore size (previously proposed to play an important role) couldn't, in and of itself, explain how mutations alter channel charge selectivity. Finally, we find that predicting the effect of mutations on charge selectivity is not generalizable across the superfamily because other residues within the protein determine charge selectivity in mutant channels that lack residues involved in charge selectivity of the wild type channel.
Issue Date:2016-06-08
Rights Information:Copyright 2016 Tyler Harpole
Date Available in IDEALS:2016-11-10
Date Deposited:2016-08

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