Elucidating the Gating Mechanism of Cys-Loop Receptors

Cys-loop receptors are membrane proteins that are key players for the fast synaptic neurotransmission. Their ion transport initiates new nerve signals after activation by small agonist molecules, but this function is also highly sensitive to allosteric modulation by a number of compounds such as anesthetics, alcohol or anti-parasitic agents. For a long time, these modulators were believed to act primarily on the membrane, but the availability of high- resolution structures has made it possible to identify several binding sites in the transmembrane domains of the ion channels. It is known that ligand binding in the extracellular domain causes a conformational earthquake that interacts with the transmembrane domain, which leads to channel opening. The investigations carried out in this thesis aim at understanding the connection between ligand binding and channel opening. I present new models of the mammalian GABAA receptor based on the eukaryotic structure GluCl co-crystallized with an anti-parasitic agent, and show how these models can be used to study receptor-modulator interactions. I also show how removal of the bound modulator leads to gradual closing of the channel in molecular dynamics simulations. In contrast, simulations of the receptor with both the agonist and the modulator remain stable in an open-like conformation. This makes it possible to extract several key interactions, and I propose mechanisms for how the extracellular domain motion is initiated. The rapid increase in the number of cys-loop receptor structures the last few years has further made it possible to use principal component analysis (PCA) to create low-dimensional descriptions of the conformational landscape. By performing PCA on the crystal structure ensemble, I have been able to divide the structures into functional clusters and sample the transitions between them using various sampling methods. The studies presented in this thesis contribute to our understanding of the gating mechanism and the functional clustering of the cys-loop receptor structures, which both are important to design new allosteric modulator drugs that influence the channel function, in particular to treat neurological disorders.QC 20160518</p

Conformational Gating Dynamics in the GluCl Anion-Selective Chloride Channel

Cys-loop receptors are central to propagation of signals in the nervous system. The gating of the membrane-spanning pore is triggered by structural rearrangements in the agonist-binding site, located some so A away from the pore. A sequential conformational change, propagating from the ligand-binding site to the pore, has been proposed to govern gating in all Cys-loop receptors. Here, we identify structural and dynamic components of the conformational gating in the eukaryotic glutamate-gated chloride channel (GluCl) by means of molecular dynamics (MD) simulations with and without the L-glutamate agonist bound. A significant increase in pore opening and accompanying hydration is observed in the presence of glutamate. Potential of mean force calculations reveal that the barrier for ion passage drops from 15 kcal/mol to 5-10 kcal/mol with the agonist bound. This appears to be explained by agonist binding that leads to significant changes in the intersubunit hydrogen-bonding pattern, which induce a slight tilt of the extracellular domain relative to the transmembrane domain in the simulations. This rearrangement is subtle, but correspond to the direction of the quaternary twist observed as a key difference between open and closed X-ray structures. While the full reversible gating is still a much slower process, the observed structural dynamics sheds new light on the early stages of how the agonist influences the extracellular domain, how the extracellular domain interacts with the transmembrane domain, and how changes in the transmembrane domain alter the free energy of ion passage.QC 20171128</p

Functional Characterization of Neurotransmitter Activation and Modulation in a Nematode Model Ligand‐gated Lon Channel

The superfamily of pentameric ligand‐gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate‐gated chloride channel (GluCl) in multiple states offer an appealing model system for structure‐function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two‐electrode voltage‐clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native‐like GluCl constructs by L‐glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site‐directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration‐dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L‐glutamate activation, consistent with computational evidence for this region\u27s role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery

Contribution of Structural Elements to Activation and Allosteric Modulation in an Anionic Ligand-Gated Ion Channel

QC 20140710</p