Allosteric modulation of pentameric ligand-gated ion channels by general anesthetics

Abstract: Pentameric ligand-gated ion channels (pLGICs) are key components of fast synaptic transmission and are targets of neuroactive drugs such as benzodiazepines, alcohol and muscle relaxants. Although early theories of general anesthesia suggested non-specific lipid interaction as the mechanism of anesthetic action, it has now become evident that they too bind to pLGICs. While general anesthetics act as positive allosteric modulators on most anion-conducting pLGICs, they inhibit cation-conducting channels. A detailed structural mechanism of how such opposite allosteric effects emerge has yet to be presented.This thesis investigates the structure-function relationship underlying the dynamics of channel activation and explores the mechanisms behind allosteric modulation by general anesthetics. Key model systems include the glutamate-gated chloride channel of C. elegans (GluCl) and the G. violaceus ligand-gated ion channel (GLIC), that show considerable structural homology to mammalian channel but with the added simplicity of homomeric assembly and accessibility to crystallization. Functional assessment is performed through recombinant expression of the channels in Xenopus oocytes, which are then used for two-electrode voltage clamp electrophysiology. These measurements are combined with recent advances in structure determination and computational simulations to propose structural mechanisms behind the functional effects.In this thesis I present the exploration and validation of the crystallographic construct GluCl as a model system to explore fundamental questions of mammalian pLGIC function. Further studies contribute to the understanding of the basis of allosteric modulation by identifying responsible binding sites for both potentiation and inhibition by general anesthetics in GLIC and substantiate a structural mechanism for these effects. The studies also offer a link between receptor- and lipid-based theories of anesthesia, and demonstrate successful discovery of new lead compounds with general anesthetic properties using virtual screening. The thesis therefore makes a contribution to the fundamental understanding of allosteric modulation in pLGICs and builds on the basis for rational drug discovery.