Molecular modus operandi of ligand-gated ion channels : Studies of trimeric P2X receptors and pentameric GABA A receptors
This thesis, by using various chemical and biological tools, focuses on the molecular modus operandi of two different superfamilies of ligand-gated ion channels: P2XRs and GABAARs. P2XR is a cation-selective ion channel gated by extracellular ATP (and is implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Here I studied the molecular mechanism underlying ATP binding and channel opening of the P2X receptors. In the ATP-binding site study, we definitely localized the ATP-binding sites in P2X2 receptor through affnity labeling. Our results thus define a large and dynamic inter-subunit ATP-binding pocket. In the “gating”†part, an inter-subunit salt bridge located at the “body” domain that regulates channel gating movement was identified by using charge reversal and charge swapping combined with double mutant cycle analysisPentameric GABAARs form chloride permeable ion channels and mediate inhibitory synaptic transmission in the central nervous system. The modulation of their action is critical for brain normal function and for various pathophysiological conditions. In the GABAARs part, using patch-clamp electrophysiology, we described the allosteric modulation of GABAARs by a series of synthetic compounds that are trans-retrochalcones belonging to the flavonoids family. We characterized their subunit-dependent positive modulations at both synaptic and extrasynaptic GABAARs. Our data reveal an original mode of action and provide a rational basis for hypothesis-driven drug discovery efforts with emphasis on the retrochalcone scaffold for treating GABAA-related central nervous system disorders.