Periodic Reporting for period 1 - nAChR PAM-to-gate (Gating mechanism and pharmacological modulation of nicotinic acetylcholine receptors) Reporting period: 2015-04-01 to 2017-03-31 Summary of the context and overall objectives of the project Neurotransmitter-gated ion channels are responsible for fast chemical neurotransmission at synapses, i.e. the specialized chemical junction enabling the communication between excitable cells like neurons. Amongst these ion channels, the superfamily of pentameric ligand-gated ion channels (pLGIC) comprises chloride-permeable GABAA (GABAARs) and Glycine receptors (GlyRs), as well as excitatory nicotinic acetylcholine (nAChRs). GABAARs and GlyRs mediate fast inhibitory synaptic transmission, and are critical to maintain the excitation / inhibition balance of neurons, which controls their activity. The most prevalent neuronal nAChRs are the α7 homopentameric receptors and the α4β2 heteropentameric receptors, and are involved in various functions such as attention and memory.Following the binding of their agonist (GABA for GABAARs, glycine for GlyRs and acetylcholine for nAChRs), these receptors undergo a conformational change leading to the opening their trasmembrane pore, enabling the flow of ions across the cell membrane, thereby producing changing the electrical activity of the cell. A wealth of structure-function studies has improved our understanding as to how pLGICs activate following the binding of agonist. However, besides their agonist-induced activation, most pLGICs display another fundamental pharmacological property: desensitization. Indeed, for most pLGICs, the sustained presence of the neurotransmitter will cause the channels to transit from the active open-channel agonist-bound conformation to a shut-channel, agonist-bound state called the desensitized state. Desensitization is thought to prevent the over-activation of receptors in pathological conditions, and can also lead to the reduction of postsynaptic current upon repetitive synaptic neurotransmitter release. The structural basis and the physiological roles of pLGICs’ desensitization remain challenging questions to tackle, with potentially far-reaching applications like the development of drugs to treat pathologies in which pLGICs are involved, such as epilepsy, Alzheimer’s disease, schizophrenia, depression, chronic pain…The overall objective of this project is to advance our understanding of the basic molecular events at play during pLGICs’ desensitization, in order to provide a better understanding of the desensitization process and provide proof-o-concepts for the development of drugs modulating the desensitization of pLGICs. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the short time-frame (10 months) of the funded project, we have advanced along three lines of experimental work.First, we have developed a series of so-called chimeric receptors containing the extracellular domain of a bacterial pLGIC, and the transmembrane domain of the α7 nAChR. Indeed, the transmembrane domain of α7 nAChRs is the target of drugs modulating this receptor’s desensitization, and the use of a chimera with the bacterial pLGIC is a molecular trick to facilitate the biophysical and structural study of the transmembrane domain of α7 nAChR. Amongst these chimeric receptors, one exhibits high level of expressions, making this construct potentially amenable to structural studies.Second, to understand the interplay between subunits of nAChRs during activation and desensitization, we have constructed a series of concatemeric receptors, whereby all five subunits are linked through short polypeptides. This allows the precise control of subunits order within the pentamer, and to introduce mutations in each subunit one by one, thus providing a very precise tool to dissect the biophysical and pharmacological properties of nAChRs. These concatemeric receptors lead to robust expression in recombinant systems (Xenopus oocytes).Third, we have constructed a series of 22 different concatemeric GABAARs, harbouring combinations of gain-of-desensitization mutations. This enables us to examine the interplay between subunits during the receptors’ desensitization. The final kinetic modeling of the experimental data is currently under progress, but we currently favour a model in which individual subunits rearrange independently during desensitization. These results should lead to a scientific publication in the near future (i.e. in 2017). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our results with the concatemeric receptors will provide evidence to discriminate between hypotheses that have been formulated 20 years ago: do the various desensitized states (as seen from functional data) reflect structurally distinct states, or merely the fact that individual subunits can rearrange independently during desensitization, thereby leading to asymmetric intermediate states?From a more industry-oriented point of view, a structural characterization of the transmembrane domain of α7 nAChR would be a great leap forward, enabling the rational design of drugs modulating this receptor with strong selectivity over other nAChRs, thereby limiting potential adverse effects. Our chimeric receptors may thus offer a welcome structural template to further current research from the pharmaceutical industry, which has developed a series of compound targeting α7 nAChR. Given the involvement of these receptors in diverse pathologies strongly impacting our societies (e.g. Alzheimer’s disease, schizophrenia, depression,…), any pharmacological treatment improving these conditions would be a welcome addition to the medical doctor’s toolbox. plgic.jpg
