Deadline for full application: December 15th, 2013
Interviews: March, 2014
Start of the Ph.D.: October 1st, 2014
Title of the PhD project: Allosteric transitions of pentameric channel receptors explored by combined biophysical and electrophysiological approaches
Name of the lab: Channel-Receptors Unit
Head of the lab: Pierre-Jean Corringer
PhD advisor: Pierre-Jean Corringer
Email address: email@example.com
Web site address of the lab: http://www.pasteur.fr/ip/easysite/pasteur/en/research/scientific-departm...
Doctoral school affiliation and University:
ED 387 : iViv : Interdisciplinaire pour le vivant, University Pierre et Marie Curie, Paris 6
Presentation of the laboratory and its research topics:
Our laboratory is interested in the fundamental mechanisms involved in synaptic communication between neurons. More specifically, we work on post-synaptic channel-receptors, which convert the binding of neurotransmitters (released from the presynaptic neuron), into the opening of their intrinsic channel to promote neuronal excitation or inhibition. We are interested in a particular family of channel receptors called “pentameric”, which includes nicotinic acetylcholine receptors, glycine and GABAA receptors (Corringer et al, 1012). Those receptors play a key role in the functioning of the brain, and are the target of several classes of therapeutic drugs such as anxiolytics (benzodiazepine), general anesthetics, anti-smoking and anti-Alzheimer compounds. They are also the target of the addictive drugs nicotine and ethanol.
The goal of our research is to understand, at the atomic resolution, the molecular mechanisms involved in pentameric channel receptor functioning. We are part of the neuroscience department, and we have extensive interaction with the department of structural biology and chemistry.
Seven years ago, our group has discovered bacterial homologs of pentameric channel receptors (Bocquet et al, 2007), paving the way for the resolution of their structure at atomic resolution by X-ray crystallography. Indeed, those proteins are integral membrane and are notoriously difficult to produce, purify and crystallize in three dimensions. We set up the production in milligram of one bacterial homolog, called GLIC, and developed a close collaboration with the unit of Marc Delarue (Institut Pasteur) allowing solving the GLIC structure at up to 2.4 angstrom resolution by X-ray crystallography (Bocquet et al, 2009, Prevost et al, 2012). This yielded structural data on three different conformations, as well as the identification of general anesthetics and alcohol binding sites (Nury et al, 2011; Sauguet et al, 2013). In the laboratory, we currently perform protein production and purification, mutagenesis, electrophysiology that is the best technique to follow the opening of the ion channel, as well as several biophysical approaches (fluorescence, surface plasmon resonance, etc).
Description of the project:
(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))
Pentameric channel receptors are allosteric proteins interconverting between different conformations: a resting conformation carrying a closed channel, an active conformation triggered by agonist and carrying an open channel and one or several desensitized conformations. In addition, functional experiments have shown that the allosteric transitions are not direct, and that several intermediate conformations are significantly populated during both activation and desensitization. Channel opening elicits cell excitation (depolarization) or inhibition (hyperpolarization) by letting specific ions to diffuse passively through their electrochemical gradient.
The project will be centered on GLIC, which is a proton-gated ion channel (Bocquet et al, 2007, Prevost et al, 2013) used as a prototype of pentameric channel receptors. GLIC expresses well in E. coli and eukaryotic cells and yields robust electrophysiological responses. Its structure has been solved in three different conformations: an open-channel conformation (Bocquet et al, 2009) and two different closed channel conformations (Prevost et al, 2012, Sauguet et al, unpublished). However, these data were collected from X-ray crystallography experiments on detergent solubilized receptors packed in three-dimensional crystals. X-ray structures can thus be considered as snapshots of protein conformation, but their assignment to particular allosteric conformation occurring at the plasma membrane remain to be explored.
The project will aim at monitoring the structural reorganization associated with activation and desensitization, in a context where the protein in embedded within the membrane and functional. More specifically, we will use the various available X-ray structures to make predictions of local and global motions that are likely to occur during the transitions of activation and desensitization. These predictions will be tested by site-directed mutagenesis of the indentified hinges regions, as well as by chemical bridging designed to stabilize particular conformations. To this aim, couples of cysteines will be introduced at key positions, and bridged either by direct disulphide bound with redox agents or by the use of bifunctional cross-linking reagents of different lengths (Prevost et al, 2012). These approaches will be further extended by the use of cross-linkers carrying an azobenzene moiety that undergo cis-trans isomerization upon light illumination (Beharry et al. (2012). Biochemistry, 51, 6421–6431). This will allow performing contraction or extension of the cross linker by remote light application, and follow online the channel activation. These experiences will be performed in two systems: 1) on the protein expressed in Xenopus oocytes which allow monitoring of the channel opening/closing events by electrophysiology in real time, and 2) on the protein expressed in E. coli, detergent purified and reconstituted in lipidic vesicles, which allows performing biochemical (native and SDS gel electrophoresis, size exclusion chromatography, etc), biophysical (fluorescence, surface plasmon resonance, microcalorimetry) as well as electrophysiological (on planar lipid bilayer) experiments.
Overall, the aim of the project will be to assign particular X-ray structures to allosteric conformations, and to propose molecular mechanisms for the allosteric reorganization underlying activation and desensitization. The experiments will also allow investigating the mechanisms of action of allosteric effectors, notably those acting within the transmembrane domain such as alcohols and general anesthetics. This will contribute to our knowledge of allosteric effector action, helping the design of potential therapeutic compounds.
Cited references from our laboratory:
Prevost MS, Delarue-Cochin S, Marteaux J, Colas C, Van Renterghem C, Blondel A, Malliavin T, Corringer PJ, Joseph D. Identification of Cinnamic Acid Derivatives As Novel Antagonists of the Prokaryotic Proton-Gated Ion Channel GLIC. J Med Chem. 56(11):4619-30 (2013)
Sauguet L, Howard R., Malherbe L. Lee U, Corringer P.J., R. Harris A., Delarue M. Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel. Nat. Commun 16;4:1697 (2013)
Corringer PJ, Poitevin F, Prevost MS, Sauguet L, Delarue M, Changeux JP. Structure and pharmacology of pentameric receptor channels: from bacteria to brain Structure. 20:941-56. (2012) review
Prevost, MS, Sauguet, L., Nury H., Van Renterghem, C., Huon, C., Poitevin, F., Baaden, M., Delarue, M., Corringer, P.J. a locally-closed conformation of a bacterial pentameric proton-gated ion channel, Nat Struct Mol Biol. 19:642-9 (2012)
Nury, H, Van Renterghem, C, Weng, Y, Tran, A, Baaden, M, Dufresne, V, Changeux, J.P., Sonner, J.M., Delarue, M, Corringer, P.J. X-ray structures of general anesthetics bound to a pentameric ligand-gated ion channel Nature, 469:428-31 (2011)
Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J.P., Delarue, M., Corringer, P.J. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation Nature, 457 :111-4 (2009)
Bocquet, N., Prado de Carvalho, L., Cartaud, J., Neyton, J., Le Poupon, C., Taly, A., Grutter, T., Changeux, J.P., Corringer, P.J. A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family, Nature, 445: 116-119 (2007)
Integral membrane protein
Expected profile of the candidate (optional):
The candidate should have a solid training in molecular biology and in biochemistry. Previous training in electrophysiology would be a plus but is not mandatory. Computer skills for the visualization and analysis of X-ray structures would be a plus but is not mandatory.