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Mapping "missing" conformations of ATP-gated P2X receptor ion channels

Reference	BB/P001076/1
Principal Investigator / Supervisor	Professor Richard Evans
Co-Investigators / Co-Supervisors	Dr Ralf Schmid
Institution	University of Leicester
Department	Molecular and Cell Biology
Funding type	Research
Value (£)	423,496
Status	Completed
Type	Research Grant
Start date	01/12/2016
End date	30/11/2019
Duration	36 months

Abstract

P2X receptors (P2XRs) comprise a distinct family of channels activated by binding extracellular ATP and have important physiological and pathophysiological roles. The crystallization of zebrafish P2X4Rs in agonist free (apo) and ATP bound open states provided a major advance in understanding the molecular basis of agonist binding and channel gating. However it is clear that there are additional fundamentally important receptor conformations where there is little or no structural information. Our cysteine mutant accessibility studies identified a "relaxed" apo state, and that desensitization involves distinct changes in the extracellular ligand binding loop as well as a desensitization gate. In addition, it is clear that a distinct "dilated-dye-permeant" pore state is present in some P2XRs. In this proposal we will use a cysteine mutagenesis based approach coupled with computer based molecular modelling to investigate the structural basis of missing receptor conformations. Mutants will be generated on the appropriate receptor background to investigate the "relaxed-apo", desensitized and dye permeant states. Changes in the extracellular region (upper vestibule, left flipper, lower body and central vestibule) will be measured predominantly by assessing the accessibility of introduced cysteine residues to a range of sulphydryl reactive compounds of varying dimensions. This will be determined for P2XRs in the apo, ATP-bound open or desensitized states. ATP evoked currents at mutants lining the channel pore are reduced by cysteine reactive compounds. Investigating the state-dependence and kinetics of any modifications for a range of different sized compounds will give insight to the dimensions and conformational changes in the pore. In all cases information from the cysteine reactive compounds will be used in an iterative cycle with molecular modelling/dynamics simulations to provide validated models of "missing" P2XR states.

Summary

Cells within the body communicate with one-another through the release of chemicals recognised by specific cell surface receptors. One example of such a chemical is ATP that binds to P2X receptors (P2XRs) and activates them. In humans there are at least 13 different types of P2XR (e.g P2X1R and P2X7R) that vary in their properties, for example how long they are able to be "turned-on/activated". P2XRs play an important role in a range of normal bodily functions e.g. in control of blood clotting and taste sensation, as well being drug targets for the treatment of pain and neurodegenerative diseases e.g. Alzheimer's disease. P2XRs are membrane proteins with parts on the outside of the cell (extracellular) that recognize the ATP molecule, a channel region that passes through the cell wall (a valve/tap that regulates the movement of positively charged ions) and a region inside the cell (intracellular) that regulates how long the receptor is "ON" for (dependent on the receptor type). In the absence of ATP the P2XR channel is closed and "OFF". ATP binding to the extracellular region leads to a change in the shape of the receptor; an "ON" signal opening the channel (movement of positive ions through it excites the cell). Recent studies have shown the 3D structure of a P2XR in the "OFF" and "ATP-ON" states and this has provided a major advance in our understanding of how this novel family of receptors works. However, these are only two snapshots of the receptor, and it is clear that additional movements in the 3D shape are important that result in distinct forms of the receptor with special properties. This proposal aims to gain 3D structural information on three distinct "missing" conformations/states of the receptor. (i) A "relaxed-OFF" form of the receptor in the absence of ATP that may be important for understanding of how drugs work to block/stop the receptor being turned on. (ii) An "ATP-CLOSED" desensitized receptor, where after opening following ATP bindingthe channel closes (i.e. turns off); a feature of the P2X1R involved in blood clotting. (iii) An "ATP-EXTRA-ON" state where the channel region gets larger and allows large molecules to enter the cell; this is particularly associated with the P2X7R and its role in inflammation and cell death. Information on these additional "missing" structures is essential to understand the fundamental mechanisms associated with the activity of this distinct family of receptors. P2XRs are made up of different amino acid "building blocks" and these can be individually changed. Of particular use for this is mutating an amino acid to cysteine; this is a chemically unique amino acid that can be targeted with a wide range of cysteine-specific compounds. These bind to the cysteine residue and change the chemical properties/size. In normal P2XRs there are no cysteine residues available for modification and cysteine-specific compounds have no effect. Therefore we can introduce cysteine mutations at defined parts of the receptor and determine the effects of cysteine reactive compounds to investigate the structure. This will be carried out in the absence of ATP (relaxed-OFF), and in the presence of ATP at desensitizing P2XRs (ATP-CLOSED) and at the ATP-EXTRA-ON P2X7R. We will test whether an introduced residue is accessible (on the receptor surface or in the channel), and by varying the size of the cysteine reactive compound measure the dimensions around that residue as well as test the effects of the modification on ATP evoked responses. These results will give molecular dimensions that will then be used in computer based studies to map the molecular changes in the receptor and provide validated 3D models of the missing receptor structures. This will provide a fundamental insight into how P2XRs work at the molecular level, understanding variations between receptors, and why genetic mutations affect receptor properties that can lead to imbalances in signalling and disease.

Impact Summary

In the 20 years since the original cloning of the first P2X receptors it has become clear that this distinct family of ligand gated ion channels have fundamental signalling roles in the body. At least one form of P2X receptor for ATP is expressed by almost every cell type at some stage and defined P2X receptor subtypes have been shown to have important biological functions. For example, P2X2 receptors are essential for taste sensation, P2X1 receptors contribute to blood clotting and P2X7 receptors play central roles in inflammatory responses. However, we have an incomplete understanding of the molecular basis of receptor properties. Our work will provide validated models of important states of the receptor where there is currently little or no structural information. This new insight into these receptors has relevance not only to the academic community but also more widely to the Pharmaceutical Industry for development of novel therapies and the general public for an increased understanding of human health and disease. The project addresses directly BBSRC objectives to support fundamental discoveries and will apply computational and modelling techniques to high-quality quantitative biological data, and use the models generated to test new hypotheses and inform experimental strategies. We will provide structural insight into fundamentally important P2X receptor states to improve our understanding of the molecular transitions of this distinct family of ion channels. The project will provide impact to the academic community, industry/healthcare and the general public. Academic impact and training. Our work will be communicated through scientific publications and conference presentations and have relevance not only to those working on P2X receptors but also those with interests in other ion channels and application of molecular modelling based approaches to biochemical data. In addition to publications we will generate movie animations illustrating key aspectsof the models that will be made available on the PIs web pages to further engage colleagues, students and the wider public with our work. Ongoing training and support will be provided to the post-doc to facilitate their career progression. In addition, undergraduate and MSc students will also be directly engaged with the project to provide them with training/laboratory experience (~ 2-3 month placements) that will have impact in their future careers. Implications for healthcare/drug development. The new structural information will have direct relevance to several Pharmaceutical companies that are developing P2X receptor compounds. For example, we are currently collaborating with Johnson & Johnson on some of their P2X7 receptor selective antagonists. We will engage with relevant Pharmaceutical companies as appropriate following consultation with the University Enterprise and Research Office to protect commercial opportunities. Our validated models will provide additional templates for understanding how drugs bind to the P2X receptor and will provide novel templates for in silico drug design/refinement. Engagement with the general public. P2X receptor research has direct relevance to physiology and pathophysiology and therefore is of interest to the general public. Although we are not directly working on disease states the information generated will have direct relevance to understanding underlying signalling mechanisms. For example, in animal models P2X1 receptor antagonists are protective against stroke and P2X7 receptors are novel analgesics. We will therefore provide (in conjunction with our research publications) focused press releases on our work explaining in lay terms our findings and their relevance to human health and disease. In addition the work will be included in Outreach work from the University, public lectures and engagement with prospective students/accompanying persons at University Open days and UCAS visits.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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