Dr Mark Young
I am interested in understanding how the 3D-structure of mammalian P2X receptors relates to their function and cell-signalling in chronic pain and inflammation.
P2X receptors are cell-surface ion channels which are activated by extracellular ATP. Activation leads to a sequence of downstream signalling events which have important consequences for nerve transmission, pain sensation, inflammation and control of smooth muscle tone. For these reasons, drugs which target P2X receptors may well have analgesic or anti-inflammatory actions. The process of drug discovery would be accelerated if high-resolution 3D-structures of mammalian P2X receptors were available, enabling structure-based drug design. In addition, an understanding, at the molecular level, of downstream signalling pathways might open up new targets for therapeutic intervention.
Work in my laboratory is currently centred on three main themes:
- The development of novel eukaryotic over-expression systems for mammalian P2X receptors in order to enable their purification and 3D-structure determination.
- The P2X7 Interactome (www.p2x7.co.uk) - understanding the molecular basis of downstream signalling following P2X receptor activation.
- Structure-based drug design using molecular models of mammalian P2X receptors.
I became interested in studying membrane protein structure-function relationships during my Biochemistry degree at the University of Bristol (1994-1997). I stayed on in Bristol for my Ph.D. and first postdoc (1997-2003) under the guidance of Prof. Mike Tanner, where I explored the interaction between the red blood cell anion transporter (band 3) and its accessory subunit, glycophorin A (GPA).
A growing interest in ion channels led me to take up postdocs with Profs. Annmarie Surprenant and Alan North at the Universities of Sheffield (2003-2005) and Manchester (2005-2007), where I worked on P2X receptor structure-function relationships. During this time, the focus of my research shifted towards the direct structural study of P2X receptors. With the aid of an Advanced Training Fellowship (2007-2010) and the mentorship of Prof. Bob Ford (University of Manchester), I determined the structure of human P2X4 at a resolution of 21Å, using electron microscopy of single protein particles and 3D reconstruction.
In September 2009 I took up the Evans-Huber Fellowship at Cardiff University, which has enabled me to set up my own research lab, where I continue to study the 3D structure and downstream signalling functions of mammalian P2X receptors, as well as looking to develop new expression systems for mammalian membrane proteins. I became a Lecturer (Teaching and Research) in September 2012.
P2X receptors are ATP-gated ion channels which play key roles in a variety of physiological processes such as synaptic transmission, taste sensation and smooth muscle control. They function in cells as trimers, with two transmembrane domains per monomer and large, glycosylated extracellular domains.
P2X receptors are important drug targets primarily because of their involvement in pain following nerve damage and inflammation. In a rat model of neuropathic pain, tactile allodynia was abolished when levels of the P2X4 receptor subtype were knocked down using antisense oligonucleotides . It is therefore likely that drugs which target P2X4 may have significant analgesic properties, but the current lack of human P2X4-selective antagonists is hampering research into its specific roles.
Our understanding of the structure of human P2X4 is limited to 21Å resolution at present , but the recent publication of the 3D crystal structures of a truncated mutant of zebrafish P2X4.1 in both the closed  and ATP-bound open state  raises hopes that crystal structures of suitably modified human P2X receptors can be determined. High-resolution structures of the ligand-binding sites of human P2X receptors, solved in the presence of ATP, would enable structure-based drug design, leading to new analgesic and anti-inflammatory therapies.
P2X receptors are also involved in inflammation. The P2X7 receptor subtype is expressed in immune cells; and in knock-out mice lacking P2X7, chronic inflammatory pain was abolished, while acute pain responses remained unchanged . P2X7 is unique among the P2X receptors in that its activation leads to the release of pro-inflammatory cytokines, and prolonged activation causes cell death . The properties of P2X7 are regulated by its long intracellular C-terminal domain, which couples ion channel activation to downstream signalling, and it has recently been suggested that targeting P2X7-mediated downstream signalling might represent a good strategy to develop more selective anti-inflammatory drugs .
Optimisation of mammalian P2X receptors for 3D-structure determination
Previously, His-tagged human P2X4 trimers (over-expressed in human cells) were purified by a combination of metal affinity chromatography and non-denaturing gel electrophoresis. Electron microscopy of single protein particles was used to determine a 3D structure at a resolution of 21Å . The dimensions and overall architecture of the low-resolution structure were highly consistent with that of the 3D crystal structure of the closed state of zebrafish P2X4.1 , and the fit of the extracellular domain was particularly good (Figure 1). We are currently exploring ways to optimise mammalian P2X receptors for structural study using a combination of mutagenesis, biochemical analysis and functional assay.
Exploring the expression of P2X receptors in other eukaryotic systems
We have also expressed P2X receptors in both baculovirus-infected insect cells  (collaboration with Dr Mark Parker, Case Western, USA) and the yeast Saccharomyces cerevisiae. Insect cell expression was successfully used in solving the structure of zebrafish P2X4.1 , so it represents a proven system for the expression of P2X receptors. We are currently developing the fruit fly Drosophila melanogaster as an expression system for membrane proteins which permits both structural and functional studies (collaboration with Dr Wynand Van der Goes Van Naters).
The yeast system has the advantages of being both cost-effective and easy to use; initial expression trials of rat P2X7 are encouraging and, once suitably optimised, this system will provide a straightforward way to screen multiple constructs for expression, folding and suitability for further structural studies.
P2X7 downstream signalling pathways and the P2X7 Interactome (www.p2x7.co.uk)
Upon activation by extracellular ATP, P2X7 couples via its long C-terminal domain to several intracellular signalling pathways, leading to recruitment of the NRLP3 inflammasome and release of pro-inflammatory cytokines . This signalling has important consequences in pain and inflammation, particularly in conditions of chronic inflammation such as arthritis and Alzheimer's disease. Although many downstream components of the signalling pathways have been characterised, the molecular basis of the first step in this process remains unknown. What is the 3D-structure of the P2X7 C-terminal domain, which proteins does it interact with, and how are these interactions regulated? In collaboration with Dr Amanda MacKenzie (University of Bath), we are currently using the isolated, purified P2X7 C-terminal domain in structural and proteomic studies to address these questions. We have also developed the P2X7 Interactome website (www.p2x7.co.uk) which lists all the P2X7-interacting proteins detected to date along with key information about their function in cell-signalling.
Structure-based drug design using molecular models of mammalian P2X receptors
Although there are no high-resolution crystal structures of mammalian P2X receptors, molecular models based upon the ATP-bound crystal structure of zebrafish P2X4  can be constructed. In collaboration with Dr Andrea Brancale we have used these molecular models to perform in silico docking of a range of drug-like compounds into the ATP-binding site, selecting those which give the best fit for functional assays using calcium uptake and electrophysiology. In this way we hope to find 'hit' compounds which modulate P2X receptor function, which can then be further modified to develop potent and selective drugs, which may be of significant therapeutic benefit in conditions of pain and inflammation.
- Tsuda M et al. (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424, 778-783
- Young MTet al. (2008) Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy. J Biol Chem 283, 26241-26251
- Kawate Tet al. (2009) Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460, 592-598
- Hattori M and Gouaux E (2012) Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485, 207-212
- Chessell IP et al. (2005) Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 114, 386-396
- Surprenant A and North RA (2009) Signaling at purinergic P2X receptors. Annu Rev Physiol 71, 333-359
- Sorge RE et al. (2012) Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat Med 18, 595-599
- Valente M et al. (2011) Expression, purification, electron microscopy, N-glycosylation mutagenesis and molecular modelling of human P2X4 and Dictyostelium discoideum P2XA. Biochim Biophys Acta 1808, 2859-2866
University of Bath
- Dr Amanda MacKenzie