Professor Richard Evans
|Tel: 0116 229 7057 Email: email@example.com|
Research Interests and Techniques
P2X receptors for ATP are ligand gated cation channels that comprise a distinct family of ligand gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven P2X receptor genes have been cloned (P2X1-7) and they form functional homo-and heteromeric channels with a range of phenotypes.
A major focus of our recent work has been to understand the molecular basis of drug action at P2X receptors. We have used site directed mutagensis to develop a model of the ATP binding site of the receptor that has been supported by the recent crystallization of the zebra fish P2X4 receptor in a closed agonist free conformation. We have also shown that the P2X1 receptor function can be regulated by G protein coupled receptors and the localised lipid environment in cells.
The P2X1 receptor is also expressed in the nervous system, generally as a heteromeric channel with other P2X receptors subunits can contribute to neuronal regulation of the auditory system and glial signalling. Work in the lab is focused on three areas:
- Site directed mutagenesis studies on P2X1 receptors to determine the antagonist binding site(s) of the receptor.
- Understanding the basis of antagonist action at P2X7 receptors.
- Measuring in real time drug binding and conformational changes at P2X1 receptors using voltage clamp fluorometry.
Two adjacent P2X receptor subunits are shown in slate blue and grey, residues predicted to form the ATP binding pocket are shown in red (P2X1 receptor numbering), the blue residues corresponds to an Arg residue in P2X7 that can be ADP ribosylated and activate the channel. Mutations that affect sensitivity to PPADs and suramin are shown in orange and yellow respectively. Based on the zebra fish P2X4 receptor structure (Kawate et al., 2009, Nature, 460, 592-598).
- Contraction studies on isolated smooth muscle preparations using organ baths, myography and video imaging microscopy.
- Patch clamp studies on smooth muscle and neurons.
- Protein purification and mass spectrometry.
- Confocal microscopy.
- Molecular biology.
- Expression of recombinant receptors in Xenopus oocytes and two electrode voltage clamp recording.
- Voltage clamp flurometry.
Present Group Members
Dr Rebecca Allsopp
Dr Alistair Fryatt
Mrs Manijeh Maleki-Dizaji
Medical Research Council (3 year project grant, start date September 2013) “Integrated mutagenesis, bio-informatic and fluorescence approaches to characterie the molecular basis of antagonist action at P2X7 receptors for ATP” £396,706.
British Heart Foundation (3 year project grant, start date November 2011) “Investigation of ligand sensitive conformational changes in cardiovascular P2X receptors with voltage-clamp fluorometry” £175,476.
Allsopp, R.C., Farmer, L.K., Fryatt, A.G. and Evans, R.J. (2013). P2X receptor chimeras highlight roles of the amino terminus to partial agonist efficacy, the carboxyl terminus to recovery from desensitization, and independent regulation of channel transitions. J. Biol. Chem. 288, 21412-21421.
El-Ajouz, S., Ray, D., Allsopp, R.C. & Evans, R.J. (2012). Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop. Br. J. Pharmacol., 165, 390-400.
Roberts, J. A., Allsopp, R. C., El Ajouz, S., Vial, C., Schmid, R., Young, M. T. and Evans, R. J. (2012) Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel. Proc Natl Acad Sci U S A, 109, 4663-4667.
Lecut, C., Faccinetto, C., Delierneux, C., van Oerle, R., Spronk, H. M., Evans, R. J., El Benna, J., Bours, V. and Oury, C. (2012) ATP-gated P2X(1) ion channels protect from endotoxemia by dampening neutrophil activation. J Thromb Haemost, 10, 453-465.
Lalo, U., Jones, S., Mahaut-Smith, M.P. and Evans, R.J. (2012) Heat Shock Protein 90 inhibitors reduce trafficking of ATP-gated P2X1 receptors and human platelet responsiveness. J. Biol. Chem 287, 32747-32754.
Roberts, J.A., Bottrill, A., Mistry, S. and Evans, R.J. (2012) Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes. J. Neurochem. 123, 725-735.
Wen, H. & Evans, R.J. (2011). Contribution of the intracellular C terminal domain to regulation of human P2X1 receptors for ATP by phorbol ester and Gq coupled mGlu1a receptors. Eur. J. Pharmacol. 654, 155-159.
Allsopp, R.C., El Ajouz, S., Schmid, R. & Evans, R.J. (2011). Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP; mapping agonist binding and channel gating. J. Biol. Chem., 286, 29207- 29217.
Jones, S., Evans, R.J. & Mahaut Smith M.P. (2011). Extracellular calcium modulates ADP-evoked aggregation through altered agonist degradation:implications for conditions used to study P2Y receptor activation. Br. J. Heamatol. 153, 83-91.
Lalo, U., Roberts, J.A. & Evans, R.J. (2011). Identification of human P2X1 receptor- interacting proteins reveals a role of the cytoskeleton in receptor regulation. J. Biol. Chem., 286, 30591-30599.
Allsopp, R. C. and Evans, R. J. (2011) The intracellular amino terminus plays a dominant role in desensitisation of ATP gated P2X receptor ion channels. J Biol Chem, 286, 44691-44701.
Lalo, U., Allsopp, R.C., Mahaut-Smith, M.P. & Evans, R.J. (2010). P2X1 receptor mobility and trafficking; regulation by receptor insertion and activation. J.Neurochem, 113, 1177-1187.
Allsopp, R.C., Lalo, U. & Evans, R.J. (2010). Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP; chimeras and point mutants identify intracellular amino terminal residues involved in lipid regulation of P2X1 receptors. J. Biol. Chem. 285, 32770-32777.
Wen H and Evans RJ. (2009). Regions of the amino terminus of the P2X1 receptor required for modification by phorbol ester and mGlur1α receptors. J. Neurochem 108, 331-340.
Roberts JA, Valente M, Allsopp RC, Watt D and Evans RJ. (2009). Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis. J. Neurochem 109, 1042-1052.
Lecut C, Frederix K, Johnson DM, Deroanne C, Thiry M, Faccinetto C, Maree R, Evans RJ, Volders PG, Bours V, Oury C. (2009). P2X1 Ion channels promote neutrophil chemotaxis through Rho kinase activation. J Immunol. 183(4), 2801-2809.
Agboh KC, Powell AJ and Evans RJ. (2009). Characterisation of ATP analogues to cross-link and label P2X receptors. Neuropharmacology 56, 230-236.
Atarashi K, Nishimura J, Shima T, Umesaki Y, Yamamoto M, Onoue M, Yagita H, Ishii N, Evans RJ, Honda K and Takeda K. (2008). ATP drives lamina propria TH17 cell differentiation. Nature 455, 808-812.
Roberts JA, Digby HR, Kara M, El Ajouz S, Sutcliffe MJ and Evans RJ. (2008). Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors. J. Biol. Chem. 283, 20126-20136.
Roberts JA and Evans RJ. (2007). Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors. J. Neurosci. 27, 4072-4082.
Harrington LS, Evans RJ, Wray J, Norling L, Swales KE, Vial C, Ali F, Carrier MJ and Mitchell JA. (2007). Purinergic P2X1 receptors mediate endothelial dependent vasodilation to ATP. Mol. Pharmacol. 72, 1132-1136.
Roberts JA and Evans RJ. (2006). Contribution of conserved polar glutamine, asparagines and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP. J. Neurochem. 96, 843-852.
Wong A, Billups B, Johnston J, Evans RJ and Forsythe I. (2006). Endogenous activation of adenosine A1 receptors but not P2X receptors during high frequency synaptic transmission at the calyx of Held. J. Neurophysiol. 95, 3336-3342.
Vial C, Fung E, Goodall AH, Mahaut-Smith MP and Evans RJ. (2006). Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts. Biochem. Biophys. Res. Comm. 343, 415-419.
Lamont C, Vial C, Evans RJ and Wier WG. (2006). P2X1 receptors mediate sympatheirc post-junctional Ca2+ transients (jCaTs) in mesenteric small arteries. Am. J. Physiol. 291, H3106-H3113.
Roberts JA and Evans RJ. (2005). Mutagenesis studies of conserved proline residues of human P2X1 receptors for ATP indicate that proline 272 contributes to channel function. J. Neurochem. 92, 1256-1264.
Vial C and Evans RJ. (2005). Disruption of lipid rafts inhibits P2X1 receptor mediated currents and arterial vasoconstriction. J. Biol. Chem. 280, 30705-30711.
Digby HR, Roberts JA, Sutcliffe MJ and Evans RJ. (2005). Contribution of conserved glycine residues to ATP action at human P2X1 receptors: mutagenesis indicates that the glycine at position 250 is important for channel function. J. Neurochem. 95, 1746-1754.
Roberts JA and Evans RJ. (2004). ATP binding at human P2X1 receptors; contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists. J. Biol. Chem. 279, 9043-9055.
Vial C, Tobin AB and Evans RJ. (2004). G-protein coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation. Biochem. J. 382, 101-110.
Watano T, Calvert JA, Vial C, Forsythe ID and Evans RJ. (2004). P2X receptor subtype specific modulation of excitatory and inhibitory input in the rat brainstem. J. Physiol. 558, 745-757.
Hechler B, Lenain N, Marchese P, Vial C, Heim V, Freund M, Cazenave J-P, Cattaneo M, Ruggeri Z, Evans RJ and Gachet C. (2003). A role of the fast ATP-gated P2X1 cation channel in the thrombosis of small arteries in vivo. J. Exp. Med 198, 661-667.
Vial C, Pitt SJ, Roberts J, Rolf MJ, Mahaut-Smith MP and Evans RJ. (2003). Lack of evidence for functional ADP-activated human P2X1 receptors supports a role for ATP during hemostasis and thrombosis. Blood 102, 3646-3651.
Inscho EW, Cook AK, Imig JD, Vial C and Evans RJ. (2003). Physiological role for P2X1 receptors in renal microvascular autoregulatory behaviour. J. Clin. Invest. 112, 1895-1905.
Mulryan K, Gitterman DP, Lewis CJ, Vial C, Leckie BJ, Cobb AL, Brown JE, Conley EC, Buell G, Pritchard CA and Evans RJ. (2000). Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors. Nature 403: 86-89.