Professor Richard Evans

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.

The primary interest of the lab is in the P2X1 receptor that is expressed in smooth muscle, platelets, neurons, glia and immune cells. We generated a P2X1 receptor knockout mouse to determine the role of the receptors and have shown that they are involved in the control of arteries in response to sympathetic nerve stimulation, autoregulation in the kidney and blood clotting. P2X1 receptors therefore are novel targets for the treatment of hypertension and stroke.

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:

1. Site directed mutagenesis studies on P2X₁ receptors to determine the ATP and antagonist binding site(s) of the receptor.
2. Lipid rafts can play a role in the regulation of P2X1 receptors and their trafficking.  We are characterising the molecular basis of regulation and P2X1 using chimeric receptors, electrophysiological recording and mobility measurements.
3. We are using the P2X1 receptor knockout mouse to study the role of these receptors in the nervous system.

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).

Techniques

Research Group and Funding

Present Group Members

Dr Rebecca Allsopp
Louise  Farmer
Mrs Manijeh Maleki-Dizaji
Dr Jon Roberts

Alistair Fryatt

Current Funding

The Wellcome Trust (five-year programme grant started August 2007):
'P2X1 receptors for ATP;  function, regulation and molecular properties' - £1,108,021

The British Heart Foundation (3 year project grant applications Prof Mahaut-Smith, Prof R J Evans and Prof A Goodall, started September 2008).  'Interactions between P2 receptor signals in the platelet' - £130,372

Selected Publications

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.