Characterisation of P2X1 receptor function, regulation and development of molecular models of drug action at ATP gated P2X receptors (360G-Wellcome-080487_Z_06_Z)

ATP gated P2X1 receptor channels play important roles in the cardiovascular system contributing to the control of arterial tone as well as blood clotting and may be novel targets for the development of anti-thrombotic drugs. The aim of this proposal is to study the neuronal role, regulation and molecular properties of P2X1 receptors. (i) To address the role of P2X1 receptors in the nervous system we will generate a fluorescent P2X1-EGFP knock-in reporter mouse to identify and characterise the receptor in live cells. (ii) P2X1 receptors are expressed in lipid rafts, phosphorylated and sensitive to the intracellular environment. We will determine the extent and mechanisms of P2X1 receptor regulation by other receptors, the contribution of the intracellular domains to channel function and identify the role of interactingproteins in modulating receptor properties. (iii) We have used a mutagenesis approach to identify conserved residues in the extracellular domain that are involved in ATP action, however the pharmacological differences between subunits suggest that variant amino acids are also important in drug binding. We aim to use cysteine scanning mutagensis and analysis of the site of binding of cross-linked ATP analogues to develop molecular models of agonist and antagonist action at P2X receptors.