Anales RANF

S8-04 STRUCTURE-FUNCTION RELATIONSHIP OF P2X7 RECEPTORS Nancy Zipfel 1 , Anja Pippel 1 , Michaela Stolz 2 , Achim Kless 3 , Günther Schmalzing 2 and Fritz Markwardt 1 1 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale, Germany; 2 Department of Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany, 3 Grünenthal GmbH, Global Drug Discovery, Departments of Molecular Pharmacology and Discovery Informatics, Aachen, Germany. The human P2X7 receptor (hP2X7R) is an ATP 4- -gated nonspecific cation channel that is expressed in cells of the immune system. By activating this receptor, ATP, released during cell damage or hypoxia, serves as danger-associated molecular pattern to induce inflammatory reactions. In comparison to other P2X receptors, P2X7R-dependent ionic currents differ in their activation kinetics by an absence of desensitization and induction of large membrane pores upon long lasting agonist applications. Despite significant advances, the gating and permeation mechanisms of the P2X7R is not fully understood. We have found in a cysteine scanning mutagenesis study that Gly338 within the second transmembrane helix (TM2) is part of the narrowest channel region, which extends from Tyr336 to Gly345. The gate and selectivity filter of hP2X7R are colocalized and primarily determined by Ser342. Here we report that substitution of Gly338 to cysteine generates a constitutively open channel that closes in response to extracellular application of ATP or the typical P2X7R agonist benzoyl ATP (BzATP). Substitution of extracellular Na + by the larger organic Tris + significantly decreased the ion conductance and shifted the reversal potential of the ATP-induced current to more negative potentials indicating that the leak current is carried by cations. Homology modeling of hP2X7R G338C during ATP application with the truncated zebrafish P2X4R X ray structure as template suggests that G338 is located near the narrowest part of the closed ion channel pore and substitution by cysteine pushes the TM2 of the three subunits apart, thus preventing a complete channel closure in the apo state. ATP application induces a conformational switch of hP2X7R G338C , allowing Cys338 to interact with Tyr40 and Tyr343 in TM1 and TM2, respectively. This decreases the pore diameter of the trihelical TM2 bundle forming the ion channel of the hP2X7R G338C , thus reducing the ion current. These findings point to a critical role of Gly338 in gating the P2X7R. Acknowledgements We thank the Deutsche Forschungsgemeinschaft (Ma1581/15-3; Schm536/9-3) for their financial support.