Role of G338 of the Second Transmembrane Domain in Gating of the Human P2X7 Receptor

Abstract

The human P2X7 receptor (hP2X7R) is an ATP-gated nonspecific cation channel. It is expressed in cells of the immune system. Via 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 by long lasting agonist applications, i.e. the gating and permeation mechanisms of P2X7 receptors are not fully understood.G338 within the second transmembrane domain (TM2) is part of the narrow ion channel pore domain. Substitution of G338 to cysteine reversed the gating behaviour of the hP2X7R channel, i.e. hP2X7G338C expressing Xenopus oocytes displayed a leak conductance which is decreased by application of ATP or by the P2X7R agonist, 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 was carried by cations.Modeling the structural changes of hP2X7G338C during ATP application with the recently published truncated zebrafish P2X4 receptor model as template we found that G338 is located at the narrowest part of the closed ion channel pore and substitution by cysteine pushes the TM2 of the three subunits apart leading to an incomplete channel closure. ATP application leads to a conformation switch of hP2X7G338C which allows an interaction of C338 with Y40 and Y343 decreasing the ion channel pore diameter and leading to a reduced ion current.These findings point to a critical role of G338 in gating the P2X7 receptor.This work was supported by the German research foundation (DFG, Ma 1581/15-1, Schm 536/9-1