Function of the second Transmembrane Domain of the Human P2X7 Receptor

Abstract

P2X7 receptors are ATP-activated cation channels involved in inflammatory and immunological reactions to cell injury. Their second transmembrane domain (TM2) is believed to line the ion channel pore. Here, we applied cysteine scanning to investigate the role of TM2 residues in the permeation and gating of the human P2X7 receptor (hP2X7R). Following heterologous expression of the hP2X7R cysteine substitution mutants in Xenopus oocytes, their function was measured by voltage-clamp in the whole-cell mode and by single-channel voltage-clamp in the outside-out configuration before and after application of methanethiosulfonate (MTS) reagents, which can covalently react with the substituted cysteines. Furthermore, we checked if the hP2X7R cysteine substitution mutants expressed in Xenopus oocytes could be labeled by extracellularly applied sulfo-Cy5-maleimide. At the putative extracellular end of the hP2X7R TM2 domain, we found cysteine substitution mutants which could be labeled by the fluorescent Cy5 dye, but the ATP-induced currents were not affected by MTS administration. This indicates that the corresponding amino acid residues are extracellularly accessible, but are neither involved in the gating nor in the permeation of the ion channel. One hP2X7R mutant (S342C) could be Cy5-labeled and the ATP-induced current was blocked by the cationic 2-aminoethyl methanethiosulfonate (MTSEA) pointing to an involvement of S342 in ion permeation or to a stabilization of the closed state of the channel. Several other cysteine mutants were dye labeled and exhibited increased ATP-induced currents after MTSEA application. This MTSEA-dependent current increase was due to an increase of the channel's open probability indicating that the corresponding amino acid residues are involved in in P2X7R channel gating. The structure-function relationship of the TM2 domain of the hP2X7R is discussed using a hP2X7R homology model based on the published X-ray structures of the zebrafish-P2X4 receptor.

Localization of the gate and selectivity filter of the full-length P2X7 receptor

“…Our combined data define the ion channel pore and gating region of P2X7R and reveal ATP 4− triggered distinct widening of the pre-TM2 region and the trihelical TM2 channel that was completely stable over time. Partial results of this work were presented in a poster at the 58th Annual Meeting of the Biophysical Society in San Francisco (28). − Maleimide.…”