Immune cells express P2X7 receptors (P2X7R) which are ATP-gated ion channels permeable to small inorganic cations. P2X7Rs are homotrimers withintracellularly located N- and C-termini, two transmembrane domains and a large extracellular domain containing with the ATP-binding site. The second transmembrane domains (TM2) were shown to be involved in permeation and gating of P2X2 and P2X4 receptor subtype channels.

Therefore, we tested the modifiability and accessibility by sulfhydryl group modifying reagents of cysteine mutated amino acids in the TM2. For this, Xenopus laevis oocytes were injected with cRNAs for the respective human P2X7R constructs and ATP-dependent currents were measured using the two microelectrode voltage clamp technique.

SeveralP2X7R mutants with cysteine substitutions in the TM2 between amino acids I331 and Y343 were found to increase the ATP-dependent current after MTSEA application. The voltage dependence of the rate of the stimulating effect of the cationic MTSEA was increased the more cytosolic the amino acids are located indicating the different position within the electric field of the membrane. Similar effects were observed at administration of the larger MTSET except for Y343C indicating that the selectivity filter is situated above (extracellular) and close to Y343. Furthermore, in contrast to the more extracellular situated amino acids, modification of S342C and Y343C does not take places without ATP-induced opening of the P2X7R ion channel pore pointing to a position of the ion channel gate just above S342.

We thank the German Research foundation (Deutsche Forschungsgemeinschaft, DFG) for financial support (grants Ma1581/15-1 and Schm536/9-1).