The human P2X7-receptor is an ATP-dependent cation channel expressed in immune cellslike macrophages and B-cells. It has a large extracellular domain, two transmembrane domains and intracellularly located N- and C-termini. The second transmembrane domain (TM2) has been shown to be involved in ion permeation and pore opening of P2X receptor channels. We tested this assumption for the P2X7 receptor subtype by single cysteine scanning mutagenesis of TM2 residues near the putative intracellular vestibule of P2X receptors, which is involved in formation of the P2X2 receptor channel pore.Xenopus laevisoocytes were injected with cRNAs for the various his-hP2X7 constructs and ATP-dependent currents were measured using the two microelectrode voltage clamp technique.

P2X7 receptor mutants with cysteine substitutions between amino acids F328 and Y343 were found to be sensitive to the extracellularly applied sulfhydryl group modifying reagent MTSEA.

Amino acids C-terminal from Y343 were unresponsive to application of MTSEA or Ag⁺ and therefore probably not involved in the formation of the ion channel pore of the human P2X7 receptor.

Applying MTSEA to the mutant hP2X7V335C increased the ATP-induced current. Single channel measurements showed that this was due to the ion channel's higher opening probability. However, the conductance of the ion channel pore is not influenced. Thus, V335 is suspected to participate in gating but not in the permeation characteristics of the ion channel pore.

Contrarily, applying MTSEA to hP2X7S342C resulted in blocking the ion channel pore. In both cases the MTSEA reaction for hP2X7V335C and hP2X7S342C was membrane voltage dependent, so the altered amino acids are localized within the electrical field of the cell membrane.