P2X7 receptors are ligand-gated ion channels activated by extracellular ATP. Upon activation, they serve as cytolytic and apoptotic receptors but also have control in cell growth and proliferation. Neither how the same receptor can play such different roles nor the pore formation phenomena are presently understood. The occupancy of binding sites could play a role in directing the receptor action towards different functions. To shed more light on binding, gating and the function of individual subunits, the study was performed on receptors with a defined number of binding sites. A concatemer strategy was used to form functional trimers of P2X7 channels. Then the point mutation K64A in the binding site was introduced in defined subunits of the concatemers. The constructs were heterologously expressed in the HEK293 cell line or in Xenopus laevis oocytes to determine the better expression system and the channels were electrophysiologically characterized with the patch-clamp technique in the whole-cell or outside-out configuration and the two-electrode voltage-clamp technique. The obtained electrophysiological data were analysed for the changes in functional expression, apparent ligand affinity, and activation and deactivation time course for each receptor. The results showed that the concatemer strategy can be used for investigation of P2X7 receptors, with outside-out patches of Xenopus oocytes as the most suitable configuration for the aims of this study. In the outside-out patches of the K64A mutant, facilitation was observed, a phenomenon still not described in this configuration. Study of the concatemers with the decreased numbers of binding sites showed that even a single ATP binding was sufficient for receptor activation, while three bound ligands were necessary for fully stabilising the open state. Together, the results suggest that each binding site contributes to the P2X7 activation gating; thus, P2X7 receptors can be described with stepwise activation.