Activation of the Rat P2X7 Receptor by Functionally Different ATP Activation Sites.

The homotrimeric P2X7 receptor (P2X7R) contains three ATP binding sites in its ectodomain. Here, we investigated the role of individual ATP activation sites in rat P2X7R (rP2X7R) using trimeric concatemers consisting of either three wild-type subunits (7-7-7) or one to three subunits with ATP binding sites knocked out by the K64A mutation. Following expression in oocytes, ATP-elicited ion currents were recorded using the two-microelectrode voltage clamp technique. The 7-7-7 concatamer exhibited a biphasic ATP concentration dependence, best fit by the sum of two Hill functions, confirming the existence of functionally distinct ATP activation sites. The activation time course of the 7-7-7 was best approximated by the sum of a fast and a slow exponential saturating activation component. Similarly, deactivation exhibited both fast and slow exponential decay. Only one Hill function was required to best fit the ATP concentration dependence of concatamers with only two or one ATP binding sites, and their deactivation time courses largely lacked the slowly deactivating components. We conclude that the binding of one ATP is sufficient for partial activation of the rP2X7R and that allosteric effects occur when all three ATP binding sites are occupied, leading to distinct functional activation sites.