Activation of the P2X7 Receptor by Functionally Different ATP Activation Sites

The homotrimeric P2X7 receptor (P2X7R) contains three ATP4- binding sites in its ectodomain. Here, we investigated the role of the individual ATP4- activation sites of the rat P2X7R (rP2X7R) using trimeric rP2X7R concatamers consisting either of three wild-type subunits (7-7-7) or concatamers with up to three subunits having knocked-out ATP binding sites (7ko-7ko-7ko). Following expression in Xenopus oocytes, ATP4--elicited ion currents were recorded using the two-microelectrode voltage clamp technique. The 7-7-7 concatamer exhibited a biphasic ATP4- concentration dependence, best fit by the sum of two Hill functions, confirming the existence of functionally distinct ATP4- 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 a fast and a slow exponential decay. Only one Hill function was required to best fit the ATP4- concentration dependence of concatamers with only two or one ATP4- binding sites, and their deactivation time courses largely lacked the slowly deactivating components. We conclude that binding of one ATP4- is sufficient for partial activation of the rP2X7R and that allosteric effects occur when all three ATP4- binding sites are occupied, leading to distinct functional activation sites.