Intermediate closed channel state(s) precede(s) activation in the ATP-gated P2X2 receptor

The molecular mechanism underlying channel opening in response to agonist binding remains a challenging issue in neuroscience. In this regard, many efforts have been recently undertaken in ATP-gated P2X receptors. Among those efforts, we have provided evidence in the P2X2 receptor that tightening of ATP sites upon agonist binding induces opening of the ion channel. Here we extend our analysis to show that the sulfhydryl-reactive ATP analog 8-thiocyano-ATP (NCS-ATP), a potent P2X2 agonist, when covalently labeled in the ATP-binding site at position Leu186 likely favors the tightening mechanism, but not the channel opening mechanism. Our data predict the existence of intermediate or preactivation state(s) trapped by NCS-ATP, in which tightening of the binding site is favored while the channel is still closed. We propose that this (these) intermediate ATP-bound state(s) prime(s) channel gating in the P2X2 receptor.