CONICET | Buscador de Institutos y Recursos Humanos

To gain insight into the way that P2X2 receptors localized at synapses might function, we explored the properties of outside-out patches containing many of these channels as ATP was very rapidly applied and removed. Using a new method to calibrate the speed of exchange of solution over intact patches, we were able to reliably produce applications of ATP lasting less than 200 microseconds. For all concentrations of ATP, there was a delay of at least 80 µs between the time when ATP arrived at the receptor and the first detectable flow of inward current. This indicates that there is an intermediate closed state between the fully liganded closed state and the open state. In response to 200 µs pulses of ATP, the time constant of the rising phase of the current was approximately 600 microseconds. Thus most channel openings occurred when no free ATP was present. The current deactivated with a time constant of approximately 60 ms. The amplitude of the peak response to a brief pulse of a saturating concentration of ATP was about 70% of that obtained during a long application of the same concentration of ATP. Thus at least 30% of the time ATP leaves a channel without producing an opening. Results can be best described by three schemes: 1) a scheme with five sequential closed states (three binding plus one flipping steps) branching to two open states that converged on a final closed state; 2) a tertiary allosteric scheme with additive couplings between binding, gating and flipping occurring at the channel level, 3) a tertiary allosteric scheme with flipping occurring at each one of three domains. These kinetic properties indicate that responses to ATP at synapses that use homomeric P2X2 receptors would be expected to greatly outlast the duration of the synaptic ATP transient produced by a single presynaptic spike. Like NMDA receptors, P2X2 receptors provide the potential for complex patterns of synaptic integration over a time scale of hundreds of milliseconds.