Single channel properties of P2X2 purinoceptors

The single channel properties of cloned P2X(2), purinoceptors expressed in human embryonic kidney (HEK;) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current-voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of similar to 30 pS at -100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore TMS similar to 150 mM I at 0 mV and voltage dependent. The binding site appeared to be similar to 0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity : K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (P-o) to a maximum of 0.6 with an EC50 of 11.2 mu M and a Hill coefficient of 2.3. Lowe ring extracellular pH enhanced the apparent affinity of the channel for ATP with a pK(a) of similar to 7.9 but did not cause a proton block; of the open channel. High pH slow ed the rise time to steps of ATP without affecting the fall rime. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states: (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates in creased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted P-o vs. ATP concentration closely matches th single channel curl-ent dose-response curve.