2 MECHANISMS FOR INWARD RECTIFICATION OF CURRENT FLOW-THROUGH THE PURINOCEPTOR P2X(2) CLASS OF ATP-GATED CHANNELS

1. The ATP receptor subunit P2X(2) was expressed in Xenopus oocytes an d human embryonic kidney (HEK) 293 cells. ATP-activated currents were studied with two-electrode voltage clamp recordings from oocytes, whol e-cell recordings from HEK 293 cells, and outside-out patch clamp reco rdings from both cell types. The steady-state current-voltage (I-V) re lation showed profound inward rectification in all recording configura tions. 2. Recordings from outside-out patches demonstrated that inward rectification does not require intracellular Mg2+ or polyamines, and that inward rectification was present when the same solution was used on both sides of the patch. 3. Voltage jump experiments were performed to evaluate the voltage dependence of channel gating. After fast volt age jumps, instantaneous current jumps were followed by substantial re laxations to the steady state. The time course of the current relaxati ons could be fitted by single exponential functions. The instantaneous I-V relation was less inwardly rectifying than the steady-state I-V r elation; however, it was not linear. 4. Single channel recordings indi cated that the single channel conductance became smaller when the memb rane potential became more positive. This decrease could quantitativel y account for inward rectification of the instantaneous I-V relation. 5. We conclude that inward rectification of P2X(2) is due to two mecha nisms: voltage-dependent gating and voltage dependence of the single c hannel conductance.