Abstract

Purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels, and this response can be altered by acute hypoxia. This study examined the effect of acute hypoxia on cloned homo- and heteromeric P2X₂ and P2X₃ receptors expressed in human embryonic kidney 293 cells. In cells expressing homomeric P2X₂ receptors, perfusion of 5 μM ATP (EC₂₅) induced an inward whole-cell current that showed little desensitization during repeated exposures under continuously normoxic conditions. Exposure to a hypoxic ATP solution (pO₂, 25-40 mm Hg) significantly reduced the whole-cell current to 49% of normoxic control. This hypoxic inhibition of P2X₂-mediated inward current was maintained across all potentials when a voltage-step protocol was applied. In contrast, currents mediated by homomeric P2X₃ receptors or heteromeric P2X_2/3 receptors were insensitive to an acute hypoxic challenge. One mechanism whereby hypoxia may modulate P2X₂ channels is via the production of reactive oxygen species (ROS). H₂O₂ (1.8 mM) reversibly reduced homomeric P2X₂ whole-cell currents to 38% of control. Furthermore, H₂O₂ attenuated the effect of hypoxia on homomeric P2X₂ whole-cell currents. Inhibitors of the mitochondrial electron transport chain that reduce (rotenone and myxothiazol) or increase (antimycin A) the production of ROS altered the magnitude of P2X₂-mediated currents. In summary, this is the first report indicating that acute hypoxia is able to regulate the activity of any ligand-gated ion channel. Furthermore, our data show that acute hypoxia selectively modulates the P2X₂ receptor and that the response of P2X₂ receptor subunits to hypoxia is mediated through the mitochondrial production of ROS.