Identifying the Ion Access Pathway to the Transmembrane Pore in P2X Receptors

P2X receptors are trimeric ion channels activated by extracellular ATP. Upon activation, P2X receptors promote inward current of cations to evoke action potentials or trigger calcium mediated signaling that are important for pain sensing, inflammation, and the synaptic transmission. However, the molecular mechanism of how extracellular ions access the transmembrane pore of P2X receptors is unknown. According to the zebrafish P2X4 crystal structure in the closed state, extracellular ions appear to be readily accessible to the pore through three identical fenestrations located right above the membrane leaflet (lateral pathway). In addition, ions may access the pore through a second possible pathway that runs through the central voids along the molecular three-fold axis of symmetry (central pathway). While this pathway is hypothetical, as the constrictions flanking the central voids are too narrow for hydrated ions to pass (∼2.3 A), agonist binding may expand these constrictions to enable ions to access the transmembrane pore. We have begun to explore the pathway ions use to move through the extracellular domain to enter the pore by inserting cysteine residues into rat P2X2 receptor channels and measuring reaction rates with a range of thiol-reactive reagents and ions. We found that MTSEA-Texas Red (MW=∼750) can access T336 in the transmembrane pore with an apparently fast modification rate when the channel is open. These results suggest a large access pathway exists between the extracellular solution and the transmembrane pore, consistent with the fenestrations observed in the crystal structure. We are currently testing whether reagents of various size may also move through the central pathway.