Ion Access Pathway to the Transmembrane Pore in P2X Receptors

P2X receptors are ATP-gated channels that mediate cation influx to evoke action potentials or trigger intracellular signaling, important in sensory signal transmission and inflammatory responses. However, the molecular mechanism of how cations penetrate the extracellular region of the receptor and access the transmembrane pore is unknown. Examination of the closed state crystal structure predicts two potential pathways: i) three fenestrations located above the membrane (lateral pathways) and ii) the central void along the molecular three-fold axis of symmetry (central pathway). By solving the Poisson-Boltzmann equation, we analyzed the electrostatics of cations along these pathways in the closed state, and found that relatively small widening (∼1 A) seemed sufficient to allow cations to penetrate the lateral pathways, while even a large displacement (> 5 A) along the central pathway remained unfavorable for cations to go through. To probe the accessibility in the open state, we systematically introduced cysteine residues into the rat P2X2 receptor along these potential access pathways and measured reaction rates with Cys-reactive reagents using patch-clamp recording techniques. Among 47 mutants examined, only I328C along the lateral pathway was modified by MTSET and MTS-TPAE, suggesting that cations do not utilize the central pathway. To further investigate the motion during activation, we cross-linked neighboring subunits by introducing disulfide-bridges at 5 different positions along the central pathway. While the disulfide-bridges at the three middle positions did not affect currents, the top pair exhibited reduction and the bottom pair showed potentiation when treated with reducing agents. These indicate that the central pathway seems unlikely to become wider, except the bottom part that is directly connected to the lateral pathways. Altogether, these results suggest that the lateral pathways seem to “breathe” during activation, allowing extracellular cations to penetrate and access the transmembrane pore.