Regulation of the P2X7 receptor permeability to large molecules by extracellular Cl^- and Na⁺

Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG⁺). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl^- (Cl^-o) regulates the outward current, whereas extracellular Na ⁺ (Na⁺_o) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R^-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG⁺ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl^-_o reduced the inward Na⁺ current by ∼35% and the outward Na⁺ current by only 10%. By contrast, in the absence of Na⁺_i and the presence of Na ⁺_o or NMDG⁺_o, the removal of Cl ^-_o reduced the inward Na⁺ or NMDG⁺ currents by 35% but the outward NMDG⁺ current by >95%. The effect of Cl^-_o was half-maximal at ∼60 mM. Reducing Cl ^-_i from 150 to 10 mM did not reproduce the effects of Cl^-_o. All currents were eliminated in P2X7R^-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl^-_o primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG⁺ were stimulated in the presence of Na⁺_o, subsequent removal of Na⁺_o resulted in a strongly outward rectifying NMDG ⁺ current, indicating maintained high selectivity for Na⁺ over NMDG⁺. During continuous incubation in Na⁺-free medium, the permeability of the P2X7Rs to NMDG⁺ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG⁺ and only then stimulated with ATP, the NMDG⁺ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na⁺_o memory" and that Na⁺_o regulates the inward permeability of P2X7Rs to large molecules. The novel regulation of P2X7R outward and inward permeability to large molecules by Cl^-_o and Na⁺_o, respectively, may have an important protective function, particularly in secretory epithelial cells.