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 similar to 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 similar to 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.