Regulation of the P2X7 Receptor Permeability to Large Molecules

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 P2X7Rmediated 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. The ionotropic P2X7 receptors (P2X7Rs) function as nonselective cation channels that are activated by high concentrations of ATP and by the relatively specific agonist 3 -O-(4benzoyl)benzoyl ATP (1). P2X7Rs participate in several cell functions. Activation of P2X7Rs in hematopoietic cells leads to cytokine release, cell permeabilization, and apoptosis (2). In bone P2X7Rs have been implicated in the generation of osteoclasts (3), and in epithelial cells they may modulate secretory and growth function (4, 5). In addition, after prolonged activation with ATP, the P2X7Rs become permeable to large molecules with molecular weights as high as 1000 (1). Association between increased permeability to large molecules and induction of apoptosis was reported in several cell types (1). Epithelial cells of secretory glands secrete ATP to both the basal and luminal spaces (6, 7) that can activate the P2 receptors in the respective membranes. Furthermore, neutrophils and epithelial cells (8), including salivary glands (9), synthesize and secrete the antimicrobial cathelicidins. The cathelicidin-derived antimicrobial peptide LL37 appears to be an activator of the P2X7Rs (10). Hence, activation of the P2X7Rs can play an important role in the inflammatory response observed in epithelial autoimmune disorders such as Sjogren’s syndrome (11). Understanding the function and regulation of P2X7Rs in epithelial cells is of intrinsic interest and may also impact the understanding of tissue function in normal and pathological states. Like many epithelial cells (1, 7), parotid acinar and duct cells express P2X7Rs (5, 12, 13). Recently, we immunolocalized the P2X7Rs in the luminal membrane of parotid acinar and duct cells (13). Similar localization was reported in the pancreatic duct (14) and uterine epithelial cells (15), whereas P2X7Rs were found at the basal pole of outer hair cells (16). The P2X7Rs showed remarkable cell-specific behavior in native cells (13). For example, the first activation of P2X7R current by ATP proceeds very slowly in parotid acinar cells, but duct cells from the same gland respond very rapidly to the first application of ATP. The P2X7Rs appear to interact differentially with the cytoskeleton in parotid acinar and duct cells (13). However, the P2X7Rs in both cell types show similar pharmacology, such as similar activation by ATP, sensitivity to external divalent ions, permeability to Na , and inhibition by Brilliant Blue G, Cu , and pyridoxal phosphate-6-azophenyl-2 ,4 -disulfonic acid tetrasodium (13). The cell-specific behavior of the receptors further highlights the need to understand the function and regulation of the P2X7Rs in native tissues. A central function of salivary glands is Ca -stimulated fluid secretion fueled by transcellular ion transport (17, 18). Salivary gland acinar cells secret isotonic NaCl-rich fluid. The duct * This work was supported by National Institutes of Health Grants DE12309 and DK38938 and Cystic Fibrosis Foundation Grant MUALLE01G0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Tel.: 214-648-2593; Fax: 214-645-6054; E-mail: [email protected]. 1 The abbreviations used are: P2X7R, P2X7 receptor; Cl o, extracellular Cl ; Cl i, intracellular Cl ; hP2X7, human P2X7; I/V, current/ voltage; mP2X7, mouse P2X7; Na o, extracellular Na ; Na i, intracellular Na ; NMDG , N-methyl-D-glucamine; NMDGCl, NMDG chloride; TEA, tetraethyl ammonium; TEACl, TEA chloride; WT, wild type. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 29, Issue of July 22, pp. 26922–26927, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.