Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO‐K1 cells

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Abstract
CHO‐K1 cells were examined for their cellular responses to the P2 receptor agonist, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase‐polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1‐P2X6 subunits. DbATP (EC50∼100 μm) evoked non‐desensitizing inward currents which reversed at ∼0mV, suggesting activation of a non‐selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO‐PRO‐1, in CHO‐K1 cells. Both responses were inhibited by NaCl and MgCl2. In 280 mm sucrose buffer, 45Ca2+ accumulation was measurable within 10–20 s of agonist addition, whereas YO‐PRO‐1 accumulation was only detectable after 8 min. ATP and ATPγS were also agonists but were less potent than DbATP, while UTP, 2‐methylthio ATP, ADP and αβmethylene ATP were inactive at concentrations up to 100 μm. DbATP increased lactate dehydrogenase release from CHO‐K1 cells, suggesting cell lysis, although this effect was only pronounced after 60–90 min. These data suggest that CHO‐K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO‐PRO‐1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors. British Journal of Pharmacology (1998) 125, 1194–1201; doi:10.1038/sj.bjp.0702205