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

Abstract

1. 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.

2. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X₇ but not P2X₁-P2X₆ subunits.

3. 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.

4. 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.

5. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60–90 min.

6. 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.

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