We have isolated a 1785-bp complementary DNA (cDNA) encoding the murine P2X U receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X U subunits, respectively. Functional properties of the heterologously expressed murine P2X U homomeric receptor broadly resembled those of the P2X U receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X U receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.z 1998 Federation of European Biochemical Societies.
1 Apparent species di erences in the responses of recombinant P2X 7 receptors to repeated application of 2'-and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) have been investigated. 2 Repeated application of 100 mM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X 7 receptors. 3 Current growth was thought to re¯ect progressive dilation of the P2X 7 ion-channel to a pore permeable to large molecules (MW5900), suggesting that channel dilation was not occurring at the rat P2X 7 receptor. However, 100 mM BzATP produced a rapid in¯ux of YO-PRO-1 (MW375) in cells expressing rat or human P2X 7 receptors. 4 There were, however, species di erences in agonist potency such that 100 mM BzATP was a supra-maximal concentration at rat, but not human or mouse, P2X 7 receptors. Importantly, when sub-maximal concentrations of BzATP or ATP were examined, current growth occurred at rat P2X 7 receptors. 5 The rate of current growth and YO-PRO-1 accumulation increased with agonist concentration and appeared more rapid at rat and human, than at mouse P2X 7 receptors. 6 The potency of BzATP and ATP was 1.5 ± 10 fold lower in naõÈ ve cells than in cells repeatedly exposed to ATP. 7 This study demonstrates that current growth occurs at mouse, rat and human P2X 7 receptors but only when using sub-maximal concentrations of agonist. Previously, current growth was thought to re¯ect the progressive increase in pore diameter of the P2X 7 receptor ion channel, however, the results of this study suggest a progressive increase in agonist potency may also contribute.
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 P2X 7 but not P2X 1 -P2X 6 subunits. 3 DbATP (EC 50 *100 mM) 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 45 calcium ( 45 Ca 2+ ) and the DNA binding dye, YO-PRO-1, in CHO-K1 cells. Both responses were inhibited by NaCl and MgCl 2 . In 280 mM sucrose bu er, 45 Ca 2+ accumulation was measurable within 10 ± 20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and abmethylene ATP were inactive at concentrations up to 100 mM. 5 DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this e ect was only pronounced after 60 ± 90 min. 6 These data suggest that CHO-K1 cells express an endogenous P2X 7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45 Ca 2+ . 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 P2X 7 receptor should be considered when these cells are used to study recombinant P2X receptors.
In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist. BzATP was more potent than ATP at the three species homologues and exhibited highest potency for rat P2X7 receptors suggesting that channel closure time was related to agonist potency. Furthermore, BzATP potency for the P2X7 receptor could be modified by changing extracellular ionic concentrations or by mutating the receptor and modifications which increased agonist potency also increased the time taken for channel closure. The dependence of channel closure time on agonist potency suggests it reflects agonist dissociation from the P2X7 receptor rather being an intrinsic property of the ion-channel. Consistent with this, our previous studies have shown that agonist potency increases after repeated agonist applications and in this study channel closure time at rat P2X7 receptors increased after repeated agonist applications. Overall these results suggest that the species differences in channel closure times reflect differences in agonist dissociation rates which arise as a consequence of the marked species differences in agonist potency.
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