Regulated expression of the rat recombinant P2X3 receptor in stably transfected CHO-K1 tTA cells

Lachnit, Wilhelm; Oglesby, Ian B; Gever, Joel R.; Gever, Marina; Huang, Chiao-Chain; Li, Xing Cheng; Hong, Jin Pyo; McGivern, Joseph G.; Ford, Anthony

doi:10.1016/s0165-1838(00)00120-x

Cited by 10 publications

(6 citation statements)

References 24 publications

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“…(Thomas et al 1998) or agonists, such as ATP, abmeATP and BzATP (Bianchi et al 1999;Zemkova et al 2004). The EC 50 of ab-meATP was in the same range as previously reported for ectopically expressed P2X 3 receptors (e.g., Bianchi et al 1999;Lachnit et al 2000;Fabbretti et al 2004). Two to three agonist binding sites were suggested for P2X receptors (Bean 1990).…”

Section: Discussionsupporting

confidence: 82%

Functional characterization of P2X₃receptors fused with fluorescent proteins

Grote

Boldogköi

Zimmer

et al. 2005

Molecular Membrane Biology

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P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X3 receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6 pS) and the fast activation kinetics of the receptor (20 ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X3 constructs carrying a fluorescent tag (ECFP or DsRed2) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X3 receptors in CNS signaling.

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Section: Discussionsupporting

confidence: 82%

Functional characterization of P2X₃receptors fused with fluorescent proteins

Grote

Boldogköi

Zimmer

et al. 2005

Molecular Membrane Biology

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“…However, affinity estimates yield values that are 100-fold to 1000-fold lower than estimates based on equilibrium binding assays (Michel et al, 1996;Lachnit et al, 2000;North and Surprenant, 2000). Our results provide a possible explanation for this inconsistency and suggest that agonist binds to at least two classes of binding sites: (1) a low-affinity (micromolar Figure 6.…”

Section: Discussionmentioning

confidence: 69%

Use-Dependent Inhibition of P2X₃Receptors by Nanomolar Agonist

Pratt¹,

Brink²,

Bergson³

et al. 2005

J. Neurosci.

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P2X 3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (Ͻ1 nM) of desensitized P2X 3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X 3 current. Sustained applications of 0.5 nM ATP inhibited Ͼ50% of current to repetitive applications of P2X 3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X 3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X 3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [ 32 P]ATP from desensitized P2X 3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC 50 for inhibition and increased the rate of recovery.

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“…Chinese hamster ovary (CHO‐K1) cells stably expressing recombinant human P2X4, rat P2X5 or human P2X7 were cultured in Ham's F12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 250 µg·mL −1 G418 (Invitrogen) and 100 µg·mL −1 hygromycin B. Expression of recombinant human P2X1 and rat P2X3 in CHOK‐K1 cells was regulated through the use of a tetracycline‐controlled transactivator (tTA) gene expression system, such that cells grown in the presence of 0.1 µg·mL −1 tetracycline did not express P2X1 or P2X3 protein subunits, but upon removal of tetracycline from the growth medium, abundant expression of P2X1 or P2X3 was achieved within 7 days (Lachnit et al ., 2000). 1321N1 Human astrocytoma cells expressing hP2X2, hP2X3 and hP2X2/3 were cultured in a base medium of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and either 275 µg·mL −1 G418 (hP2X2), 0.25 µg·mL −1 puromycin (hP2X3), or 300 µg·mL −1 G418 and 0.25 µg·mL −1 puromycin (hP2X2/3).…”

Section: Methodsmentioning

confidence: 99%

AF‐353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

Gever

Soto

Henningsen

et al. 2010

British J Pharmacology

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114

109

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Background and purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. Experimental approach: The antagonistic potencies (pIC50) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC50 estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t1/2 = 1.63 h) and plasma-free fraction (98.2% protein bound). Conclusions and implications:The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.

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Regulated expression of the rat recombinant P2X3 receptor in stably transfected CHO-K1 tTA cells

Cited by 10 publications

References 24 publications

Functional characterization of P2X₃receptors fused with fluorescent proteins

Functional characterization of P2X₃receptors fused with fluorescent proteins

Use-Dependent Inhibition of P2X₃Receptors by Nanomolar Agonist

AF‐353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

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Regulated expression of the rat recombinant P2X3 receptor in stably transfected CHO-K1 tTA cells

Cited by 10 publications

References 24 publications

Functional characterization of P2X3receptors fused with fluorescent proteins

Functional characterization of P2X3receptors fused with fluorescent proteins

Use-Dependent Inhibition of P2X3Receptors by Nanomolar Agonist

AF‐353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

Contact Info

Product

Resources

About

Functional characterization of P2X₃receptors fused with fluorescent proteins

Functional characterization of P2X₃receptors fused with fluorescent proteins

Use-Dependent Inhibition of P2X₃Receptors by Nanomolar Agonist