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Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop.

ABSTRACT: The cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor.Chimeras and point mutations in the cysteine-rich head region were made between human P2X1 and P2X2 receptors. Mutant receptors were expressed in Xenopus oocytes and P2X receptor currents characterized using two-electrode voltage clamp.The chimera replacing the region between the third and fourth conserved cysteine residues of the P2X1 receptor with the corresponding part of P2X2 reduced NF449 sensitivity a thousand fold from an IC(50) of ?1 nM at the P2X1 receptor to that of the P2X2 receptor (IC(50) ?1 µM). A similar decrease in sensitivity resulted from mutation of four positively charged P2X1 receptor residues in this region that are absent from the P2X2 receptor. These chimeras and mutations were also involved in determining sensitivity to the antagonist suramin. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity.These data indicate that a cluster of positively charged residues at the base of the cysteine-rich head region can account for the highly selective antagonism of the P2X1 receptor by the suramin derivative NF449.

SUBMITTER: El-Ajouz S

PROVIDER: S-EPMC3268193 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop.

El-Ajouz S S Ray D D Allsopp R C RC Evans R J RJ

British journal of pharmacology 20120101 2

<h4>Background and purpose</h4>The cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor.<h4>Experimental approach</h4>Chimeras and point mutations in the cysteine-rich head region were made between h ...[more]

PMID: 21671897

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Project description:Activation and desensitization kinetics of the rat P2X1 receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in <0.5 s. Sustained exposure to 1-100 nM ATP led to a profound desensitization of P2X1 receptors. At steady-state, desensitization could be described by the Hill equation with a K1/2 value of 3.2 +/- 0.1 nM. Also, the ATP dependence of peak currents could be described by a Hill equation with an EC50 value of 0.7 microM. Accordingly, ATP dose-effect relationships of activation and desensitization practically do not overlap. Recovery from desensitization could be described by a monoexponential function with the time-constant tau = 11.6 +/-1.0 min. Current transients at 10-100 nM ATP, which elicited 0.1-8.5% of the maximum response, were compatible with a linear three-state model, C-O-D (closed-open-desensitized), with an ATP concentration-dependent activation rate and an ATP concentration-independent (constant) desensitization rate. In the range of 18-300 nM ATP, the total areas under the elicited current transients were equal, suggesting that P2X1 receptor desensitization occurs exclusively via the open conformation. Hence, our results are compatible with a model, according to which P2X1 receptor activation and desensitization follow the same reaction pathway, i.e., without significant C to D transition. We assume that the K1/2 of 3.2 nM for receptor desensitization reflects the nanomolar ATP affinity of the receptor found by others in agonist binding experiments. The high EC50 value of 0.7 microM for receptor activation is a consequence of fast desensitization combined with nonsteady-state conditions during recording of peak currents, which are the basis of the dose-response curve. Our results imply that nanomolar extracellular ATP concentrations can obscure P2X1 receptor responses by driving a significant fraction of the receptor pool into a long-lasting refractory closed state.

Dataset Information

Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop.

Publications

Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop.

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