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Br J Pharmacol
2012 Jan 01;1652:390-400. doi: 10.1111/j.1476-5381.2011.01534.x.
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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
,
Ray D
,
Allsopp RC
,
Evans RJ
.
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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.
Figure 1. Contribution of the cysteine-rich head region to P2X1 receptor function and antagonist action. (A) Amino acid line-up for human P2X1, P2X2 and the Dictyostelium P2X receptor for the region between cysteine residues 1–6 that are conserved in most P2X receptors. Deletions from the P2X1 receptor equivalent to that missing in the Dictyostelium receptor (P2XA, DDB0168616) are shown (P2X1 receptor sequence-linking cysteines 1–6, Del and with the Dictyostelium sequence as a linker, DelD). (B) Homology model of the P2X1 receptor based on the zebrafish P2X4 receptor structure. The three subunits are shown in black, grey and pink. The cysteine-rich head region of the black subunit is shown in green. Residues that are predicted to form the ATP-binding pocket are shown in red. (C) ATP (100 µM, 3 s application indicated by bar) evoked rapidly desensitizing currents at P2X1 WT receptors but was ineffective at the P2X1 Del mutant. (D) P2X1 WT, Del and DelD mutant receptors are expressed at equivalent levels. Upper panel shows levels of sulphoNHS-LC biotin labelled P2X1 receptors and the lower panel shows total P2X1 receptor levels (representative of three experiments). (E) Effects of NF449 (10 nM) on current evoked by an EC90 concentration of ATP (3 s application indicated by bar) at WT P2X1, WT P2X2 and chimeric P2X1 Cys2(1–6) receptors. (F) NF449 concentration-dependent inhibition of ATP (EC90 concentration) evoked currents for WT P2X1, WT P2X2 and chimeric P2X1 Cys2(1–6) receptors (n = 3–6), pIC50 values were 9.07 ± 0.11, 5.86 ± 0.09 and 6.54 ± 0.07 and Hill slopes were 0.98 ± 0.13, 1.61 ± 0.08 and 1.38 ± 0.07 respectively.
Figure 2. NF449 sensitivity at the P2X1 receptor chimeras swapping parts of the cysteine-rich head region. (A) Sequence alignment of the cysteine-rich loop region of human P2X1 and P2X2 receptors showing the crossover points of the subdivided chimeras. (Bi) Cartoon of the pairing of cysteine disulphide bonds in the head region. (Bii) Homology model of the head region showing subdivision with the chimeras; P2X1 Cys2(1–3) chimera (blue), P2X1 Cys2(3–4) chimera (purple) and P2X1 Cys2(4–6) chimera (brown). (C) Inhibition of ATP-evoked responses (EC90 concentration) by NF449 (10 nM) at the chimeras dividing the head region. NF449 was applied 5 min before the application of ATP and then co-applied with ATP. (D) Concentration-dependent inhibition curves of the chimeras by NF449 to an EC90 concentration of ATP. WT P2X1 and P2X2 receptor responses are shown with dotted lines for comparison (n = 3).
Figure 3. NF449 sensitivity at P2X1 receptor mutants. (A) Sequence alignment of the residues 132–149 in the P2X1 and P2X2 receptor (P2X1 numbering). (B) Homology model of the P2X1 receptor zoomed in on the head region showing the location of the proposed ATP-binding pocket in red and the charged residues K136 (blue), K138 (purple), R139M (pink) and K140L (orange). (C) Inhibition of ATP-evoked currents (EC90 concentration of ATP) in the absence (control) and presence of NF449 (∼IC50 concentration). NF449 was applied 5 min before the application of ATP and then co-applied with ATP. (D) NF449 concentration-dependent inhibition curves of the P2X1 receptor mutants (against an EC90 concentration of ATP). WT P2X1 and P2X2 receptor responses are shown with dotted lines for comparison (n = 3).
Figure 4. Suramin sensitivity of P2X receptors, chimeras and mutants. Suramin concentration-dependent inhibition curves against an EC90 concentration of ATP for (A) P2X1 WT, P2X2 WT receptors and the P2X1 Cys2(1–6) chimera (B) The subdivided chimeras and (C) P2X1 receptor mutants. In (B) and (C), WT P2X1 and P2X2 receptor responses are shown with dotted lines for comparison. n = 3–4 for all data.
Figure 5. NF449 and suramin sensitivity at P2X2 receptor chimeras and mutants. (A) Sequence alignment of the human P2X1 and P2X2 receptors from the residues 132 to 149. (B) NF449 sensitivity of the X2-KLKMK(3) and D136K P2X2 receptor mutants. (C) NF449 sensitivity of the P2X2 Cys1(3–4) chimera and X2-KLKRK(4) P2X2 receptor mutant. (D) Suramin sensitivity of the P2X2 Cys1(3–4) chimera and P2X2 receptor mutants. For (B) to (D), the antagonists were tested on an EC90 concentration of ATP. WT P2X1 and P2X2 receptor responses are shown with dotted lines for comparison. n = 3–4 for all data.
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