Amino acid residues constituting the agonist binding site of the human P2X3 receptor

Abstract

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.

FIGURE 1.

Current responses to α,β-meATP of the WT hP2X3 receptor and its NBS mutants in HEK293 cells. A, AA residues of the supposed nucleotide binding segments (NBS1–4). Bold characters, conserved AAs; underlined characters, substitution by Ala. B, panel a, whole-cell current responses, induced by α,β-meATP (0.03–30 μm), were recorded with the patch clamp technique at a holding potential of −65 mV. Increasing concentrations of the agonist were locally superfused for 2 s with 5- or 7-min intervals as indicated. Panel b, increases in the intracellular calcium concentration ([Ca²⁺]_i) were induced by α,β-meATP (0.3–300 μm). Increasing concentrations of the agonist were locally superfused for 5 s with 15-min intervals. The cells were labeled with the calcium-sensitive fluorescent dye Fura-2, and fluorescence ratio (FR_340/380) measurements were made with a dual wavelength spectrometer (alternating excitation at 340 and 380 nm). The changes in FR were used as a measure of [Ca²⁺]_i. Representative tracings are shown in both panels a and b. C, panel a, α,β-meATP (30 μm)-induced charge transfer measured by the patch clamp technique and fluorescence ratio determined by Ca²⁺ imaging as a function of time. The time courses of these responses were similar. Two representative experiments are shown out of a total of three (current response) and four ([Ca²⁺]_i transients) similar ones (2-s application time). pC, picocoulombs. Panel b, the current response to α,β-meATP (30 μm) and the differentiated F_340/380/dt also have similar time courses. The recordings from panel a were replotted in panel b.

FIGURE 2.

Current responses to α,β-meATP in HEK293 cells transfected with the WT hP2X3 receptor and its mutants. Whole-cell patch clamp recordings were made as described in the legend for Fig. 1. Concentration-response curves for α,β-meATP were constructed both for the WT hP2X3 receptor and for its point mutants, where the indicated AAs in their nucleotide binding segments were replaced by Ala (in Fig. 1A, see underlined one-letter coding of AAs). Ala was sequentially introduced to substitute individual AAs in NBS1 (a), NBS2 (b), NBS3 (c), and NBS4 (d). Each symbol indicates the mean ± S.E. of 5–13 cells.

FIGURE 3.

Increases of intracellular calcium by α,β-meATP in HEK293 cells transfected with the WT hP2X3 receptor and its mutants. FR measurements were made as described in the legend for Fig. 1 and were taken as a measure of [Ca²⁺]_i. Concentration-response curves for α,β-meATP were constructed both for the WT hP2X3 receptor and for its point mutants, where the indicated AAs in their nucleotide binding segments were replaced by Ala (in Fig. 1A, see underlined one-letter coding of AAs). Ala was sequentially introduced to substitute individual AAs in NBS1 (a), NBS2 (b), NBS3 (c), and NBS4 (d). Each symbol indicates the mean ± S.E. of 9–20 cells.

FIGURE 4.

Summary of α,β-meATP concentration-response curves for the hP2X3 receptor and its NBS mutants in HEK293 cells. Curves presented in Figs. 2 and 3, and supplemental Fig. 1 were fitted as described under “Experimental Procedures” to obtain the EC₅₀ and E_max (I_max or ΔFR_max) values. On the left-side graph, the EC₅₀ values of the indicated mutants are expressed in μm. On the right-side graph, the E_max values of the same mutants are expressed in pA and ΔFR, respectively. Current (a) and FR (b) measurements are indicated for each single and double mutant. Each symbol indicates mean ± S.E. of 4–13 (a) and 9–20 (b) cells. With several mutants, no clear maximum of the concentration-response curve was reached, and therefore neither the EC₅₀ nor the E_max values could be reliably determined. In such cases, the EC₅₀ values were not indicated, and the E_max values were replaced with the effect of the highest agonist concentration tested (300 μm; designated by a thick line). *, p < 0.05; statistically significant difference from the respective value measured with the WT receptor.

FIGURE 5.

Original recordings of α,β-meATP-induced currents at hP2X3 receptors or its NBS mutants. The expression systems of the HEK293 cell (a, left panel; experimental conditions were as described in the legend for Fig. 1) or the Xenopus leavis oocyte (b, right panel; holding potential, −60 mV; agonist application was for 10 s every 1 min) were used. a, α,β-meATP (100 μm) effects are shown at those mutants only, where either the amplitude or the time course of the current responses appeared to be modified in comparison with the WT receptor. b, α,β-meATP (100 μm) effects are shown at nine selected mutants only, where the amplitude and/or the time course of current responses appeared to be modified in comparison with the WT receptor. These mutants were tested for assembly and surface expression in the oocyte system in the following experiments (Fig. 6B). Representative recordings for 5–14 experiments are shown.

FIGURE 6.

Sensitivity to α,β-meATP and assembly and surface expression of hP2X3 mutants in X. laevis oocytes. A, typical two-electrode voltage clamp current traces recorded from oocytes expressing the hP2X₃ receptor or its selected NBS mutants as indicated. α,β-meATP (100 μm) caused smaller current amplitudes at all nine mutants tested than at the WT receptor. Mutants K63A, K276A, and K299A were unable to mediate any current response. Each symbol indicates mean ± S.E. of 5–6 oocytes. *, p < 0.05; statistically significant difference from the WT receptor. B, [³⁵S]methionine-labeled oocytes were chased for 24 h and surface-labeled with the membrane-impermeant fluorescent Cy5 dye prior to protein purification by non-denaturing nickel-nitrilotriacetic acid chromatography. B, panel a, oligomeric state of the P2X3 proteins as visualized by blue native PAGE and ³⁵S phosphorimaging. The ovals schematically illustrate the migration positions of the non-denatured trimeric state and dimeric and monomeric states produced by partial or complete denaturation with SDS treatment, respectively. Panel b, aliquots of the same samples shown in panel a were denatured with reducing SDS-PAGE sample buffer, resolved by SDS-urea-PAGE, and visualized in their Cy5-labeled surface form by Typhoon fluorescence scanning. Each mutant was analyzed at least twice with identical results.

FIGURE 7.

Model of the hP2X3 receptor. A, panel a, extracellular loop of the hP2X3 trimer. The individual subunits are labeled by different colors. Panel b, detailed view of the supposed binding site at the interface of two neighboring subunits containing four binding segments (NBSs). Color coding of the amino acid residues is: blue, NBS1; red, NBS2; yellow, NBS3; green, NBS4. B, NBS1–2 and NBS3–4 are located at opposite sites of a single subunit.