Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors

Abstract

P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of the P2X receptors is a flexible loop structure, and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which are pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide cross-linking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.

Keywords: P2X receptors; conformational change; gating; ion channel; molecular simulations; physical couplings; protein domain; receptor structure-function; single channel recording.

Figure 1.

Bound ATP-evoked allosteric changes associated with channel opening of P2X4 receptors. A, allosteric changes essential for the channel activation of P2X4 receptors. The white dotted lines denote the outward flexing of two lower body domains and the concomitant expansion of the central vestibule of P2X4 receptors. The gray and red arrows indicate the conformational changes after ATP binding and the cation-permeating pathway, respectively. B, superposition of resting (blue) and open (red) conformations of P2X4 receptor and zoom-in view of the expansion of central vestibule. The gray arrows indicate the movements of the DF, LF, and lower body domains associated with the expansion of central vestibule.

Figure 2.

Intersubunit physical contacts fostered by the LF domain at the open state are essential for rP2X4 activation. A, 3-D homology model of rP2X4. LF domain locates in the interface between two subunits and is surrounded by the head, DF, and two lower body domains. B and C, zoom-in views of the 3-D structure of N (B) and C termini (C) at resting (upper) and open (lower) states of P2X4 receptor illustrate the switching of H-bonds (red dotted lines) between key residues in the LF, DF, and lower body domains before and after ATP binding. D, representative raw traces of P2X4's responses to saturated ATP (1–1.5 mm for R278A, D280A, R282A, P290A, and R203A; 100 μm for WT and other mutants). E, pooled data of ATP-evoked maximal current amplitudes in P2X4 with alanine replacements on the residues of LF domain (mean ± S.E., n = 4–20). *, p < 0.05; **, p < 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test.

Figure 3.

Intersubunit physical contacts fostered by the LF domain at the open state are essential for zfP2X4 activation. A, zoom-in view of the 3-D structure of the LF domain at the open state of zfP2X4 receptors (PDB code 4DW1) illustrates the H-bonds (red dotted lines) between key residues in the LF, DF, and lower body domains after ATP binding. B and C, representative raw traces (B) and pooled data (C) of WT zfP2X4 and various mutant (R281A, D283A, K285D, and R206L) responses to ATP (1 mm, mean ± S.E., n = 3–7). *, p < 0.05; **, p < 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test. D, amino acid sequence alignment of the LF and lower body domains in different P2X subtypes. In the amino acid sequence alignment, the residues were numbered according to the amino acid sequence of rP2X4 receptors, and N, C, and M refer to the N and C termini and middle region of the LF domain, respectively.

Figure 4.

Effects of mutants on the channel functions of rP2X4 receptors. A and B, representative Western blotting (A) and mean values (B) of the membrane expression of P2X4 with alanine replacements transfected in HEK-293 cells. At least three experiments were performed for each mutant: *, p < 0.05; **, p < 0.01 versus WT, one-way ANOVA with Bonferroni post hoc test. C, protein samples extracted from transfected HEK-293 cells separated by non-reducing Western blotting. Monomeric, dimeric, and trimeric receptors are indicated by triangle arrows on the left. Molecular mass markers are shown on the right. Similar results were observed in at least three independent experiments. D and E, representative Western blotting (D) and mean values (E) of rP2X4 protein for WT, R203A, R278A, D280A, and R282A. Cells were treated with 20 μg/ml CHX in a time-course experiment as indicated. The results were observed in at least three independent experiments for statistical analysis. F and G, representative traces (F) and mean values (G) of the responses of WT rP2X4 and various mutants to ATP (100 μm, mean ± S.E., n = 3–7). *, p < 0.05; **, p < 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test. H and I, representative Western blotting (H) and mean values (I) of the membrane protein expression for WT, R282W, and R203K. At least three experiments were performed for each mutant: *, p < 0.05; **p < 0.01 versus WT, one-way ANOVA with Bonferroni post hoc test. J, effects of mutations on the ATP-EC₅₀ of rP2X4. The solid line is a fit of the Hill equation to the ATP-dependent activation. Each point represents the mean ± S.E. of four measurements.

Figure 5.

Impaired intersubunit physical couplings significantly influence channel gating of P2X4 receptors. A, N-O distances of Arg-282…Glu-245 (CV1) and Arg-203…Val-288 (CV2) at the resting (left) and open (right) states. Two measured distances were defined as collective variables (CVs) of metadynamics. B and C, 3-D projection of free-energy (kcal mol⁻¹) surface showing the lowest free-energy paths passing from open to resting state with (B) or without (C) bound-ATP. CV_O and CV_R indicates CVs at the open and resting states, respectively. The gray dashed lines and white arrowheads depict the paths passing from resting to open state. D–F, single channel currents recorded from outside-out patches at −120 mV in responses to ATP (100 μm) for the WT (D), R203A (E), and R282A (F). Full opening (O) and closing (C) are indicated by black and yellow lines, respectively. Right panel summarizes corresponding all-points histograms fitted to the sum of two Gaussians. y axis denotes the ratio of the number of events to the number of bins (the bin number is set to 320). Similar results were obtained in four other independent patches. G, single channel currents recorded from outside-out patches at −120 mV in responses to ATP and following ATP-IVM co-application for the mutants R203A, R282A, and WT rP2X4. Similar results were obtained in at least three other independent recordings. H, mean amplitude of the unitary current of WT rP2X4 and mutants in the presence of ATP and IVM.

Figure 6.

Introducing different intersubunit metal bridges to weaken or strengthen intersubunit physical couplings at the open state. A, zoom-in view of the constructed zinc-bridge model of P2X4^{His-286/V288H} based on the open structure provides the details of the distances (blue dotted line) between Zn²⁺ and the coordinating NE2 or oxygen atoms from His-286, His-288, Pro-207, and Ile-209. The black circles denote the Cα atoms of residues His-288, Arg-278, and Ile-205 of P2X4^{His-286/V288H}. The Cα atoms of residues Arg-278 and Ile-205 align horizontally, whereas the Cα atoms of His-288 and Arg-278 form an angle to Arg-278 and Ile-205, suggesting a tilting posture of the LF domain. The degree of the angle correlates with the intersubunit physical couplings between DF and two lower body domains established by deformed LF domain at the open state. Red arrow indicates the downward motion of LF domain of P2X4^{His-286/V288H} after Zn²⁺ application. B and C, sample traces (B) and summarized (C, mean ± S.E., n = 4–12) effects of extracellular Zn²⁺ treatment on ATP (100 μm, saturating)-evoked currents of WT and mutant receptors. **, p < 0.01 versus P2X4 WT; #, p < 0.05 versus control, one-way ANOVA followed by Bonferroni post hoc test. D, dose-response curves of Zn²⁺ in P2X4^{His-286/V288H}, P2X4^{His-286/V288H/P207A}, and P2X4^{His-286/V288H/I209A} fitted into Hill Equation 1 (solid line, IC₅₀ = 68.4 ± 10, 39.4 ± 2, and 129.8 ± 17 μm, for P2X4^{His-286/V288H}, P2X4^{His-286/V288H/P207A}, and P2X4^{His-286/V288H/I209A}, respectively). Data points are mean ± S.E. of 5–10 measurements. E, time evolutions of the tilting angle of LF domain along the horizontal line (measured by angle formed by Cα atoms of residues 288, 278, and 205) during MD simulations on WT, P2X4^{His-286/V288H}, and P2X4^{His-286/V288H/I209C}. Zn²⁺ binding significantly decreased the tilt angle of the LF domain in P2X4^{His-286/V288H} but increased in mutant His-286/V288H/I209C when compared with that of WT P2X4 receptor. F, zoom-in view of the constructed zinc-bridge model of P2X4^{His-286/V288H/I209C} based on the open structure showing details of the distances (light-blue dotted line) between Zn²⁺ and the coordinating NE2, sulfur, and oxygen atoms of His-286, His-288, and Cys-209.

Figure 7.

Effect of Zn²⁺ on the unitary rP2X4^{His-286/V288H/I209C} currents. A and B, representative current recordings from excised outside-out membrane expressing single channel (A) or multiple channels (B) exposed to ATP and the following ATP-Zn²⁺ co-application for the mutant His-286/V288H/I209C. Full opening (O) and closing (C) are indicated by black and yellow lines, respectively. y axis denotes the ratio of the number of events to the number of bins (the bin number is set to 320). Similar results were obtained in at least three other independent recordings.

Figure 8.

Restraining the LF domain from fostering physical couplings via intersubunit disulfide. A, intersubunit disulfide bonds between the LF and lower body domains immobilize LF domain at the resting state and prevent the establishment of intersubunit physical couplings at the open state. B and C, zoom-in view of the C_β−C_β distances (green dotted line) between Ser-201 and the key residues of the middle region of the LF domain at the resting (B) and open (C) states. D, Western blotting results support the formation of intersubunit disulfide bonds between S201C (in the lower body domain of one subunit) and D283C/L284C (in the LF domain of another subunit) in homooligomeric P2X4 receptors. The cells transfected with WT or cysteine substitution mutants were lysed in buffer with or without β-ME (1%, 10 mm) as indicated. Positions corresponding to the size of monomeric, dimeric, and trimeric P2X4 subunits were labeled with arrowheads, respectively. E and F, representative currents recorded from cells transfected with S201C/D283C and S201C/L284C (E) and WT P2X4 receptors (F). Cells were voltage-clamped at −60 mV and currents were evoked by ATP (10 μm, 3 s) at 2-min intervals. DTT (10 mm) and H₂O₂ (0.3%) were applied as the schematic indicated. G, pooled data from the experiments in E and F. y axis denotes the ratio of ATP-evoked current after DTT treatment normalized by current before DTT application (mean ± S.E., n = 3–12). **, p < 0.01 after DTT application versus before DTT application, paired t test. H and I, representative recordings to effects of DTT (10 mm) and H₂O₂ (0.3%) on lower concentrations (H) and saturated (I) ATP-sensing of P2X4^S201C/D283C. J, normalized P2X4^S201C/D283Cand P2X4^S201C/L284C currents evoked by saturated ATP. y axis denotes the ratio of ATP-evoked current after DTT treatments normalized by current before DTT applications (mean ± S.E., n = 3). **, p < 0.01 after versus before, paired t test.

Figure 9.

Formation of intersubunit disulfide perturbs the conformation of the middle region of the LF domain at the open state. A, zoom-in view of the constructed zinc-bridge model of P2X4^S201C/D283C based on the open structure exhibits the details of the C_β–C_β distances (green dotted line) of Cys-201 and Cys-283 and the distances measured between Zn²⁺ and the coordinating sulfur atoms from Cys-201 and Cys-283 (yellow dotted line). B and C, sample traces (B) and summarized (C, mean ± S.E., n = 5–9) effects of extracellular Zn²⁺ treatment on ATP (100 μm, saturated)-evoked remaining currents of WT and S201C/D283C. **, p < 0.01 after versus before Zn²⁺ application, paired Student's t test. D, state-dependent cross-linking of rP2X4^S201C/D283C. DTT (+/−) indicate with (+) or without (−) the treatment of DTT (10 mm for 10 min) after the cell surface biotinylation; ATP (+/−) indicate with (+) and without (−) a treatment of ATP (100 μm for 1 min) before the cell lysis; β-ME (+/−) indicate the presence (+) and absence (−) of β-ME (1%, 10 mm) in the loading buffer.

Figure 10.

Middle region of the LF domain is a functional structural element in the channel activation of rP2X4 receptors. A, superposition of the closed (purple) and open (pink) conformations of rP2X4. Yellow arrow denotes the motion of the LF domain from closed to ATP-bound open state. The residues of the middle region of the LF domain are displayed as sticks for emphasis. B, representative traces of currents evoked by saturated ATP (1–1.5 mm for Δ283–286; 100 μm for WT and the rest of the mutants). C, pooled data for maximal current amplitude evoked by saturated ATP of mutations on the middle region of the LF domain (mean ± S.E., n = 4–20). *, p < 0.05; **, p < 0.01 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test. D and E, representative Western blotting (D) and pooled data (E) illustrating the membrane expressions of WT and Δ283–286 in HEK-293 cells. The results were observed in at least three independent experiments.

Figure 11.

Weakening intersubunit physical coupling via covalent modifications. A, schematic structure of covalent modification on Cys-194 of P2X4^W194C perturbs the H-bonds between Arg-278, Asp-280, and Arg-282 at the open state. B, reagents introduced additional charged or/and bulky groups into Cys-194 and Cys-201 of P2X4^W194C and P2X4^S201C, respectively. C and D, sample traces (C) and summarized (D, mean ± S.E., n = 3–5) effects of applications of MTSEA (1 mm), MTSES (1 mm), DTNB (1 mm), and NPM (1 mm) inhibited ATP-evoked currents of P2X4^WT, P2X4^S021C, or P2X4^W194C, which were rescued following application of DTT except for NPM treatments. **, p < 0.01 after versus before covalent modifications. Cells were voltage-clamped (amphotericin-perforated patch clamp) at −60 mV, and currents were evoked by ATP (100 μm, 15 s) at 8-min intervals. E and F, sample traces (E) and summarized (F, mean ± S.E., n = 3) effects of post MTSEA (1 mm) treatment on ATP (100 μm, saturated)-evoked remaining currents of P2X4^WT, P2X4^S021C, or P2X4^W194C. *, p < 0.05; **, p < 0.01 after versus before MTSEA application, paired Student's t test. G and H, sample traces (G) and summarized effects (H) (mean ± S.E., n = 4–20) of mutations on the maximal amplitude of currents caused by saturated ATP (100 μm). *, p < 0.05 versus WT (dashed line), one-way ANOVA with Bonferroni post hoc test.

Figure 12.

Illustration of intersubunit physical couplings established by the LF domain at the open state. The established physical couplings integrate the DF, lower body, and LF domains into a structural element stringently required by the channel gating of rP2X4 receptors. The movements of extracellular domain and TM domain were referred by light-blue arrows. The black dotted lines connecting pink dots denote H-bonds or hydrophobic interactions between the key residues. Only two of three subunits, where subunit A and B are colored in green and purple, respectively, are shown for the clarity.