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Figure 1. Inhibition of ATP-activated P2X4-mediated currents by t-DCA. (A and B) Left panels: Representative whole-cell current traces recorded in human P2X4-expressing oocytes. ATP (10 µM) and t-DCA (250 µM) were present in the bath solution as indicated by open and filled bars, respectively. Zero current level is indicated by a dotted horizontal line. Right panels: Summary of results from similar experiments as shown in the left panels (A: n = 14, N = 1; B: n = 20, N = 2). Lines connect data points obtained in the same experiment. To quantify current responses, the integral of the inward current elicited by ATP application below the baseline was determined for each response. The current integral values of the second and third ATP-activated responses were normalized to the current integral of the first response in each individual recording (normalized current integral). ***, Second normalized responses in A and B are significantly different (P < 0.001). n.s., not significant. Third normalized responses in A and B are not significantly different; Student’s ratio t test. (C) Representative whole-cell current trace recorded in a human P2X4-expressing oocyte. ATP (10 µM) and t-DCA (250 µM) were present in the bath solution as indicated by open and filled bars, respectively. Zero current level is indicated by a dotted horizontal line (n = 25, N = 2). (D) Representative whole-cell current trace recorded in a control oocyte. ATP (100 µM) was present in the bath solution as indicated by open bar. Zero current level is indicated by a dotted horizontal line (n = 25, N = 2).
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Figure 2. DCA in tauro-conjugated or unconjugated form has the strongest inhibitory effect on P2X4 among the bile acids tested. (A–F) Left panels: Representative whole-cell current traces recorded in human P2X4 expressing oocytes. ATP (10 µM) and bile acids (A: t-DCA; B: DCA; C: t-CDCA; D: CDCA; E: t-CA; F: CA; all in a concentration of 250 µM) were present in the bath solution as indicated by open and filled bars, respectively. Zero current level is indicated by a dotted horizontal line. Right panels: Summary of results from similar experiments as shown in the corresponding left panels. The ATP-activated current integral was calculated in each recording as described in Fig. 1 and normalized to the mean current integral determined in matched control oocytes (i.e., in the absence of bile acids) from the corresponding batch of oocytes (normalized current integral). Mean ± SEM values and individual data points are shown (20 ≤ n ≤ 21; N = 2); ***, *, Significantly different, P < 0.001 and P < 0.05, respectively; n.s., not significant; Student’s ratio t test.
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Figure 3. Concentration-dependent inhibition of ATP-activated P2X4 currents by t-DCA. (A) Representative P2X4-mediated whole-cell current traces recorded in individual matched oocytes from the same batch of oocytes in the absence of t-DCA or in the presence of t-DCA in the indicated concentrations. ATP (10 µM) and t-DCA were present in the bath solution as indicated by open and filled bars, respectively. Zero current level is indicated by a dotted horizontal line. (B) Concentration–response relationship of the inhibitory effect of t-DCA on ATP-activated P2X4 currents. Summary of normalized ATP-activated P2X4 current responses from similar experiments as shown in A. The current integral of each recording was normalized to the mean current integral obtained under control conditions (i.e., in the absence of t-DCA) for the corresponding batch of oocytes. Data are means ± SEM (25 ≤ n ≤ 26; N = 3) and were fitted to Eq. 1. IC50, half maximal inhibitory concentration.
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Figure 4. Concentration-dependent stimulation of P2X4 by ATP under control conditions and in the presence of t-DCA in the bath solution. (A and B) Representative P2X4-mediated whole-cell current traces recorded in matched oocytes from the same batch stimulated by ATP applied in the indicated concentrations in the absence (A) or presence (B) of t-DCA (250 µM) in the bath solution. ATP and t-DCA were present in the bath solution as indicated by open and filled bars, respectively. Zero current level is indicated by a dotted horizontal line. (C) Summary of results from similar experiments as shown in A and B and concentration–response curves of ATP-dependent stimulation of P2X4 currents recorded under control conditions (control, black symbols) or in the presence of t-DCA (t-DCA, red symbols). The current integral of each recording was normalized to the mean current integral obtained with 500 µM ATP applied under control conditions (i.e., in the absence of t-DCA) for the corresponding batch of oocytes (normalized current integral). Data are means ± SEM (n = 12, N = 2) and were fitted to Eq. 1.
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Figure 5. t-DCA slows down the ATP-mediated activation of ensemble P2X4 currents and facilitates the spontaneous inactivation of the channel. (A) Representative continuous current trace recorded at a holding potential of −70 mV from an outside-out patch obtained from a control oocyte. Application of ATP (100 µM) is indicated by an open bar. (B and C) Representative continuous current traces recorded at a holding potential of −70 mV from outside-out patches obtained from an oocyte expressing P2X4. Application of ATP (100 µM) and t-DCA (250 µM) is indicated by open and filled bars, respectively. Linear fit of initial phase of ATP-activated currents and exponential decay fit of the subsequent spontaneous current decrease were calculated as described in Materials and methods and delineated in red. To quantify the responses, the current integral (Q, pC), the slope of the initial P2X4-mediated ensemble inward current increase (sact, pA/s), and the time constant of the spontaneous P2X4 ensemble current inactivation (τinact, s) were determined for each recording. (D–F) Summary of results from similar experiments as shown in B and C. Data are means ± SEM and individual data points (n = 9, N = 5). ***, *, Significantly different, P < 0.001 and P < 0.05, respectively (Student’s t test).
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Figure 6. ATP-activated P2X4-mediated current response with single-channel events recorded from an outside-out patch. Representative continuous current trace recorded at a holding potential of −70 mV from an outside-out patch of a P2X4-expressing oocyte. Application of ATP (100 µM) is indicated by an open bar. The insets show the indicated fragments of the current trace on an expanded time scale. Binned current amplitude histograms are shown on the right side of the insets and were obtained from the corresponding parts of the trace to calculate single-channel current amplitude (i). The current level at which all channels are closed (C) was determined at the beginning of the trace before ATP application. Dotted lines in the insets indicate channel open levels (1–7).
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Figure 7. t-DCA stabilizes the closed state of P2X4. (A and B) Examples of continuous current traces recorded at a holding potential of −70 mV from outside-out patches in which ATP activated only one P2X4 channel. Application of ATP (100 µM) and t-DCA (250 µM) is indicated by open and filled bars, respectively. The insets show the middle portions (∼20 s) of the current traces during ATP exposure on an expanded time scale as indicated. Binned current amplitude histograms obtained from these portions are shown on the right side of the corresponding insets. They were used to determine the closed level of the channel (C) and the channel open level (1) indicated by the dotted lines and to calculate single-channel current amplitude (i). Channel open probability (Po) was estimated from the time integral of inward current exceeding the channel closed level divided by the product of i and total duration of the analyzed portion. (C and D) Summary of results from similar experiments as shown in A and B. Data are means ± SEM and individual data points (n = 7/group, N = 5). **, Significantly different, P < 0.01; n.s., not significant; Student’s t test. (E and F) Dwell-time histograms generated from combined dwell-time data obtained from current portions as marked in (A) and (B) using all experiments summarized in (C) and (D).
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Figure 8. Homology model of the transmembrane domains of human P2X4. A homology model of human P2X4 was generated based on the crystal structure of D. rerio P2X4.1 in the closed state. A molecular surface representation of human P2X4 (side view) is shown in the left panel. To visualize the individual subunits of the P2X4 homotrimer, they are colored in red, green, and blue. The putative location of the plasma lipid bilayer (outer and inner leaflet) is schematically depicted with DPPC molecules in sticks representation. The inset shows the channel transmembrane domains on an expanded scale in cartoon representation using the same color code for the individual subunits. Amino acid residues that were substituted by alanine and tested experimentally are indicated with arrows and shown in stick representation with carbons in green, sulfur in yellow, nitrogens in blue, and oxygens in red. DPPC, dipalmitoylphosphatidylcholine.
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Figure 9. D331, M336, and W46 are critically involved in t-DCA–mediated inhibition of P2X4. (A–F) Left panels: Representative whole-cell current traces recorded in oocytes expressing human WT (A) or mutant (B: E51A; C: D331A; D: W50A; E: M336A; F: W46A) P2X4. ATP (10 µM) and t-DCA (250 µM) were present in the bath solution as indicated by open and filled bars, respectively. Zero current level is indicated by a dotted horizontal line. Right panels: Summary of results from similar experiments as shown in the corresponding left panels. Current integrals were normalized as described in Fig. 2. Values are means ± SEM and individual data points are shown (A: n = 65; N = 7; B: n = 28; N = 3; C: n = 25; N = 3; D: n = 21; N = 2; E: n = 20; N = 2; F: n = 21; N = 2); ***, **, *, Significantly different, P < 0.001, P < 0.01, and P < 0.05, respectively; n.s., not significant; Student’s ratio t test. (G) Basal ATP-activated currents of mutant P2X4 channels compared with WT P2X4 from the same experiments as shown in A–F. The ATP-activated current integral was calculated in each recording and was normalized to the mean current integral determined in matched control oocytes (i.e., expressing WT P2X4) from the corresponding batch of oocytes (normalized to WT). Data are means ± SEM. ***, **, Significantly different compared with WT, P < 0.001 and P < 0.01, respectively; n.s., not significant; one-way ANOVA with Bonferroni post hoc test. (H) Relative effects of t-DCA on ATP-activated P2X4 currents (negative values indicate inhibition; positive values indicate stimulation) in oocytes expressing WT P2X4 or different P2X4 mutant channels. The current integral calculated in each recording was normalized as described in Fig. 2. Data are means ± SEM of the normalized current integrals in the presence of t-DCA from the same experiments as shown in A–F. ***, Significantly different compared with WT, P < 0.001; n.s., not significant; one-way ANOVA with Bonferroni post hoc test.
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Figure 10. Docking simulation predicts a putative t-DCA binding site in the intersubunit interface of P2X4 transmembrane domains in the closed state of the channel. (A and B) For docking simulations, the same homology model of human P2X4 in the closed state was used as shown in Fig. 8. Amino acid residues forming the putative bile acid binding site are shown in molecular surface representation without (A) and with (B) docked t-DCA molecules. An overlay of 30 out of 100 similar t-DCA docked modes is depicted in yellow stick representation. Our functional studies suggested that the amino acid residues indicated with arrows are involved in mediating the inhibitory effect of t-DCA on P2X4. (C) Homology model of human P2X4 in the open state showing the same channel region as in A and B.
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