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. 2018 Apr 10;9:1354. doi: 10.1038/s41467-018-03728-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Effects of gate-inhibitory mutations in rP2X3Rs on rASIC3/rP2X3R function. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a, b Analysis of rP2X3 double cysteine mutants with disulfide trapping. CHO cells were transfected with the cysteine mutants rP2X3 (I200C/V274C), rP2X3 (K201C/V274C) or co-transfected with rASIC3wt and rP2X3 (K201C/V274C) in a 1:1 ratio. a Current traces induced by α,β-meATP (10 µM) applied repeatedly for 2 s with 5 min intervals before, during and after the co-application of ditiothreitol (DTT; 1 mM). b Percentage changes of α,β-meATP-induced current responses were calculated with respect to the mean of the second and third currents during DTT application. Means ± S.E.M. of the indicated number of experiments. *P < 0.05; statistically significant difference between the current amplitudes during and after DTT application (Student’s t-test). c–f Concentration–response curves for protons and α,β-meATP in CHO cells co-transfected with the indicated receptors. c Effect of α,β-meATP (0.3–300 µM) on rP2X3Rwt, and rASIC3wt/rP2X3Rwt in the absence or presence of DTT. d Effect of α,β-meATP on rP2X3R (K201C/V274C) and rASIC3/rP2X3R (K201C/V274C) in the absence or presence of DTT. e Effect of protons (pH 7.0–5.5) on rASIC3wt and rASIC3wt/rP2X3Rwt in the absence or presence of DTT. f Effect of protons (pH 7.0–5.5) on rASIC3wt and rASIC3wt/rP2X3R (K201C/V274C) in the absence or presence of DTT. Means ± S.E.M. of the indicated number of experiments. *P < 0.05; statistically significant difference of the I_mean values at the highest agonist concentration from the rP2X3Rwt and rASIC3wt curves, respectively (one-way ANOVA followed by the Holm–Sidak test). g Interaction between rASIC3wt and rP2X3R (K201C/V274C) in the absence and presence of DTT. Currents were elicited by α,β-meATP (10 µM) for 2 s with 5 min intervals before, during and after the pH shift from 7.4 to 6.5. h Percentage changes of α,β-meATP-induced current responses were calculated with respect to the second current before the pH shift. Means ± S.E.M. of the indicated number of experiments. *P < 0.05; statistically significant difference between the current amplitudes before and after the pH shift from 7.4 to 6.5 (Student’s t-test). The scale labels for the vertical bars were 200 pA (a, g). The scale labels for the horizontal bars were 20 s (a) and 50 s (g)