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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. 4 — Changes in the Ca²⁺ concentration modify rASIC3 and rP2X3R currents in CHO cells. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a, b Concentration–response curves for protons in CHO cells transfected with rASIC3. a Inhibitory effect of an increase or decrease of the Ca²⁺ concentration in the external medium ([Ca²⁺]_o). b Inhibitory effect of an increase or decrease of the free Ca²⁺ concentration in the patch pipette solution ([Ca²⁺]_i). c, d Concentration-response curves for α,β-meATP in CHO cells transfected with rP2X3. c No effect of changes in [Ca²⁺]_o. d No effect of changes in [Ca²⁺]_i. 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 (one-way ANOVA followed by the Holm–Sidak test). e, f CHO cells transfected with both rASIC3 and rP2X3 in a 1:1 ratio. Modulation of the unilateral interaction between rASIC3 and rP2X3Rs, by changes in [Ca²⁺]_o (e) and [Ca²⁺]_i (f); the experimental protocol is identical to that shown in Fig. 2a. *P < 0.05; statistically significant difference from the current amplitudes measured in normal extra- and intracellular Ca²⁺. ^§P < 0.05; statistically significant difference between currents at pH 6.7 or 6.5 at different Ca²⁺ concentrations (one-way ANOVA followed by the Bonferroni test). Concentration–response relationships for protons (g) and α,β-meATP (h) in CHO-rASIC3/rP2X3R cells, in the absence and presence of J-8 (30 µM). Means ± S.E.M. of the indicated number of experiments. *P < 0.05; statistically significant difference between the I_mean values at the highest agonist concentration (Student’s t-test). i Potentiation of the α,β-meATP (30 µM) current by co-application with a J-8 (30 µM)-containing, low pH (6.5) external medium, but inhibition in the absence of J-8 by acidification; representative tracings. j Percentage changes of α,β-meATP-induced current responses were calculated with respect to the second current amplitude 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 in the presence of J-8. ^§P < 0.05; statistically significant difference between current amplitudes at pH 6.5 in the presence or absence of J-8 (Mann–Whitney test). The scale labels for the vertical and horizontal bars in (i) were 1 nA and 50 s, respectively