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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. 1 — Concentration–response curves for agonists at various ASICs and P2X3Rs expressed in CHO cells. a–c Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Both types of agonists were applied at the indicated increasing concentrations for 2 s onto CHO cells transfected with rASIC3 (a, upper panel) or rP2X3 (a, lower panel). The intervals between individual applications were kept at 2 min (protons) or 5 min (α,β-meATP) in order to avoid the decrease of the subsequent current amplitudes by desensitization. Drug application procedures in this and all subsequent panels of this figure were identical. CHO cells expressing rASIC3 (b) or rP2X3 (c) were treated with amiloride (AMI; 10 µM) or A-31749 (10 µM) while constructing concentration–response curves. In this and all subsequent panels of this figure, means ± S.E.M. of the indicated number of experiments are shown. Broken lines represent curves obtained in response to agonist application to CHO cells containing ASIC3 or P2X3Rs only, in the absence of any additional drug. d–f CHO cells were transfected with rASIC3 and rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with amiloride (10 µM) or A-31749 (10 µM) while constructing pH- or concentration–response curves. g–i CHO cells were transfected with hASIC3 alone or together with hP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with A-31749 (10 µM) while constructing pH- or concentration–response curves. j–l CHO cells were transfected with rASIC1a or rASIC2a alone or together with rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton-induced currents through rASIC1a (j, upper panel, k) or rASIC2a (j, lower panel, l)-containing receptors. The holding potential was −65 mV in all experiments. *P < 0.05; statistically significant difference of the I_mean values from the respective curves designated by broken lines at the highest agonist concentrations (one-way ANOVA followed by the Holm–Sidak test, or Student’s t-test, as appropriate). The scale labels for the vertical bars were 10 nA (a, upper panel), 2 nA (a, lower panel), 2 nA (d), 500 pA (g), 2 nA (j, upper panel) and 10 nA (j, lower panel). The scale labels for the horizontal bars were 20 s (a, d, g, j)