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. 2019 Mar 4;2:88. doi: 10.1038/s42003-019-0338-1

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© The Author(s) 2019

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. 3 — Activation of TRPV1 induces strong CDI in Orai1. a Perforated patch measurements in HEK293 cells expressing Orai1-GFP and STIM-DsRed. The sequential protocol consisted in the application of extracellular solution followed by a second control application of extracellular solution (ES) and finally thapsigargin. The second ES application will be replaced by either capsaicin or cytidine triphosphate (CTP) in the following experiments. In all cases, capsaicin or CTP were incubated for 3 min and then the cells were washed with fresh ES for 1–2 min prior to TG stimulation (TG was present for the remaining duration of the experiment). b Relative current illustrating the activation by applying a voltage step from 0 to −100 mV for cells expressing Orai1 and TRPV1 pre-stimulated with capsaicin (cap, black), Orai1 only with thapsigargin (red), and Orai1 and TRPV1 pre-stimulated with capsaicin in cells incubated with BAPTA-AM (green). c Perforated patch measurements using the sequential protocol pre-simulating with capsaicin in cells expressing Orai1-GFP and TRPV1-CFP. The middle panel shows the currents elicited by capsaicin stimulation (TRPV1). Panel to the right shows the subsequent activation of Orai1 with TG. d Relative current illustrating the activation of current by applying a voltage step from 0 to −100 mV for cells expressing Orai1 and activated by TG (red), cells expressing Orai1 and P2X₄ and pre-stimulated with CTP (black) and cells expressing Orai1 and P2X₄ and pre-stimulated with CTP and previously incubated with BAPTA-AM (green). Lines show mean ± standard deviation from at least 31 cells. e Perforated patch measurements using the sequential protocol in a cell expressing Orai1 and P2X₄. f Relative current illustrating the activation for cells expressing Orai1 wild type (WT) with pre-stimulation with capsaicin (black) and cells expressing the CDI-resistant mutant Orai1 Y80W with (red) or without pre-stimulation (green). g Perforated patch measurements using the sequential protocol in cells expressing Orai1 Y80W and TRPV1. h Orai1 currents relative to mutant Orai1 Y80W at different voltages. Cells pre-stimulated with CTP (black circle), with capsaicin (open circle) or cells not exposed to capsaicin, only TG (NT). All experiments show the mean ± standard deviation from at least 20 cells