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. 2019 Jul 19;10:3223. doi: 10.1038/s41467-019-11207-8

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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. 1 — Orai2 is required for IP₃R-dependent Ca²⁺ release from internal stores. a Schematic depiction of the hippocampus with the three regions CA1, CA3, and the dentate gyrus (DG). CA1 tissue was extracted for quantitative reverse transcription-PCR. b Result of Orai PCR analysis in the mouse hippocampus (P18). Left: Mean expression levels of the three Orai homologs relative to the housekeeping gene Gapdh (n = 6 mice, p = 0.024 (analysis of variance)). Gray dashes represent individual values in this and other figures. Right: Real-time monitoring of the fluorescence emission of SYBR Green I during the PCR amplification of Orai1-3 cDNA. Dashed line: noise band. c Analogous PCR analysis in single CA1 pyramidal neurons (PNs) harvested from acute hippocampal slices. Orai1: n = 21, Orai2: n = 19, Orai3: n = 15 cells; p = 0.002 (Orai1–Orai2), p = 0.001 (Orai2–Orai1). d Left: DHPG was pressure-applied to somata of whole-cell patch-clamped CA1 PNs filled through the patch pipette with OGB-1. Black traces: Ca²⁺ transients in CA1 PNs in a wild-type mouse (left) in response to repeated applications of DHPG (500 µM, 200 ms) at 2-min intervals. Red traces: Analogous experiments in an Orai2−/− mouse. e Mean amplitudes of DHPG-evoked Ca²⁺ transients in wild-type and Orai2−/− mice (n = 62 and 33 cells, respectively; p = 9.84 × 10⁻²⁴). f Left: Both ultraviolet light pulses and local DHPG puffs were applied to somata of whole-cell patch-clamped CA1 PNs filled with OGB-1 and NPE (“caged”)-IP₃ through the patch pipette. Black traces: Fluorescence transients resulting from photolysis of caged IP₃ (left) and local DHPG puff application (right) in the same cell in a wild-type mouse. Red traces: Analogous experiment in an Orai2−/− mouse. g Left: Correlation plot of relative fluorescence changes in response to IP₃-uncaging (y axis) against relative fluorescence changes in response to DHPG applications (x axis) in the same cells in the wild type. Right: Mean amplitudes of relative fluorescence changes in response to both treatments in c in both genotypes (n = 9 cells in wild type and 7 cells in Orai2−/− mice, p = 1.74 × 10⁻⁴, p = 3.5 × 10⁻⁴). * - significant (p < 0.05), ** - very significant (p < 0.01), *** - highly significant (p < 0.001)