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Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta
Upon Ca(2+) store depletion, Orai1 channels cluster and open at endoplasmic reticulum–plasma membrane (ER–PM) junctions in signaling complexes called puncta. Little is known about whether and how Orai1 channel activity may vary between individual puncta. Previously, we developed and validated optica...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478869/ https://www.ncbi.nlm.nih.gov/pubmed/32589186 http://dx.doi.org/10.1085/jgp.201812239 |
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author | Dynes, Joseph L. Yeromin, Andriy V. Cahalan, Michael D. |
author_facet | Dynes, Joseph L. Yeromin, Andriy V. Cahalan, Michael D. |
author_sort | Dynes, Joseph L. |
collection | PubMed |
description | Upon Ca(2+) store depletion, Orai1 channels cluster and open at endoplasmic reticulum–plasma membrane (ER–PM) junctions in signaling complexes called puncta. Little is known about whether and how Orai1 channel activity may vary between individual puncta. Previously, we developed and validated optical recording of Orai channel activity, using genetically encoded Ca(2+) indicators fused to Orai1 or Orai3 N or C termini. We have now combined total internal reflection fluorescence microscopy with whole-cell recording to map functional properties of channels at individual puncta. After Ca(2+) store depletion in HEK cells cotransfected with mCherry-STIM1 and Orai1-GCaMP6f, Orai1-GCaMP6f fluorescence increased progressively with increasingly negative test potentials and robust responses could be recorded from individual puncta. Cell-wide fluorescence half-rise and -fall times during steps to −100 mV test potential indicated probe response times of <50 ms. The in situ Orai1-GCaMP6f affinity for Ca(2+) was 620 nM, assessed by monitoring fluorescence using buffered Ca(2+) solutions in “unroofed” cells. Channel activity and temporal activation profile were tracked in individual puncta using image maps and automated puncta identification and recording. Simultaneous measurement of mCherry-STIM1 fluorescence uncovered an unexpected gradient in STIM1/Orai1 ratio that extends across the cell surface. Orai1-GCaMP6f channel activity was found to vary across the cell, with inactive channels occurring in the corners of cells where the STIM1/Orai1 ratio was lowest; low-activity channels typically at edges displayed a slow activation phase lasting hundreds of milliseconds. Puncta with high STIM1/Orai1 ratios exhibited a range of channel activity that appeared unrelated to the stoichiometric requirements for gating. These findings demonstrate functional heterogeneity of Orai1 channel activity between individual puncta and establish a new experimental platform that facilitates systematic comparisons between puncta composition and activity. |
format | Online Article Text |
id | pubmed-7478869 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-74788692021-03-07 Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta Dynes, Joseph L. Yeromin, Andriy V. Cahalan, Michael D. J Gen Physiol Article Upon Ca(2+) store depletion, Orai1 channels cluster and open at endoplasmic reticulum–plasma membrane (ER–PM) junctions in signaling complexes called puncta. Little is known about whether and how Orai1 channel activity may vary between individual puncta. Previously, we developed and validated optical recording of Orai channel activity, using genetically encoded Ca(2+) indicators fused to Orai1 or Orai3 N or C termini. We have now combined total internal reflection fluorescence microscopy with whole-cell recording to map functional properties of channels at individual puncta. After Ca(2+) store depletion in HEK cells cotransfected with mCherry-STIM1 and Orai1-GCaMP6f, Orai1-GCaMP6f fluorescence increased progressively with increasingly negative test potentials and robust responses could be recorded from individual puncta. Cell-wide fluorescence half-rise and -fall times during steps to −100 mV test potential indicated probe response times of <50 ms. The in situ Orai1-GCaMP6f affinity for Ca(2+) was 620 nM, assessed by monitoring fluorescence using buffered Ca(2+) solutions in “unroofed” cells. Channel activity and temporal activation profile were tracked in individual puncta using image maps and automated puncta identification and recording. Simultaneous measurement of mCherry-STIM1 fluorescence uncovered an unexpected gradient in STIM1/Orai1 ratio that extends across the cell surface. Orai1-GCaMP6f channel activity was found to vary across the cell, with inactive channels occurring in the corners of cells where the STIM1/Orai1 ratio was lowest; low-activity channels typically at edges displayed a slow activation phase lasting hundreds of milliseconds. Puncta with high STIM1/Orai1 ratios exhibited a range of channel activity that appeared unrelated to the stoichiometric requirements for gating. These findings demonstrate functional heterogeneity of Orai1 channel activity between individual puncta and establish a new experimental platform that facilitates systematic comparisons between puncta composition and activity. Rockefeller University Press 2020-06-26 /pmc/articles/PMC7478869/ /pubmed/32589186 http://dx.doi.org/10.1085/jgp.201812239 Text en © 2020 Dynes et al. http://www.rupress.org/terms/https://creativecommons.org/licenses/by-nc-sa/4.0/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Article Dynes, Joseph L. Yeromin, Andriy V. Cahalan, Michael D. Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
title | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
title_full | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
title_fullStr | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
title_full_unstemmed | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
title_short | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
title_sort | cell-wide mapping of orai1 channel activity reveals functional heterogeneity in stim1-orai1 puncta |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478869/ https://www.ncbi.nlm.nih.gov/pubmed/32589186 http://dx.doi.org/10.1085/jgp.201812239 |
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