Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta
| العنوان: | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta |
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| المؤلفون: | Joseph L. Dynes, Andriy V. Yeromin, Michael D. Cahalan |
| المصدر: | The Journal of general physiology, vol 152, iss 9 The Journal of General Physiology |
| بيانات النشر: | Rockefeller University Press, 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | In situ, ORAI1 Protein, Physiology, 1.1 Normal biological development and functioning, Medical Physiology, Gating, Endoplasmic Reticulum, Article, Molecular Physiology, Underpinning research, Humans, Stromal Interaction Molecule 1, Total internal reflection fluorescence microscope, Chemistry, ORAI1, Endoplasmic reticulum, Cell Membrane, HEK 293 cells, STIM1, Fluorescence, Neoplasm Proteins, HEK293 Cells, nervous system, Biophysics, Calcium, Cellular Physiology, Generic health relevance |
| الوصف: | The authors use a fluorescent genetically encoded Ca2+ indicator, GCaMP6f, fused to Orai1 to visualize Ca2+ influx through store-operated Orai1 channels. Combined with whole-cell recording of transfected human cells, this reporter reveals heterogeneity of channel activation and kinetics in STIM1-Orai1 puncta within the same cell. Upon Ca2+ 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 Ca2+ 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 Ca2+ 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 |
| وصف الملف: | application/pdf |
| تدمد: | 1540-7748 0022-1295 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::df17ea319f2c684ec24654c3b1abf888 https://doi.org/10.1085/jgp.201812239 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....df17ea319f2c684ec24654c3b1abf888 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15407748 00221295 |
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