Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 50, Pages 17404-17409Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0408030101
Keywords
calcium; cameleon
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Funding
- NIGMS NIH HHS [R01 GM060554, F32 GM 067488-01, F32 GM067488, GM 60554] Funding Source: Medline
- NINDS NIH HHS [R01 NS027177, R37 NS027177, NS 27177] Funding Source: Medline
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The endoplasmic reticulum (ER) serves as a cellular storehouse for Ca2+, and Ca2+ released from the ER plays a role in a host of critical signaling reactions, including exocytosis, contraction, metabolism, regulation of transcription, fertilization, and apoptosis. Given the central role played by the ER, our understanding of these signaling processes could be greatly enhanced by the ability to image [Ca2+](ER) directly in individual cells. We created a genetically encoded Ca2+ indicator by redesigning the binding interface of calmodulin and a calmodulin-binding peptide. The sensor has improved reaction kinetics and a K-d ideal for imaging Ca2+ in the ER and is no longer perturbed by large excesses of native calmodulin. Importantly, it provides a significant improvement over all previous methods for monitoring [Ca2+](ER) and has been used to directly show that, in MCF-7 breast cancer cells, the antiapoptotic protein B cell lymphoma 2 (Bcl-2) (i) lowers [Ca2+](ER) by increasing Ca2+ leakage under resting conditions and (ii) alters Ca2+ oscillations induced by ATP, and that acute inhibition of Bcl-2 by the green tea compound epigallocatechin gallate results in an increase in [Ca2+](ER) due to inhibition of Bcl-2-mediated Ca2+ leakage.
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