Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor.

Abstract

The endoplasmic reticulum (ER) serves as a cellular storehouse for Ca(2+), and Ca(2+) 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 [Ca(2+)](ER) directly in individual cells. We created a genetically encoded Ca(2+) 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 Ca(2+) 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 [Ca(2+)](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 [Ca(2+)](ER) by increasing Ca(2+) leakage under resting conditions and (ii) alters Ca(2+) oscillations induced by ATP, and that acute inhibition of Bcl-2 by the green tea compound epigallocatechin gallate results in an increase in [Ca(2+)](ER) due to inhibition of Bcl-2-mediated Ca(2+) leakage.

• Authors: Amy E Palmer, Can Jin, John C Reed, Roger Y Tsien
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 101 Issue 50 Pg. 17404-9 (Dec 14 2004) ISSN: 0027-8424 [Print] United States
• PMID: 15585581 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Bacterial Proteins
• Calmodulin
• Luminescent Proteins
• Proto-Oncogene Proteins c-bcl-2
• Tea
• citrine protein, bacteria
• Adenosine Triphosphate
• Catechin
• epigallocatechin gallate
• Calcium

Topics

• Adenosine Triphosphate (pharmacology)
• Bacterial Proteins (genetics, metabolism)
• Biosensing Techniques (instrumentation, methods)
• Calcium (metabolism)
• Calmodulin (genetics, metabolism, pharmacology)
• Catechin (analogs & derivatives, pharmacology)
• Cell Line, Tumor
• Endoplasmic Reticulum (metabolism)
• Fluorescence
• Gene Expression Regulation, Neoplastic
• Genetic Engineering
• Humans
• Luminescent Proteins (genetics, metabolism)
• Molecular Sequence Data
• Mutation (genetics)
• Proto-Oncogene Proteins c-bcl-2 (metabolism)
• Tea (chemistry)