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

Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17404-9. doi: 10.1073/pnas.0408030101. Epub 2004 Dec 7.


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.

Publication types

  • 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.

MeSH terms

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


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

Associated data

  • GENBANK/AY796115