Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor.
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.
Palmer
Amy E
AE
Department of Pharmacology and Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0647, USA.
Jin
Can
C
Reed
John C
JC
Tsien
Roger Y
RY
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005453:Fluorescence; D015972:Gene Expression Regulation, Neoplastic; D005818:Genetic Engineering; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013662:Tea / Q000737:chemistry
D001426:Bacterial Proteins; D002147:Calmodulin; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013662:Tea; C434479:citrine protein, bacteria; D000255:Adenosine Triphosphate; D002392:Catechin; C045651:epigallocatechin gallate; D002118:Calcium