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

TLDR

The endoplasmic reticulum stores Ca²⁺ and its release regulates diverse signaling pathways such as exocytosis, contraction, metabolism, transcription, fertilization, and apoptosis. The study aims to enable direct imaging of ER Ca²⁺ in single cells to deepen insight into these signaling processes. A genetically encoded Ca²⁺ sensor was engineered by redesigning the calmodulin–calmodulin‑binding peptide interface to improve binding kinetics and avoid interference from excess native calmodulin. The sensor, with faster kinetics and an optimal Kd, outperforms previous probes and was used to demonstrate that Bcl‑2 reduces ER Ca²⁺ by increasing leakage and altering ATP‑induced oscillations, while epigallocatechin gallate blocks this leakage, raising ER Ca²⁺ levels.

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.