Characterization of the ER-Targeted Low Affinity Ca(2+) Probe D4ER.
Calcium ion (Ca(2+)) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca(2+) store and the free Ca(2+) concentration ([Ca(2+)]) within its lumen ([Ca(2+)]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca(2+) sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca(2+) probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca(2+), low dynamic range, and an affinity for the cation that is too high for the levels of [Ca(2+)] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca(2+) affinity and dynamic range for monitoring [Ca(2+)] variations within the ER. As an example, resting [Ca(2+)]ER have been evaluated in a known paradigm of altered ER Ca(2+) homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca(2+) affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca(2+) content in cells expressing mutated PS2, compared to controls.
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy. e.greotti@gmail.com.|Neuroscience Institute-Italian National Research Council (CNR), Padua 35121, Italy. e.greotti@gmail.com.
Elisa
E
Greotti
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy. andreakuancie.wong@gmail.com.
Andrea
A
Wong
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy. tullio.pozzan@unipd.it.|Neuroscience Institute-Italian National Research Council (CNR), Padua 35121, Italy. tullio.pozzan@unipd.it.|Venetian Institute of Molecular Medicine, Padua 35121, Italy. tullio.pozzan@unipd.it.
Tullio
T
Pozzan
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy. diana.pendin@unipd.it.|Neuroscience Institute-Italian National Research Council (CNR), Padua 35121, Italy. diana.pendin@unipd.it.
Diana
D
Pendin
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy. paola.pizzo@unipd.it.|Neuroscience Institute-Italian National Research Council (CNR), Padua 35121, Italy. paola.pizzo@unipd.it.
Paola
P
Pizzo
Calcium; Cameleon; Endoplasmic reticulum; FRET-based probe; Presenilin