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Sensors 2016, 16(9), 1419; doi:10.3390/s16091419

Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

1,2,†
,
1,†,‡
,
1,2,3
,
1,2,* and 1,2
1
Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, Padua 35121, Italy
2
Neuroscience Institute—Italian National Research Council (CNR), Padua 35121, Italy
3
Venetian Institute of Molecular Medicine, Padua 35121, Italy
*
Author to whom correspondence should be addressed.
Academic Editors: Niko Hildebrandt and Igor Medintz
Received: 5 July 2016 / Revised: 26 August 2016 / Accepted: 30 August 2016 / Published: 2 September 2016
(This article belongs to the Special Issue FRET Biosensors)
View Full-Text   |   Download PDF [2890 KB, uploaded 2 September 2016]   |  

Abstract

Calcium ion (Ca2+) 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 Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ 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 Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] 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 Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ 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 Ca2+ content in cells expressing mutated PS2, compared to controls. View Full-Text
Keywords: Calcium; Endoplasmic reticulum; Cameleon; FRET-based probe; Presenilin Calcium; Endoplasmic reticulum; Cameleon; FRET-based probe; Presenilin
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MDPI and ACS Style

Greotti, E.; Wong, A.; Pozzan, T.; Pendin, D.; Pizzo, P. Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER. Sensors 2016, 16, 1419.

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