Characterization of the ER-Targeted Low Affinity Ca2 Probe D4ERReportar como inadecuado


Characterization of the ER-Targeted Low Affinity Ca2  Probe D4ER


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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

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





Autor: Elisa Greotti 1,2,†, Andrea Wong 1,†,‡, Tullio Pozzan 1,2,3, Diana Pendin 1,2,* and Paola Pizzo 1,2

Fuente: http://mdpi.com/



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