{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T05:54:43Z","timestamp":1771048483435,"version":"3.50.1"},"reference-count":37,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2016,9,2]],"date-time":"2016-09-02T00:00:00Z","timestamp":1472774400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>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, F\u00f6rster 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\u2019s 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.<\/jats:p>","DOI":"10.3390\/s16091419","type":"journal-article","created":{"date-parts":[[2016,9,2]],"date-time":"2016-09-02T10:01:56Z","timestamp":1472810516000},"page":"1419","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":36,"title":["Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3702-4789","authenticated-orcid":false,"given":"Elisa","family":"Greotti","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, Via U. Bassi 58\/B, Padua 35121, Italy"},{"name":"Neuroscience Institute\u2014Italian National Research Council (CNR), Padua 35121, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0181-9408","authenticated-orcid":false,"given":"Andrea","family":"Wong","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, Via U. Bassi 58\/B, Padua 35121, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0498-2641","authenticated-orcid":false,"given":"Tullio","family":"Pozzan","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, Via U. Bassi 58\/B, Padua 35121, Italy"},{"name":"Neuroscience Institute\u2014Italian National Research Council (CNR), Padua 35121, Italy"},{"name":"Venetian Institute of Molecular Medicine, Padua 35121, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2827-935X","authenticated-orcid":false,"given":"Diana","family":"Pendin","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, Via U. Bassi 58\/B, Padua 35121, Italy"},{"name":"Neuroscience Institute\u2014Italian National Research Council (CNR), Padua 35121, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6077-3265","authenticated-orcid":false,"given":"Paola","family":"Pizzo","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, Via U. Bassi 58\/B, Padua 35121, Italy"},{"name":"Neuroscience Institute\u2014Italian National Research Council (CNR), Padua 35121, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2016,9,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1038\/nrm1155","article-title":"Calcium signalling: Dynamics, homeostasis and remodelling","volume":"4","author":"Berridge","year":"2003","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1007\/s00018-011-0845-9","article-title":"Intracellular organelles in the saga of Ca2+ homeostasis: Different molecules for different purposes?","volume":"69","author":"Zampese","year":"2012","journal-title":"Cell Mol. 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