title: A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.
abstract: Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd, Baltimore, Maryland, 21224, United States of America.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Henderson
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd, Baltimore, Maryland, 21224, United States of America.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Baldwin
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, 02138, United States of America.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Werley
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, 02138, United States of America.
authors.forename: Stefano
authors.initials: S
authors.lastname: Boccardo
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd, Baltimore, Maryland, 21224, United States of America.
authors.forename: Leslie R
authors.initials: LR
authors.lastname: Whitaker
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd, Baltimore, Maryland, 21224, United States of America.
authors.forename: Xiaokang
authors.initials: X
authors.lastname: Yan
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia, 20147, United States of America.
authors.forename: Graham T
authors.initials: GT
authors.lastname: Holt
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia, 20147, United States of America.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Schreiter
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia, 20147, United States of America.
authors.forename: Loren L
authors.initials: LL
authors.lastname: Looger
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, 02138, United States of America; Department of Physics, Harvard University, Cambridge, Massachusetts, 02138, United States of America; Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, 02138, United States of America; Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, 02138, United States of America.
authors.forename: Adam E
authors.initials: AE
authors.lastname: Cohen
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia, 20147, United States of America.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Kim
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd, Baltimore, Maryland, 21224, United States of America.
authors.forename: Brandon K
authors.initials: BK
authors.lastname: Harvey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000166:cytology; D009474:Neurons / Q000166:cytology; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D002118:Calcium