title: Fast GCaMPs for improved tracking of neuronal activity.
abstract: The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca(2+)-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca(2+)-chelating residues. Second, we find that off-responses to Ca(2+) are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca(2+) and by slow binding to the C-lobe at lower Ca(2+). We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of K(D), allowing coexpression of multiple variants to span an expanded range of Ca(2+) concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Xiaonan R
authors.initials: XR
authors.lastname: Sun
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Badura
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Diego A
authors.initials: DA
authors.lastname: Pacheco
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lynch
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Eve R
authors.initials: ER
authors.lastname: Schneider
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Taylor
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Ian B
authors.initials: IB
authors.lastname: Hogue
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Lynn W
authors.initials: LW
authors.lastname: Enquist
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Mala
authors.initials: M
authors.lastname: Murthy
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Samuel S-H
authors.initials: SS
authors.lastname: Wang
mesh_terms: D000161:Acoustic Stimulation; D000595:Amino Acid Sequence; D000818:Animals; D001307:Auditory Perception / Q000502:physiology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium