title: Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics.
abstract: Faithful reporting of temporal patterns of intracellular Ca(2+) dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca(2+) signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca(2+) affinities and accelerated kinetics by weakening the Ca(2+)-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca(2+) decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca(2+) dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca(2+) at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca(2+) kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.
authors.affiliation: Institute of Cardiovascular and Cell Science, St George's, University of London, London SW17 0RE, UK.
authors.forename: Nordine
authors.initials: N
authors.lastname: Helassa
authors.affiliation: Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston SC 29425, USA.
authors.forename: Xiao-hua
authors.initials: XH
authors.lastname: Zhang
authors.affiliation: Institute of Cardiovascular and Cell Science, St George's, University of London, London SW17 0RE, UK.|MRC National Institute for Medical Research, London NW7 3RJ, UK.
authors.forename: Ianina
authors.initials: I
authors.lastname: Conte
authors.affiliation: Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston SC 29425, USA.
authors.forename: John
authors.initials: J
authors.lastname: Scaringi
authors.affiliation: Laboratoire de Physiologie Cérébrale, Centre National de la Recherche Scientifique and Université Paris Descartes, 75006 Paris, France.
authors.forename: Elric
authors.initials: E
authors.lastname: Esposito
authors.affiliation: Laboratoire de Physiologie Cérébrale, Centre National de la Recherche Scientifique and Université Paris Descartes, 75006 Paris, France.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bradley
authors.affiliation: Institute of Cardiovascular and Cell Science, St George's, University of London, London SW17 0RE, UK.|MRC National Institute for Medical Research, London NW7 3RJ, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Carter
authors.affiliation: Laboratoire de Physiologie Cérébrale, Centre National de la Recherche Scientifique and Université Paris Descartes, 75006 Paris, France.
authors.forename: David
authors.initials: D
authors.lastname: Ogden
authors.affiliation: Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston SC 29425, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Morad
authors.affiliation: Institute of Cardiovascular and Cell Science, St George's, University of London, London SW17 0RE, UK.
authors.forename: Katalin
authors.initials: K
authors.lastname: Török
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D051381:Rats
chemical_list: D002147:Calmodulin; D049452:Green Fluorescent Proteins; D002118:Calcium