title: Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.
abstract: Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fudisplaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6furevealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fuis applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.
authors.affiliation: Molecular and Clinical Sciences Research Institute, St George's, University of London, Cranmer Terrace, London SW17 0RE, UK.
authors.forename: Nordine
authors.initials: N
authors.lastname: Helassa
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Borbala
authors.initials: B
authors.lastname: Podor
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Alan
authors.initials: A
authors.lastname: Fine
authors.affiliation: Molecular and Clinical Sciences Research Institute, St George's, University of London, Cranmer Terrace, London SW17 0RE, UK.
authors.forename: Katalin
authors.initials: K
authors.lastname: Török
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D056547:CA1 Region, Hippocampal / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism*; D004558:Electric Stimulation; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D017966:Pyramidal Cells / Q000166:cytology / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D046509:Tissue Culture Techniques
chemical_list: D002147:Calmodulin; D005456:Fluorescent Dyes; D010455:Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium