Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

Helassa, N ORCID: 0000-0003-3743-1886, Podor, B, Fine, A and Török, K (2016) Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. Scientific Reports, 6 (1). 38276-.

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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 GCaMP6fu displaying 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, GCaMP6fu revealed 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 (t1/2 of 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 GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.

Item Type:	Article
Uncontrolled Keywords:	Biophysics, Neuroscience
Depositing User:	Symplectic Admin
Date Deposited:	05 Jun 2018 09:32
Last Modified:	19 Jan 2023 01:32
DOI:	10.1038/srep38276
Open Access URL:	https://www.nature.com/articles/srep38276
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3022104

Available Versions of this Item

• Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. (deposited 25 Jul 2017 11:48)
  • Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. (deposited 26 Jul 2017 11:20)
    • Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. (deposited 31 Jul 2017 08:04)
      • Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. (deposited 05 Jun 2018 09:32) [Currently Displayed]