Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator

Abstract

Intracellular Ca2+ regulates numerous proteins and cellular functions and can vary substantially over submicron and submillisecond scales, so precisely localized fast detection is desirable. We have created a 1-kDa biarsenical Ca2+ indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca2+] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca2+, with a Kd of 100 μM, <1-ms kinetics and good Mg2+ rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca2+ waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled α1C L-type calcium channels shows fast-rising depolarization-evoked Ca2+ transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca2+ buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca2+ from a tiny minority of clustered open channels. With high Ca2+ buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca2+ domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca2+] dynamics.

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Figure 1: In vitro and intracellular titration of CaGF fluorescence.
Figure 2: CaGF reports calcium dynamics of L-type calcium channel activation.
Figure 3: Ca2+ transients in response to three 20-ms depolarization pulses.
Figure 4: CaGF shows rapid kinetics.
Figure 5: Simulation of CaGF signals.

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Acknowledgements

We wish to thank M. Ellisman (University of California, San Diego) for providing the cx43-TC construct, B. Martin (University of California, San Diego) for the cx43-GFP-4C construct, J. Adams for the use of his Applied Photophysics stopped-flow instrument, D. Keller for participating in the initial experiments with CaGF on the L-type channel, P. Steinbach for assistance in numerous microscope-related challenges, Q. Xiong for conducting the fluorescent-activated cell sorting experiments, W. Li for acquiring the 13C NMR spectrum and C. Lopreore for advice on finite difference models. This work was supported by the Howard Hughes Medical Institute and US National Institutes of Health (NIH) grants NS27177 and GM72033 to R.Y.T. Additional support was provided (to R.A.K. and T.J.S.) by the US National Science Foundation–sponsored Center for Theoretical Biological Physics (grants PHY-0216576 and PHY-0225630), and by grants NIH NS0044306 and NIH GM068630.

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Authors

Contributions

S.R.A. and R.Y.T. designed CaGF. S.R.A. synthesized and characterized CaGF. O.T. and R.Y.T. designed the connexin 43 experiments. O.T., R.W.T. and R.Y.T. designed the calcium channel experiments. R.W.T. provided the α1C cDNA and cell line expressing the other Ca2+ channel subunits. O.T. and R.M.M. created the constructs and final cell lines, and O.T. performed the imaging, electrophysiology and data analysis. R.A.K. and T.J.S. designed the simulations and R.A.K. implemented them. O.T., S.R.A., R.A.K. and R.Y.T. prepared the manuscript.

Corresponding author

Correspondence to Roger Y Tsien.

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Competing interests

R.Y.T. and S.R.A. are co-inventors on patents assigned to the University of California covering biarsenical dyes and tetracysteine motifs.

Supplementary information

Supplementary Fig. 1

Tetracysteine-tagged L-type calcium channel imaged using TIRF-M. (PDF 82 kb)

Supplementary Fig. 2

Patch clamp stimulus waveforms. (PDF 28 kb)

Supplementary Fig. 3

Representative ensemble whole-cell currents exhibit similar amplitudes and kinetics, regardless of whether cells had untagged α1C channels, tagged 4N-GFP-α1C channels without CaGF labeling, or tagged 4N-GFP-α1C channels labeled by CaGF. (PDF 34 kb)

Supplementary Video 1

CaGF reports the spread of calcium. (AVI 3948 kb)

Supplementary Video 2

Spatial and temporal display of Ca2+ transients triggered by patch clamp activation of L-type calcium channels. (AVI 1947 kb)

Supplementary Video 3

A patch pipette was filled with 15 mM Ca2+ that diffused into the cell on the right following the break of the cell membrane to establish a whole-cell configuration. (AVI 3107 kb)

Supplementary Video 4

Spatial and temporal display of four Ca2+ transients triggered by patch clamp activation of L-type calcium channels. (AVI 2308 kb)

Supplementary Methods (PDF 162 kb)

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Tour, O., Adams, S., Kerr, R. et al. Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator. Nat Chem Biol 3, 423–431 (2007). https://doi.org/10.1038/nchembio.2007.4

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