Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator
- 17 June 2007
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Chemical Biology
- Vol. 3 (7), 423-431
- https://doi.org/10.1038/nchembio.2007.4
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.Keywords
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