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

Abstract:

:Intracellular Ca(2+) 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 approximately 1-kDa biarsenical Ca(2+) indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca(2+)] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca(2+), with a K(d) of approximately 100 microM, <1-ms kinetics and good Mg(2+) rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca(2+) waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled alpha(1C) L-type calcium channels shows fast-rising depolarization-evoked Ca(2+) transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca(2+) buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca(2+) from a tiny minority of clustered open channels. With high Ca(2+) buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca(2+) domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca(2+)] dynamics.

journal_name

Nat Chem Biol

journal_title

Nature chemical biology

authors

Tour O,Adams SR,Kerr RA,Meijer RM,Sejnowski TJ,Tsien RW,Tsien RY

doi

10.1038/nchembio.2007.4

subject

Has Abstract

pub_date

2007-07-01 00:00:00

pages

423-31

issue

7

eissn

1552-4450

issn

1552-4469

pii

nchembio.2007.4

journal_volume

3

pub_type

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