Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator.
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.
Tour
Oded
O
Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA.
Adams
Stephen R
SR
Kerr
Rex A
RA
Meijer
Rene M
RM
Sejnowski
Terrence J
TJ
Tsien
Richard W
RW
Tsien
Roger Y
RY
D002118:Calcium / Q000032:analysis*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling; D002460:Cell Line; D002478:Cells, Cultured; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D049408:Luminescent Agents / Q000737:chemistry; D008954:Models, Biological; D009930:Organic Chemicals / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
D020746:Calcium Channels, L-Type; D018031:Connexin 43; D049408:Luminescent Agents; D009930:Organic Chemicals; D011994:Recombinant Proteins; C071336:calcium green; D002118:Calcium