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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Augustine, G.J., Santamaria, F. & Tanaka, K. Local calcium signaling in neurons. Neuron 40, 331–346 (2003).
Rizzuto, R. & Pozzan, T. Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol. Rev. 86, 369–408 (2006).
Yamada, W.M. & Zucker, R.S. Time course of transmitter release calculated from simulations of a calcium diffusion model. Biophys. J. 61, 671–682 (1992).
Parnas, H., Segel, L., Dudel, J. & Parnas, I. Autoreceptors, membrane potential and the regulation of transmitter release. Trends Neurosci. 23, 60–68 (2000).
Llinás, R., Sugimori, M. & Silver, R.B. Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677–679 (1992).
Aharon, S., Bercovier, M. & Parnas, H. Parallel computation enables precise description of Ca2+ distribution in nerve terminals. Bull. Math. Biol. 58, 1075–1097 (1996).
Sugimori, M., Lang, E.J., Silver, R.B. & Llinas, R. High-resolution measurement of the time course of calcium-concentration microdomains at squid presynaptic terminals. Biol. Bull. 187, 300–303 (1994).
Marsault, R., Murgia, M., Pozzan, T. & Rizzuto, R. Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells. EMBO J. 16, 1575–1581 (1997).
Schneggenburger, R. & Neher, E. Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406, 889–893 (2000).
Simon, S.M. & Llinas, R.R. Compartmentalization of submembrane calcium activity during calcium influx and its significance in transmitter release. Biophys. J. 48, 485–498 (1985).
Naraghi, M. & Neher, E. Linearized buffered Ca2+ diffusion in microdomains and its implications for [Ca2+] at the mouth of a calcium channel. J. Neurosci. 17, 6961–6973 (1997).
Stanley, E.F. Single calcium channels and acetylcholine release at a presynaptic nerve terminal. Neuron 11, 1007–1011 (1993).
Borst, J.G.G. & Sakmann, B. Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem. J. Physiol. (Lond.) 506, 143–157 (1998).
DiGregorio, D.A., Peskoff, A. & Vergara, J.L. Measurement of action potential-induced presynaptic calcium domains at a cultured neuromuscular junction. J. Neurosci. 19, 7846–7859 (1999).
Zou, H., Lifshitz, L.M., Tuft, R.A., Fogarty, K.E. & Singer, J.J. Imaging Ca2+ entering the cytoplasm through a single opening of a plasma membrane cation channel. J. Gen. Physiol. 114, 575–588 (1999).
Zenisek, D., Davila, V., Wan, L. & Almers, W. Imaging calcium entry sites and ribbon structures in two presynaptic cells. J. Neurosci. 23, 2538–2548 (2003).
Demuro, A. & Parker, I. “Optical patch-clamping”: single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels. J. Gen. Physiol. 126, 179–192 (2005).
Rizzuto, R., Simpson, A.W., Brini, M. & Pozzan, T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358, 325–327 (1992).
Brini, M. et al. Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin. EMBO J. 12, 4813–4819 (1993).
Montero, M. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J. 14, 5467–5475 (1995).
Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).
Palmer, A.E., Jin, C., Reed, J.C. & Tsien, R.Y. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc. Natl. Acad. Sci. USA 101, 17404–17409 (2004).
Palmer, A.E. et al. Ca2+ indicators based on computationally redesigned calmodulin-Peptide pairs. Chem. Biol. 13, 521–530 (2006).
Mank, M. et al. A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change. Biophys. J. 90, 1790–1796 (2006).
Lee, M.Y. et al. Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. Proc. Natl. Acad. Sci. USA 103, 13232–13237 (2006).
Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).
Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).
Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).
Kuhn, M.A. in Fluorescent Chemosensors for Ion and Molecule Recognition (ed. Czarnik, A.W.) 147–161 (American Chemical Society, Washington, DC, 1993).
Levy, L.A., Murphy, E., Raju, B. & London, R.E. Measurement of cytosolic free magnesium ion concentration by 19F NMR. Biochemistry 27, 4041–4048 (1988).
Naraghi, M. T-jump study of calcium binding kinetics of calcium chelators. Cell Calcium 22, 255–268 (1997).
Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).
Regini, J.W. & Elliott, G.F. The effect of temperature on the Donnan potentials in biological polyelectrolyte gels: cornea and striated muscle. Int. J. Biol. Macromol. 28, 245–254 (2001).
Peracchia, C. Chemical gating of gap junction channels; roles of calcium, pH and calmodulin. Biochim. Biophys. Acta 1662, 61–80 (2004).
Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R. & Tsien, R.Y. Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508 (2003).
Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).
Rampe, D. & Lacerda, A.E. A new site for the activation of cardiac calcium channels defined by the nondihydropyridine FPL 64176. J. Pharmacol. Exp. Ther. 259, 982–987 (1991).
Church, P.J. & Stanley, E.F. Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons. J. Physiol. (Lond.) 496, 59–68 (1996).
Guia, A., Stern, M.D., Lakatta, E.G. & Josephson, I.R. Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance. Biophys. J. 80, 2742–2750 (2001).
Luo, D., Sun, H., Xiao, R.P. & Han, Q. Caffeine induced Ca2+ release and capacitative Ca2+ entry in human embryonic kidney (HEK293) cells. Eur. J. Pharmacol. 509, 109–115 (2005).
Shuai, J. & Parker, I. Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution. Cell Calcium 37, 283–299 (2005).
Navedo, M.F. et al. Constitutively active L-type Ca2+ channels. Proc. Natl. Acad. Sci. USA 102, 11112–11117 (2005).
Piedras-Renteria, E.S. et al. Increased expression of alpha(1A) Ca2+ channel currents arising from expanded trinucleotide repeats in spinocerebellar ataxia type 6. J. Neurosci. 21, 9185–9193 (2001).
Shistik, E., Ivanina, T., Puri, T., Hosey, M. & Dascal, N. Ca2+ current enhancement by α2/δ and β subunits in Xenopus oocytes: contribution of changes in channel gating and α1 protein level. J. Physiol. (Lond.) 489, 55–62 (1995).
Grabner, M., Dirksen, R.T. & Beam, K.G. Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca2+ channels expressed in dysgenic myotubes. Proc. Natl. Acad. Sci. USA 95, 1903–1908 (1998).
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.
R.Y.T. and S.R.A. are co-inventors on patents assigned to the University of California covering biarsenical dyes and tetracysteine motifs.
Tetracysteine-tagged L-type calcium channel imaged using TIRF-M. (PDF 82 kb)
Patch clamp stimulus waveforms. (PDF 28 kb)
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)
CaGF reports the spread of calcium. (AVI 3948 kb)
Spatial and temporal display of Ca2+ transients triggered by patch clamp activation of L-type calcium channels. (AVI 1947 kb)
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)
Spatial and temporal display of four Ca2+ transients triggered by patch clamp activation of L-type calcium channels. (AVI 2308 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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