Cell Calcium 39 (2006) 471–473

Commentary

DM-nitrophen AM is caged magnesium

Graham C.R. Ellis-DaviesDepartment of Pharmacology and Physiology, Drexel University College of Medicine,

245 N. 15th Street, Philadelphia, PA 19102, USA

Available online 17 April 2006

Abstract

DM-nitrophen is a photolabile derivative of EDTA and therefore functions as either caged Ca2+ or caged Mg2+. Several papers have beenpublished recently which use the AM ester to load intact cells with DM-nitrophen; under these conditions it is caged Mg2+. In this commentary,I give a short history of the development and application of caged Ca2+ probes, explaining why EGTA and BAPTA-based Ca2+ cages are tobe preferred for the photorelease of Ca2+ under normal intracellular conditions.© 2006 Elsevier Ltd. All rights reserved.

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The study of Ca2+ signalling was revolutionized in 1980hen Roger Tsien made quin2 [1]. Not satisfied with mereassive observation of [Ca2+], Tsien realized that a full under-tanding of Ca2+-signalling cascades could only be achievedy development of probes that enabled photomanipulation ofhe cation. Therefore, he made the first Ca2+-selective cagen 1986, called nitr-2 [2], shortly after the introduction ofura-2 [3]. However, nitr-2 was deficient in some of its prop-rties, therefore the Tsien lab made several other Ca2+ cages,he most widely used of which is called nitr-5 [4]. Theserobes use the original caged ATP photochemistry [5], toeduce the Ca buffering capacity of BAPTA by 40-fold. Inde-endently of the Tsien group, I took a different approacho caging Ca2+ and synthesized photolabile derivatives ofGTA and EDTA [6]. The EDTA derivative was called DM-itrophen, and has been commercially available for manyears from Calbiochem (Molecular Probes call this moleculeMNP–EDTA). The full binding constants of DM-nitrophen

or Ca2+, Mg2+, Ba2+, and protons were published in 19897]. These values are fairly similar to the parent chelator,DTA, except that the pKa of one nitrogen atom is lower due

the study of excitation–secretion coupling. The Ca2+ sensorsof secretory machinery work very quickly, and to accomplishthis, they possess fairly (or very) low affinities for Ca2+, whichin turn require high [Ca2+]. Photolysis of DM-nitrophen isthe best way to do this, as it enables the uncaging of largequantities of Ca2+.

Since the development of caged Ca, literally hundreds ofstudies have been published using these probes. The real pio-neer in their use was Bob Zucker (see ref. [8] for reviews).The 1993 study by Neher and Zucker [9] was important,as they measured Ca2+-driven membrane fusion, and thuswere closer to the Ca2+ sensor in the signalling cascade thanearlier measurements of postsynaptic current. Almers andco-workers published analogous work on melanotrophs atthe same time [10]. Both groups used flash photolysis ofDM-nitrophen to stimulate secretion. Cells were loaded withknown concentrations of DM-nitrophen, Ca2+ and Ca2+ dye(no Mg2+ or Mg2+ATP) via a patch pipette, and whole-cellcapacitance measurements were used to follow Ca2+-drivensecretion quantitatively.

Lipp and Niggli were the first to accomplish 2-photon

o the electron withdrawing capacity of the adjacent chro-

ophore. Thus, at pH 7.2, DM-nitrophen has affinities fora2+ and Mg2+ of 5 nM and 2.5 �M, respectively, and irradia-

ion decreases the Ca2+ affinity 600,000-fold, to about 3 mM.

uncaging of any signalling molecule in living cells [11].They photolyzed DM-nitrophen in acutely isolated cardiacmyocytes, on the surface of the SR, to initiate CICR, anddepending on the laser intensity this resulted in Ca2+ sparksotD

his large change in affinity has proved extremely useful in

E-mail address: ged@drexel.edu.

143-4160/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2006.02.002

r waves. They used whole-cell patch clamp to dialyzehe cell with a known concentration of caged Ca2+ (i.e.M-nitrophen:Ca2+ complex), and like Neher, Zucker and

472 G.C.R. Ellis-Davies / Cell Calcium 39 (2006) 471–473

Table 1Properties of all the calcium cages

Ca Kd (nM) Product Kd (mM) Mg Kd (mM) φ ε (M−1 cm−1) φ·ε Rate calcium release (s−1) 2PCS (GM)

DM-nitrophen [7] 5 3.0 0.0025 0.18 4300 774 38000 0.01nitr-5 [4] 145 0.0063 8.5 0.012 5500 66 2500a NDb

NP-EGTA [12] 80 1.0 9.0 0.23 975 224 68000 0.001azid-1 [25] 230 0.12 8.0 1.0 33000 33000 500 1DMNPE-4 [23] 48 2.0 7 0.09 5120 461 45000 0.01NDBF-EGTA [24] 100 2.0 15 0.7 18400 12880 20000 0.6

Notes and abbreviations: φ, quantum yield; ε, molar extinction coefficient; 2PCS, 2-photon cross section.a Rate of appearance of organic photoproduct, Ca2+ probably released at same rate.b Probably 0.01, by analogy with DM-nitrophen; DMNPE-4 and NDBF-EGTA will become commercially available in 2006 from www.poseidonprobes.com.

Almers, no Mg was included in the internal solution becauseof the known high affinity of DM-nitrophen for this cation.

With the development of Ca2+-selective cages basedupon EGTA [12,13], I collaborated with both the Almersand Niggli groups in studies that compared DM-nitrophenwith NP-EGTA [14] and DMNPE-4 [15] using UV and2-photon uncaging. In the latter paper, we showed thatMg2+ has a profound effect on the ability of photolysisof DM-nitrophen to uncage Ca2+. These data are con-sistent with the cation binding properties of this probe[7]. Indeed, calculations suggest that under “typicalintracellular conditions” (viz. 100 nM Ca2+, 1 mM Mg2+,5 mM Mg2+ATP, pH 7.2), DM-nitrophen is 97% loadedwith magnesium. Thus, experiments that do not preciselycontrol the relative divalent cation concentrations (e.g.the AM-ester loading technique) are fated not to allowefficient Ca2+ uncaging from photolysis of DM-nitrophen.There have been at least six papers published recentlythat claim to effect intracellular uncaging of Ca2+ in cellsloaded with DM-nitrophen AM (from Molecular Probes),either by UV or 2-photon photolysis [16–21]. I feel that thedivalent cation binding properties of DM-nitrophen (sum-marized in Table 1 and ref. [22]—download this reference athttp://www.pages.drexel.edu/∼ge24/publications/index.html)must call in to question all that has been concluded in thesestudies that involves the use of caged Ca2+. I am writingtpaEcapp

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[3] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+

indicators with greatly improved fluorescence properties, J. Biol.Chem. 260 (1985) 3440–3450.

[4] S.R. Adams, J.P.Y. Kao, G. Grynkiewicz, A. Minta, R.Y. Tsien,Biologically useful chelators that release Ca2+ upon illumination, J.Am. Chem. Soc. 110 (1998) 3212–3220.

[5] J.H. Kaplan, B. Forbush, J.F. Hoffman, Rapid photolytic release ofadenosine 5′-triphosphate from a protected analogue: utilization bythe Na:K pump of human red blood cell ghosts, Biochemistry 17(1978) 1929–1935.

[6] G.C.R. Ellis-Davies, J.H. Kaplan, A new class of photolabile chela-tors for the rapid release of divalent cations: generation of caged Caand caged Mg, J. Org. Chem. 53 (1988) 1966–1969.

[7] E. Grell, E. Lewitzki, H. Ruf, E. Bamberg, G.C.R. Ellis-Davies, J.H.Kaplan, P. De Weer, Caged-Ca2+: a new agent allowing liberationof free Ca2+ in biological systems by photolysis, Cell. Mol. Biol.35 (1989) 515–522.

[8] R.S. Zucker, Calcium and transmitter release at nerve terminals,Biochem. Soc. Trans. 21 (1993) 395–401;R.S. Zucker, Exocytosis: a molecular and physiological perspective,Neuron 17 (1996) 1049–1055.

[9] E. Neher, R.S. Zucker, Multiple calcium-dependent processesrelated to secretion in bovine chromaffin cells, Neuron 10 (1993)21–30.

[10] P. Thomas, J.G. Wong, W. Almers, Millisecond studies of secretionin single rat pituitary cells stimulated by flash photolysis of cagedCa2+, EMBO J. 12 (1993) 303–306.

[11] P. Lipp, E. Niggli, Fundamental calcium release vents revealedby two-photon photolysis of caged calcium in guinea-pig cardiacmyocytes, J. Physiol. 508 (1998) 801–809.

[12] G.C.R. Ellis-Davies, J.H. Kaplan, Nitrophenyl-EGTA, a photolabile

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his comment to alert the scientific community to theroblems associated with the use of DM-nitrophen AMs a caged Ca2+ probe in intact cells. I advise the use ofGTA-based [12,23,24] or BAPTA-based [4,25] calciumages, as these chelators have very low affinities for Mg2+

nd high affinities for Ca2+; their use permits the selectivehotomanipulation of Ca2+ in the presence of normal andhysiological [Mg2+].

eferences

[1] R.Y. Tsien, New calcium indicators and buffers with high selectivityagainst magnesium and protons: design, synthesis and properties ofprototype structures, Biochemistry 19 (1980) 2396–2404.

[2] R.Y. Tsien, R.S. Zucker, Control of cytoplasmic calcium with pho-tolabile tetracaroxylate 2-nitrobenhydrol chelators, Biophys. J. 50(1986) 843–853.

chelator that selectively binds Ca2+ with high affinity and releasesit rapidly upon photolysis, Proc. Natl. Acad. Sci. U.S.A. 91 (1994)187–191.

13] G.C.R. Ellis-Davies, Synthesis of photolabile EGTA derivatives,Tetrahedron Lett. 39 (1998) 953–957.

14] T.D. Parsons, G.C.R. Ellis-Davies, W. Almers, Millisecond studies ofcalcium-dependent exocytosis in pituitary melanotrophs: comparisonof the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen, Cell Calcium 19 (1996) 185–190.

15] F. DelPrincipe, M. Egger, G.C.R. Ellis-Davies, E. Niggli,Two-photon and UV-laser flash photolysis of the Ca2+ cage,dimethoxynitrophenyl-EGTA-4, Cell Calcium 25 (1998) 85–91.

16] M.F. Leite, A.D. Burgstahler, M.H. Nathanson, Ca2+ waves requiresequential activation of inositol trisphosphate receptors and ryan-odine receptors in pancreatic acini, Gastroenterology 122 (2002)415–427.

17] M.F. Leite, K. Hirata, T. Pusl, A.D. Burgstahler, K. Okazaki, J.M.Ortega, A.M. Goes, M.A. Prado, D.C. Spray, M.H. Nathanson,Molecular basis for pacemaker cells in epithelia, J. Biol. Chem.277 (2003) 16313–16323.

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[18] W. Echevarria, M.F. Leite, M.T. Guerra, W.R. Zipfel, M.H.Nathanson, Regulation of calcium signals in the nucleus by a nucle-oplasmic reticulum, Nat. Cell Biol. 5 (2003) 440–446.

[19] S.J. Mulligan, B.A. MacVicar, Calcium transients in astrocyte end-feet cause cerebrovascular constrictions, Nature 431 (2004) 195–199.

[20] T. Takano, G.F. Tian, W. Peng, N. Lou, W. Libionka, X. Han, M.Nedergaard, Astrocyte-mediated control of cerebral blood flow, Nat.Neurosci. 9 (2006) 260–267.

[21] P. Marius, M.T. Guerra, M.H. Nathanson, B.E. Ehrlich, M.F. Leite,Calcium release from ryanodine receptors in the nucleoplasmic retic-ulum, Cell Calcium 39 (2006) 65–73.

[22] G.C.R. Ellis-Davies, Development and application of caged calcium,Methods Enzymol. 360 (2003) 226–238.

[23] G.C.R. Ellis-Davies, R.J. Barsotti, Tuning caged calcium: photolabileanalogues of EGTA with improved optical and chelation properties,Cell Calcium 35 (2006) 75–83.

[24] A. Momotake, N. Lindegger, E. Niggli, R.J. Barsotti, G.C.R. Ellis-Davies, The nitrodibenzofuran chromophore—a new caging groupfor ultra efficient photolysis in living cells, Nat. Methods 3 (2006)35–41.

[25] S.R. Adams, V. Lev-Ram, R.Y. Tsien, A new caged Ca2+, azid-1,is far more photosensitive than nitrobenzyl-based chelators, Chem.Biol. 4 (1997) 867–878.