Proc. Natl. Acad. Sci. USAVol. 91, pp. 187-191, January 1994Physiology

Nitrophenyl-EGTA, a photolabile chelator that selectively bindsCa2+ with high affinity and releases it rapidly upon photolysisGRAHAM C. R. ELLIS-DAVIES AND JACK H. KAPLAN*Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104-6085

Communicated by Robert E. Forster II, September 20, 1993 (received for review June 11, 1993)

ABSTRACT The synthesis and properties of a caged cal-cium are described. The compound is an ortho-nitrophenylderivative of EGTA. It is synthesized in 10 steps and with 24%overall yield. The photosensitive chelator, nitrophenyl-EGTA,has a Kd value for Ca2+ of 80 nM and for Mg2+ of 9 mM. Uponexposure to UV radiation (-350 nm), the chelator is cleaved,yielding iminodiacetic acid photoproducts with low Ca affinity(Kd = 1 mM). The quantum yield of photolysis of nitrophenyl-EGTA in the presence of Ca2+ is 0.23 and in the absence ofCa2+ is 0.20. In experiments with chemically skinned skeletalmuscle fibers, a fully relaxed fiber equilibrated with nitrophe-nyl-EGTA-Ca2+ complex, in the presence of 1 mM free Mg2+,maximally contracted after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension wasachieved in 18 ms at 15C. Nitrophenyl-EGTA provides a toolfor the investigation of the mechanism of Ca2+-dependentphysiological processes, since under conditions of normal in-tracellular Ca2+ and Mg2+ concentrations, only Ca2+ is boundby the photolabile chelator and on illumination released rapidlyand in high photochemical yield.

Ca2+ is an important second messenger for a wide variety ofphysiological and biochemical processes, such as musclecontraction, neurotransmitter release, ion-channel gating,exocytosis, etc. The essential role of Ca2+ release andsequestration in intracellular communication has also beenhighlighted by the growing appreciation of the importance ofinositol phospholipid metabolism in signaling. A techniquefor the controlled, localized, and rapid increase in Ca2+concentration would provide a tool that would enable thestudy of the kinetics and regulatory and structural mecha-nisms of such processes to be undertaken. Two approachesto this problem have been taken (for review, see ref. 1). Thefirst, developed by Tsien and coworkers (2), involves reduc-ing the Ca2+-buffering capacity ofa 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid derivative (BAPTA) bydecreasing the electron-donating capacity of one of thecoordinating ligands on illumination after the photoexpulsionof a small molecule from the chelator. This strategy has ledto two readily available photosensitive buffers, nitr-5 andnitr-7 (2). Our approach is conceptually different in that wehave designed photosensitive derivatives of chelators withknown high affinity for Ca2+ that upon illumination arebifurcated, producing two moieties with known low affinityfor Ca2+, and thus the bound Ca2+ is released (Fig. 1).1-(2-Nitro-4,5-dimethoxyphenyl)-N,N,N' ,N'-tetrakis[(oxy-carbonyl)methyl]-1,2-ethanediamine (DM-nitrophen) is acommercially available (Calbiochem) photosensitive deriva-tive of EDTA (3, 4) representative of this approach, whichhas found wide application during the last several years as acaged Ca (5-14) and caged Mg (15). The distinct advantage ofnitr-5 and nitr-7 compared to DM-nitrophen is that they areCa2+-selective chelators whereas DM-nitrophen has chela-

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tion properties similar to EDTA from which it is derived. Thecomparative advantages of DM-nitrophen are that its Ca2+affinity is very high before photolysis and very low afterphotolysis, thus ensuring a good photochemical yield ofliberated Ca2+.

In this report we describe a photosensitive Ca2+ chelator,nitrophenyl-EGTA (NP-EGTA, 1), that binds Ca2+ selectivelywith high affinity (80 nM) and upon photolysis is bifurcatedproducing iminodiacetic acid photoproducts (see Fig. 1) witha 12,500-fold lower affinity for Ca2+. NP-EGTA possesses thedesired properties of Ca2+ selectivity in combination with arapid high photochemical yield of liberated Ca2W.

METHODS, RESULTS, AND DISCUSSIONSynthesis of NP-EGTA. NP-EGTA was synthesized in 10

steps and 24% overall yield as outlined in Fig. 2.o-Nitrophenylbromoacetic acid ethyl ester (2). A solution

of o-nitrophenylacetic acid (19.0 g, 105 mmol) and ethanol(46.0 g, 1 mol) withp-toluenesulfonic acid catalyst in benzene(150 ml) was heated at reflux for 8 h, and water was removedfrom the reaction mixture by azeotropic distillation. Thesolution was extracted with a Na2CO3 solution (100 ml), andthe organic phase was concentrated in vacuo to give 19.8 g(95%, 100% based on recovered starting material) of nitro-ester as a yellow solid, m.p. 64-66°C, Rf = 0.25. A mixtureof nitroester (2.0 g, 9.56 mmol) and N-bromosuccinimide(1.98, 11.0 mmol) with benzoyl peroxide catalyst in carbontetrachloride (50 ml) was heated at reflux for 48 h. Themixture was filtered and concentrated in vacuo. Flash chro-matography purification (elution with 20%o ethyl acetate inhexane) gave 2.48 g (90%) of compound 2 as a yellow liquid:'H NMR (250 MHz, CDC13) 8 8.20 (ddd, J = 8.9, 8.1, 1.8 Hz,2H), 7.70 (td, J = 8.1, 1.8 Hz, 1H), 7.53 (td, J = 8.9, 1.8 Hz,1H), 6.07 (s, 1H), 4.29 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1Hz, 3H). Rf = 0.36. (All Rf values are given for the solventsystem used for purification.)o-Nitrophenylbromoacetaldehyde dimethyl acetal (3). To

a solution of compound 2 (3.0 g, 10.4 mmol) in dichlo-romethane (130 ml) at -78°C was added diisobutyl aluminumhydride (10.7 mmol from a 1 M solution in hexanes) dropwiseover 25 min. The reaction mixture was stirred a further 10 minand then poured into a concentrated aqueous solution ofsodium tartrate (100 ml). This mixture was stirred at roomtemperature overnight. The organic layer was separated anddried. Montmorillonite clay K 10 impregnated with trimethylorthoformate (9.0 g) was added and the reaction mixture wasstirred for 1 h. The clay was removed by filtration throughCelite and the solution was concentrated in vacuo andpurified by flash chromotography (elution with 25% ethylacetate in hexane). The combined yield from two such

Abbreviations: NP-EGTA, nitrophenyl-EGTA; caged Pi, 1-(o-nitrophenyl)ethyl phosphate; DM-nitrophen, 1-(2-nitro-4,5-dimeth-oxyphenyl)-N,N,N',N'-tetrakis[(oxycarbonyl)methyl]-1,2-ethane-diamine.*To whom reprint requests should be addressed.

187

188 Physiology: Ellis-Davies and Kaplan

N- -- :- --,C ---N hv /HN _2 _NO7hvCt 1 + HN

O*N * 02C CO2-b

FIG. 1. Photorelease of Ca2+ from NP-EGTA. Ca2+ is liberated from the chelator-cation complex by lysis of the chelator backbone. Thetwo iminodiacetic acid photoproducts have a 12,500-fold lower affinity for Ca2+ and produce a net alkalinization of unbuffered solutions as theiramine nitrogens become protonated when Ca2+ is released.

reactions was 5.83 g (95%) of compound 3 as a yellow oil: 'HNMR (250 MHz, CDC13) 8 7.93 (dd, J = 8.0,1.5 Hz, 1H), 7.83(dd, J = 8.0, 1.5 Hz, 1H), 7.62 (dt, J = 8.0, 1.5 Hz, 1H), 7.44(dt, J = 8.0, 1.5 Hz, 1H), 5.54 (d, J = 5.5 Hz, 1H), 4.67 (d,J = 5.5 Hz, 1H), 3.45 (s, 3H), 3.35 (s, 3H). Rf = 0.31.

o-Nitrophenylbromoacetaldehyde bis-2-(2-chloroethoxy)-ethyl acetal (4). A solution ofcompound 3 (1.52 g, 5.20 mmol)and 2-(2-chloroethoxy)ethanol (5.18 g, 41.6 mmol) in anisole(20 ml) with pyridinium p-toluenesulfonate catalyst washeated at 110°C for 7 h. The solution was concentrated invacuo and purified by flash chromotography (elution with25% ethyl acetate in hexane) to give 1.96 g (79%) of com-pound 4 as a yellow oil: 'H NMR (250 MHz, CDC13) 8 7.94(dd, J = 8.0, 1.4 H, 1H), 7.80 (dd, J = 8.0, 1.4 Hz, 1H), 7.60(td, J = 8.0, 1.4 Hz, 1H), 7.43 (td, J = 8.0, 1.4 Hz, 1H), 5.87(d, J = 5.5 Hz, 1H), 4.99 (d, J = 5.5 Hz, 1H), 3.91-3.50 (ni,16 H). Rf = 0.24.2-(2-Chloroethoxy)ethyl 2-o-nitrophenyl-2-bromoethyl

ether (5). A solution of compound 4 (2.59 g, 5.45 mmol) andmonochloroborane-methyl sulfide complex (2.41 g, 21.8mmol) in diethyl ether (30 ml) was heated at reflux for 24 h.Water was added; the reaction mixture was heated for 5 minand then extracted with dichloromethane. The organic phasewas dried, concentrated in vacuo, and subjected to flashchromotography on silica gel (elution with 25% ethyl acetatein hexane) to give 1.60 g (84%) ofcompound 5 as a yellow oil:'H NMR (250 MHz, CDC13) 8 7.85 (dd, J = 8.1, 1.3 Hz, 2H),7.63 (td, J = 8.1, 1.3 Hz, 1H), 7.45 (td, J = 8.1, 1.3 Hz 1H),5.82 (t, J = 6.7 Hz, 1H), 4.04 (d, J = 6.7 Hz, 1H), 3.76-3.56(m, 8H). IR (CHC13, cm-') 3020, 2970,2910,2880, 1530, 1355,1120. High-resolution mass spectrum: C12 H15 ClBr NO4requires 350.9873, M + NH' observed 369.0217. Majorfragments: 272 (23%) and 274 (8%), ArNO2CHCH2O(CH2)2-O(CH2)2CI; 228 (96%) and 230 (95%), ArNO2CHBrCH2; 137

OH

022

(100%o) and 139 (33%), CH2 O(CH2)2 O(CH2)2 Cl; 107 (100%)and 109 (33%), (CH2)2 O(CH2)2 Cl. Rf = 0.39.

2-(2-Azaethoxy)ethyl 2-o-nitrophenyl-2-azaethyl ether. Asolution of compound 5 (0.309 g, 0.876 mmol) in dimethylfor-mamide (10 ml) was heated at 110°C with sodium azide (0.171g, 2.64 mmol) for 5 h. Water was added and the product wasextracted into ether. The organic phase was dried, concen-trated in vacuo, and subjected to flash chromatography onsilica gel (elution with 25% ethyl acetate in hexane) to give0.275 g (99%) ofthe diazide as a yellow oil: 'HNMR (250 MHz,CDC13) 8 7.97 (dd, J = 8.0, 1.0 Hz, 1 H), 7.72-7.62 (m, 2H),7.48 (m, 1H), 5.51 (dd, J = 8.0, 4.7 Hz, 1H), 3.86 (dd, J = 9.5,3.7 Hz, 1H), 3.79-3.65 (m, 7H), 3.38 (t, J = 4.8 Hz, 2H). IR(CHC13, cm-') 3010, 2930, 2880, 2105, 1530, 1355, 1290, 1255,1125. Rf = 0.39.2-(2-Aminoethoxy)ethyl 2-o-nitrophenyl-2-amino ether (6).

A solution of the diazide (2.41 g, 7.61 mmol) and triphe-nylphosphine (12.0 g, 45.7 mmol) in tetrahydrofuran washeated at reflux for 1.5 h. NaOH (2 M, 40 ml) was added andthe reaction mixture was heated for 1.5 h. On cooling, 1 MHCI (100 ml) was added and the reaction mixture extractedwith dichloromethane. The pH of the aqueous phase wasadjusted to 12 with NaOH(solid) and then the product wasextracted into dichloromethane. This organic phase wasdried, concentrated in vacuo, and purified by flash chroma-tography on silica gel [elution with MeOH/CH2CI2/Et3N,5:4:1 (vol/vol)] to give 1.54 g (75%) ofcompound 6 as viscousyellow oil: 'H NMR (250 MHz, CDC13) 8 7.86 (dd, J = 8.0,1.0 Hz, 1H), 7.77 (dd, J = 8.0, 1.4 Hz, 1H), 7.59 (dt, J = 7.8,1.4 Hz, 1H), 7.36 (dt, J = 8.2, 1.4 Hz, 1H), 4.68 (dd, J = 8.0,3.7 Hz, 1H), 3.73-3.52 (m, 8H), 2.99 (t, J = 5.2 Hz, 2H). IR(CHC13, cm-') 3380, 3300, 2960, 1530, 1360. Rf = 0.43.

4-(2-Nitrophenyl)-3,13-bis[(ethoxycarbonyl)methyll-6,9-dioxa-3,12-diazatetradecanedioic acid diethyl ester. A solu-tion of compound 6 (1.54 g, 5.72 mmol), ethyl bromoacetate

Br

CO (vi) BH2ClISMe2

NO25

(vii) NaN3(vili) Ph3P. NaOH

NH2

0_..'/° O.._oN NH2 (lx) BrCH2CO2Et

(x) NaOHNO2

6

Br

N02

4

N202C CO2Na

KN)

INO2 rNm

NaO2C CO2Na

nftrophenyl-EGTA

FIG. 2. Synthesis of NP-EGTA.

Br Br

OEt ~ L OMe(i) EtOH N (1i1) Dibal b3(11) NBS No20 (Iv) (MeO)3CH NO2

2(v) HOCH2CH20CH2CH2CI

Proc. Natl. Acad Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994)

(9.52 g, 57.0 mmol), Nal (8.55 g, 57.0 mmol), and 1,2,2,6,6-pentamethylpiperidine (4.44 g, 28.6 mmol) in acetonitrile (60ml) was heated at reflux for 14 h. Flash chromatographicpurification on silica gel (elution with 50%o ethyl acetate inhexane) gave 2.29 g (65%) of NP-EGTA tetraethyl ester as ayellow oil: 1H NMR (250 MHz, CDCl3) 8 7.97 (dd, J = 8.0, 1.5Hz, 1H) 7.74 (dd, J = 8.0, 1.4 Hz, 1H) 7.55 (dt, J = 8.5, 1.4Hz, 1H) 7.37 (dt, J = 8.5, 1.4 Hz, 1H) 4.91 (dd, J = 6.6, 3.4Hz, 1H) 4.21-4.06 (m, 8H) 3.80 (dd, J = 11.2, 6.6 Hz, 1H)3.71-3.56 (m,7H) 2.91 (t, J = 5.6 Hz, 2H) 1.29-1.19 (m, 12H).13C NMR (62.9 MHz, CDCl3) 8 171.4, 171.2, 150.0, 135.5,132.5, 130.4, 123.8, 73.0, 70.2, 70.1, 69.9, 58.9, 55.7, 53.5, 52.8,14.1. IR (CHCl3, cm-') 3040, 2990, 2950, 2910, 2880, 1740,1530, 1375, 1355, 1200. UV (EtOH) 6250 = 4.14 x 103M-1 cm-1. High-resolution mass spectrum: C28 H43 N3 012requires 613.2847; M + H+ 614.2902 was observed. Rf = 0.13.NP-EGTA (1). A solution of NP-EGTA tetraethyl ester

(0.599 g, 0.971 mmol) and NaOH (4.4 mmol) in 50% aqueousethanol was heated at 60°C for 18 h. Complete hydrolysis ofthe tetraester to give NP-EGTA, compound 1, was effectedas shown by 1H NMR (250 Hz, 2H20) 8 7.86 (t, J = 6.8 Hz,2H) 7.72 (t, J = 7.4 Hz, 1H) 7.53 (t, J = 7.9 Hz, 1H) 4.95 (t,J = 6.0 Hz, 1H) 4.00 (dd, J = 9.0, 4.5 Hz, 1H) 3.83 (dd, J =10.4, 4.2 Hz, 1H) 3.77-3.59 (m, 4H) 3.47 (t, J = 5.7 Hz, 2H)3.32-3.08 (m, 8H) 2.68 (t, J = 5.7 Hz, 2H).

Spectral Properties. The absorption spectra of NP-EGTAwithout and with saturating Ca2+ are shown in Fig. 3, spectraa and b, respectively. The extinction coefficients at 260 and347 nm are 5.52 x 103 M-1 cm-1 and 974 M-1-cm-1, respec-tively, in Ca2+-free buffer (40 mM Hepes/100 mM KCI, pH7.2). Saturating Ca2+ produces a 10% decrease in the absorp-tion at 260 nm. The absorption spectrum changes to thatshown in Fig. 3, spectrum c, upon complete photolysis in thepresence of Ca2+. Fluorescence emission spectra of NP-EGTA show that this probe is virtually nonfluorescent (e.g.,fluo-3 saturated with Ca2+ is 2.1 x 105-fold more fluorescentat 530 nm than NP-EGTA is at 400 nm; data not shown), ashas been reported for DM-nitrophen (16).Quantum Yield. The quantum yield of NP-EGTA photol-

ysis was determined by comparison to that of 1-(o-nitrophenyl)ethyl phosphate (caged Pi), which has been de-termined (17) to have a quantum yield of photolysis of 0.54.The time course of disappearance of the caged compoundswas followed by HPLC analysis of the reaction mixture (Fig.4). The amount of NP-EGTA or caged Pi remaining in thereaction mixture was normalized for each HPLC analysis bycomparison to a photochemically inert internal standard,inosine (100 ,uM). HPLC analysis indicated that after 15 s,15.1% of the caged Pi had been photolyzed (Fig. 4a). Underthe same conditions (i.e., flux density), 10.3% of NP-EGTA

1.00

0.80

u

0.60.0f0

a 0.40

0.20

a

c

350 400Wavelength, nm

FIG. 3. Absorption spectra of NP-EGTA. The absorption spec-trum of a solution of NP-EGTA (66.5 AM) in buffer (40 mMHepes/100 mM KCI, pH 7.2) is shown (spectrum a). The spectrumis altered (spectrum b) on addition of saturating Ca2+ (0.20 mM).Photolysis of this sample for 300 s with the filtered output of a 1-kWHg arc lamp yielded photoproducts with spectrum c. Spectra weremeasured using a Perkin-Elmer Lambda 6.

0.4

. 0.2-0

.0

0.0-

0.4-

e

., 0.2-0

0.0 -

a

.. ....... ... I.

caged-Pi

inosdne l...I 1 ,

10 20 30

0.4

NP-EGTA

0.2

inosine

....A.... - 0.0--

10 20 30Time, min

10 20 30

FIG. 4. Quantum yield of NP-EGTA photolysis. The filteredoutput of 1000-W high-pressure Hg arc lamp was used to photolyzesolutions ofNP-EGTA (1.77 mM, A350 = 0.165) or caged P1 (3.12 mM,A350 = 0.105) in buffer (40 mM Hepes/100 mM KCI, pH 7.2) in acuvette with a pathlength of 1.0 mm. Each solution also containedinosine (100 ,uM) as the internal standard. (a) HPLC analysis ofphotolysis of caged Pi. On the left, t = 0 s; on the right, t = 15 s. (b)HPLC analysis of the photolysis of the NP-EGTA-Ca2+ complex.On the left, t = 0 s; on the right, t = 15 s. HPLC was performed usinga Novapak (Waters) C18 reverse-phase HPLC column and twoWaters 501 pumps fitted with a Waters 680 automated control moduledelivering 2 ml/min. Pump A delivered 100o H20 + 2.5% trifluoro-acetic acid and pump B delivered 80% acetonitrile/20%o (vol/vol)H20 + 0.052% trifluoroacetic acid. The gradient was for 0-5 min100o A, for 5-20 min a linear change to 80%6 A, and for 20-40 min80%o A. The eluent was monitored by a Waters 990 photodiode arraydetector and chromatograms are shown at 260 nm.

was photolyzed when saturating [Ca2+] (3.0 mM) was present(Fig. 4b). Thus, by taking into account the different A350values of NP-EGTA and caged Pi, the quantum yield ofphotolysis of the NP-EGTA-Ca2+ complex is 0.23. In Ca2+-free conditions, HPLC analysis (data not shown) indicatedthat 8.91% of NP-EGTA was photolyzed, implying that thequantum yield of photolysis of cation-free chelator is 0.20;therefore, the Ca2+-free chelator is photolyzed with approx-imately the same efficiency as the NP-EGTA-Ca2+ complex.This is about six times greater than nitr-5 or nitr-7 (2) andabout the same as DM-nitrophen (4).

Cation Binding and Release. The affinity of NP-EGTA forCa2+ was obtained by titration of the chelator with incre-

Physiology: Ellis-Davies and Kaplan 189

.; .....

190 Physiology: Ellis-Davies and Kaplan

mental additions of Ca2+. [Ca2+Ifree (and hence [Ca2+Ibound byNP-EGTA) was measured using fluo-3, a Ca2+-selectiveindicator that shows a fluorescence emission increase whenCa2+ binds (18). The increases in Ca2+-indicator fluorescenceare shown in Fig. Sa. Simultaneous measurement of [Ca2+Ifreeusing a Ca-selective electrode prepared according to Am-mann et al. (20) gave identical estimates of [Ca2+I. A Scat-chard analysis (Fig. Sa Inset) of these data indicates the Kdvalue of NP-EGTA for Ca2+ is 80 nM. This is lower than theparent EGTA chelator (150 nM at pH 7.2) but is slightlyhigher than nitr-7 (54 nM; ref. 2) and is considerably higherthan the EDTA-derived molecule, DM-nitrophen (5 nM; ref.4). The properties of these four chelators are shown in Table1.For any chelator-Ca2+ complex to function effectively as

a caged Ca, the prephotolysis affinity should be high for tworeasons. First, it is crucial that the resting or prephotolysis[Ca2+] is very low, i.e., that it is nonactivating or subthresh-old for the biological system being investigated. The Kcavalue and the total amounts of Ca2+ and chelator determine[Ca2+]free. Second, the percentage of chelator loaded withCa2+ before photolysis is important because this is a factorthat determines how much Ca2+ is liberated upon photolysis,as Ca2+ can only be photoreleased if it is complexed. Also,if the chelator is fully loaded, then the photoreleased Ca2+will not be rebound by an unphotolyzed unloaded chelator;i.e., step increases rather than pulses of Ca2+ will be pro-duced (see refs. 21 and 22).The Kd value of the photoproducts also influences the

amount of Ca2+ released upon photolysis. The very efficientrelease ofCa2+ from NP-EGTA (and DM-nitrophen) is a resultof the fact that the iminodiacetic acid photoproducts (Fig. 1)have Kd'values for Ca2+ in the millimolar range. Thus, thechange in afiTnity for Ca2+ upon photolysis is =12,500-fold(nitr-7 changes by 56-fold; ref. 2). Table 2 compares the free[Ca2+] before and after 509o photolysis of four currently

1000-a 8000

4000800

0 200 400 600[Cal bound, uM

600-

520 540 560Wavelength, nm

Table 1. Properties of NP-EGTA, DM-nitrophen, nitr-5,and nir-7

Kd(Ca2+), M Kd(Mg2+), QuantumChelator Before After M yield

NP-EGTA 8.0 x 10-8 1.0 x 10-3 9.0 X 10-3 0.23DM-nitrophen 5.0 x 10-9 3.0 x 10-3 2.5 x 10-6 0.18Nitr-5 1.45 x 10-7 6.3 x 10-6 8.5 x 10-3 0.035Nitr-7 5.4 x 10-8 3.0 x 10-6 5.4 x 10-3 0.042Kd values before and after photolysis are given for pH 7.2 and

100-150 mM ionic strength. The Kd value for Mg2+ is given onlybefore photolysis.

available photosensitive chelators (NP-EGTA, DM-nitro-phen, nitr-5, and nitr-7) at three concentrations of Ca2+. Thus,resting [Ca2+] in a frog skeletal muscle fiber (0.19-0.28 ,uM,ref. 23) can be achieved with [chelator] and [Ca2+] at 2mM and1.50 mM, respectively, for NP-EGTA and nitr-7. When 50%ophotolysis is accomplished, only 3 uM Ca2+ is released bynitr-7, whereas 186 ,tM Ca2+ is liberated using NP-EGTA. Tohave a prephotolysis [Ca2+] of 0.145 ,uM, using nitr-5 as thephotosensitive buffer, only =1.0mM Ca2+ can be present (seeTable 2), and upon 50%o photolysis, 0.80 ,uM Ca2+ is released.To activate full contraction of a frog skeletal muscle fiber,[Ca2+]frt, should rise to -5 ,uM (24); however, the totalamount of Ca2+ that must be released to attain this [Ca2N] istwo orders of magnitude higher than this value, as the Ca2+-buffering capacity of the muscle fiber is -350 ,uM (25).Therefore, large changes in the Kd value upon photolysis arenecessary to evoke sufficient Ca2+ release from a caged Ca toproduce full tension in a skeletal muscle fiber.A clear advantage of the nitr series of photosensitive

chelators compared to DM-nitrophen is their high selectivityfor Ca2+ over Mg2+ (e.g., for nitr-7, Kmg = 5.4 mM).NP-EGTA possesses similar discrimination properties. Theselectivity of NP-EGTA for Ca2+ vs. Mg2+ is demonstrated

540 560Wavelength, nm

FIG. 5. NP-EGTA divalent cation affinities. (a) Emission spectra of fluo-3 (10 ,uM, excitation at 490 nm) resulting from titration at 20°C ofNP-EGTA (0.665 mM in 40 mM Hepes/100 mM KCI, pH 7.2) with 0.10 mM incremental additions of Ca2+. (Inset) Scatchard analysis (usinga Kd value of 500 nM for fluo-3; ref. 19) of these data. (b) Emission spectra of fluo-3 under the same conditions as in a. Lower curve, [Ca2+]= 0; intermediate curve, 0.55 mM Ca2+ and 0.55 mM Ca2+ + 1.0 mM Mg2+ (Mg2+ had no effect on the fluo-3 signal); uppermost curve, 0.55mM Ca2+ + 1.0 mM Mg2+ following irradiation with a frequency-doubled ruby laser. Spectra were measured using a Perkin-Elmer LS-50 with5-mm slit widths.

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 191

Table 2. [Ca2+]free before and after photolysis at three ratios of chelator/Ca2+,tOaW for NP-EGTA, DM-nitrophen, nitr-5, and nir-7

[Ca]free, JUM2 mM/1.0 mM 2 mM/1.50 mM 2 mM/1.80 mM

Chelator Before After ACa Before After ACa Before After ACaNP-EGTA 0.080 5.13 4.33 0.240 186 186 0.717 318 317DM-nitrophen 0.0050 1.73 1.625 0.015 312 312 0.045 510 510Nitr-5 0.145 0.952 0.807 0.434 6.68 6.25 1.30 22.8 21.5Nitr-7 0.054 0.402 0.348 0.162 3.16 3.0 0.485 11.5 11.0Change in [Ca2+]free is shown as ACa, assuming 50% photolysis of the chelator. Calculations were performed for pH 7.2 and 100-150 mM

ionic strength using the Kd values given in Table 1.

by the lack of effect of the addition of 1 mM Mg2+ to 0.665mM NP-EGTA plus 0.55 mM Ca2+ (see Fig. 5b). There is noincrease in the fluo-3 signal, indicating that NP-EGTA doesnot bind Mg2+ significantly in the millimolar concentrationrange. [In fact, titration of the low-affinity Mg2+ indicatormag-fura-red (20 ,AM; determined Kd value for Mg, 4.3 mM)in the absence and presence of NP-EGTA (10 mM) gave anestimate of 9 mM for the Kd value of NP-EGTA for Mg2+.This was found to be the same as that of EGTA under theconditions used: 40 mM Hepes/100 mM KCl, pH 7.2, at25°C.] Irradiation of this sample using a frequency-doubledruby laser (emission, 347 nm) liberates Ca2+ from the NP-EGTA-Ca2+ complex as shown by the increase in the fluo-3signal (Fig. Sb). Thus, at intracellular [Ca2+] and [Mg2+] onlyCa2+ is bound to the chelator and liberated upon photolysis.Rapid Ca2+ Release in Rabbit Skeletal Muscle Fiber. Pho-

tolysis of NP-EGTA-Ca2+ and DM-nitrophen-Ca2+ com-plexes was used to release Ca2+ rapidly in chemically skinnedpsoas fibers from rabbit skeletal muscle. Rapid developmentof maximal tension was elicited under two conditions: (i)[NP-EGTA-Ca2+] = 1.9 mM, [ATP-Mg2+] = 5 mM, and[Mg2+]free = 1 mM and (ii) [DM-nitrophen-Ca2+] = 1.4 mM,[ATP-Mg2+] = 0.62 mM, and [Mg2+]free = 40 AM (Fig. 6).The time to achieve half-maximal tension was 17.9 ms withNP-EGTA and 18.1 ms with DM-nitrophen at 15°C (Fig. 6).We have shown (21) that Ca2+ release from the DM-nitrophen-Ca2+ complex is very rapid (n/2 c 180 ,s), andtherefore, the photorelease process is not rate-limiting forcontraction in skeletal muscle fibers. The fact that photore-lease ofCa2+ from NP-EGTA-Ca2+ and DM-nitrophen-Ca2+

I

L-

LJ

50 ms

FIG. 6. Ca-induced contraction of skinned skeletal muscle fibers.The arrow indicates the time of a pulse from a frequency-doubledruby laser (347 nm). For DM-nitrophen, before photolysis, themuscle-bathing solution contained 100 mM Tes, 27.91 mM 1,6-diaminohexane-N,N,N',N'-tetraacetic acid (HDTA), 3.0 mM Na2ATP, 20 mM creatine phosphate, 10 mM glutathione, 2.0 mMDM-nitrophen, 1.19mM MgCl2, and 1.60 mM CaCl2. The NP-EGTAsolution contained 100 mM Tes, 25.35 mM HDTA, 5.60 mM Na2-ATP, 20 mM creatine phosphate, 10 mM glutathione, 2.10 mMNP-EGTA, 7.0 mM MgCl2, and 1.9 mM CaCl2. The pH of bothsolutions was 7.1; the ionic strength was 0.2 M. Tension developmentwas measured as in ref. 26.

elicits identical rapid tension transients in skeletal muscleindicates that release of Ca2+ from NP-EGTA is also notrate-limiting. Furthermore, under the conditions of photoly-sis (100-mJ pulse from a frequency-doubled ruby laser), theNP-EGTA-Ca2+ complex releases sufficient Ca2+ to gener-ate full tension in the muscle fiber. It is interesting that thereare no major effects on the rate of the development oftensionor on the maximal contraction due to a change in [Mg]free from40 ,uM to 1 mM in these experiments.

In summary, we have developed and characterized aCa2+-specific chelator that has high affinity for Ca2+ andupon photolysis rapidly releases the complexed Ca2+ withgood quantum efficiency. We anticipate that NP-EGTA willbe useful for studying intracellular processes involvingchanges in [Ca2+]. Furthermore, in conjunction with DM-nitrophen, systematic studies on the role of Mg2+ in theseprocesses can also be undertaken.We thank Drs. Yale Goldman and Taylor Allen for performing with

us the muscle fiber experiments, Dr. Steve Baylor for help with theaffinity measurements and the calculations for Table 2, and Dr. BobBarsotti for help with the spectral measurements in Figs. 3 and 5.This work was supported by National Institutes of Health GrantsGM39500 and HL30315 to J.H.K.1. Kaplan, J. H. (1990) Annu. Rev. Physiol. 52, 897-914.2. Adams, S. R., Kao, J. P. Y., Grynkiewicz, G., Minta, A. & Tsien, R. Y.

(1988) J. Am. Chem. Soc. 110, 3212-3220.3. Ellis-Davies, G. C. R. & Kaplan, J. H. (1988)J. Org. Chem. 53,1966-1969.4. Kaplan, J. H. & Ellis-Davies, G. C. R. (1988) Proc. Natl. Acad. Sci.

USA 85, 6571-6575.5. Morad, M., Davies, N. W., Kaplan, J. H. & Lux, H. D. (1988) Science

241, 842-844.6. Niggli, E. & Lipp, P. (1993) Biophys. J. 55, 400a (abstr.).7. Naebauer, M., Ellis-Davies, G. C. R., Kaplan, J. H. & Morad, M. (1989)

Am. J. Physiol. 256, H916-H920.8. Delaney, K. R. & Zucker, R. S. (1990) J. Physiol. (London) 426, 473-498.9. Rapp, G., Poole, K. J. V., Maeda, Y., Kaplan, J. H., Ellis-Davies,

G. C. R., McCray, J. A. & Goody, R. S. (1990)Bunsenges. Phys. Chem.93, 410-424.

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man, Y. E., Martyn, D. A. & Gordon, A. M. (1993) Biophys. J. 64, 135a(abstr.).

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Physiology: Ellis-Davies and Kaplan