Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.     

The crystal structures of semi-synthetic aequorins

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.     

Isolation and properties of various molecular forms of aequorin.

The photoprotein aequorin emits light by an intramolecular reaction when a trace of Ca2+ is added. The samples of aequorin that were purified by the conventional methods of column chromatography were separated by high-performance liquid chromatography into eight molecular forms (isoaequorins), which were designated aequorins A-H. Aequorins A, C and F were obtained in crystalline states. A wide range of properties were studied with aequorins A-F, which were essentially pure. These six isoaequorins showed relatively small differences in their spectroscopic properties, but their values of A0.1%/1 cm, 280 were found to be close to 3.0, about 10% more than the previously reported value of 2.70-2.71 that was obtained with the samples of conventionally purified aequorin. The Mr values ranged from 20,100 (aequorin F) to 22,800 (aequorin A), the luminescence activities ranged from 4.35 X 10(15) photons/mg (aequorin A) to 5.16 X 10(15) photons/mg (aequorin F), and the first-order reaction rate constants of luminescence ranged from 0.95 s-1 (aequorin A) to 1.33 s-1 (aequorin F). As regards sensitivity to Ca2+, aequorin D was the most sensitive, having a sensitivity about 0.4-0.5 pCa unit above that of the least sensitive kind (aequorin A).     

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.     

Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.

Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.     

Effect of calcium chelators on the Ca2+-dependent luminescence of aequorin.

The luminescence of aequorin, a useful tool for studying intracellular Ca2+, was recently found to be inhibited by the free EDTA and EGTA that are present in calcium buffers. In the present study we have examined the effect of the free forms of various chelators in the calibration of [Ca2+] with aequorin. Free EDTA and EGTA in low-ionic-strength solutions strongly inhibited the Ca2+-triggered luminescence of aequorin, causing large errors in the calibration of [Ca2+] (approx. 2 pCa units), whereas in solutions containing 150mM-KCl, errors were relatively small (0.2-0.3 pCa units). Citric acid in low-ionic-strength solutions and [(carbamoylmethyl)imino]diacetic acid in high-ionic-strength solutions showed no inhibition and did not cause detectable error in the calibration of [Ca2+], indicating that they are better chelators than EDTA and EGTA for use with aequorin.     

Calcium-sensitive photoproteins

The recombinant Ca2+ sensitive photoprotein aequorin was the first probe used to measure specifically the Ca2+ concentration, [Ca2+], inside the intracellular organelles of intact cells. Aequorin-based methods offer several advantages: (i) targeting of the probe is extremely precise, thus permitting a selective intracellular distribution; (ii) the use of wild-type and low Ca2+-affinity aequorins allows covering a large dynamic range of [Ca2+], from 10(-7) to 10(-3)M; (iii) aequorin has a low Ca2+ buffering effect and it is nearly insensitive to changes in Mg2+ or pH; (iv) it has a high signal-to-noise ratio; (v) calibration of the results in [Ca2+] is made straightforward using a simple algorithm; and (vi) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine is necessary to generate the functional photoprotein and this procedure requires at least 1h; (iii) in the case of aequorin targeted to high Ca2+ compartments, because of the high rate of aequorin consumption at steady-state, only relatively brief experiments can be performed and, because of the steepness of the Ca2+-response curve, the calibrated [Ca2+] values may not reflect the real mean in cells or compartments with dyshomogeneous behavior; and (iv) expression of targeted aequorins requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones.     

The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.     

Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration.

The photoprotein aequorin isolated from the jellyfish Aequorea emits blue light in the presence of Ca2+ by an intramolecular process that involves chemical transformation of the coelenterazine moiety into coelenteramide and CO2. Because of its high sensitivity to Ca2+, aequorin has widely been used as a Ca2+ indicator in various biological systems. We have replaced the coelenterazine moiety in the protein with several synthetic coelenterazine analogues, providing semi-synthetic Ca2+-sensitive photoproteins. One of the semi-synthetic photoproteins, derived from coelenterazine analogue (II) (with an extra ethano group), showed highly promising properties for the measurement of Ca2+, namely (1) the rise time of luminescence in response to Ca2+ was shortened by approx. 4-fold compared with native aequorin and (2) the luminescence spectrum showed two peaks at 405 nm and 465 nm and the ratio of their peak heights was dependent on Ca2+ concentration in the range of pCa 5-7, thus allowing the determination of [Ca2+] directly from the ratio of two peak intensities. Coelenterazine analogue (I) (with a hydroxy group replaced by an amino group) was also incorporated into apo-aequorin, yielding a Ca2+-sensitive photoprotein, which indicates that an electrostatic interaction between the phenolate group in the coelenterazine moiety and some cationic centre in apo-aequorin is not important in native aequorin, contrary to a previous suggestion.     

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.     

Bioluminescence of the Ca-binding photoprotein, aequorin, after histidine modification

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His¹⁶⁹ may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.     

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.     

Halistaurin, phialidin and modified forms of aequorin as Ca2+ indicator in biological systems.

Two kinds of aequorin-type photoproteins, i.e., halistaurin and phialidin, and four kinds of modified forms of aequorin, i.e., products of acetylation, ethoxycarbonylation, fluorescamine-modification and fluorescein labelling, were prepared. The modified forms of aequorin were more sensitive to Ca2+ than was aequorin in their Ca2+-triggered luminescence reactions, whereas halistaurin and phialidin were less sensitive. The emission maxima of luminescence were all within a wavelength range 450-464 nm, except for fluorescein-labelled aequorin, which emitted yellowish light (lambda max. 520 nm). A new technique of measuring Ca2+ concentration is suggested.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

Ionized calcium concentrations in squid axons

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.     

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]_i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]_i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]_i is high, but there is a region of [Ca]_i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]_i change.     

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]i is high, but there is a region of [Ca]i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]i change.     

Intracellular calibration of the fluorescent calcium indicator Fura-2

We present the techniques we have used and the problems we have encountered in our laboratories in the in vivo calibration of the fluorescent Ca2(+)-indicator Fura-2. These techniques include the use of potentiometric methods for the precise control and determination of Ca2+ levels in bathing solutions, in association with methods for the equilibration of internal and external solutions with ionophores (Br-A23187, ionomycin, monensin and nigericin). A by-product of these techniques has been the development of a simple procedure that utilizes Fura-2 as a general indicator of ionized Ca2+ concentrations within the physiological range (pCa 7.5 to 5.5), in other experimental solutions. The major advantages of this relatively simple procedure are that it is (i) rapidly performed, (ii) independent of the total EGTA concentration within each experimental solution, (iii) independent of the absolute EGTA purity, and (iv) unaffected by a large number of potentially interfering cations (i.e. Mg2+, H+, K+, Na+) within the test solutions.     

A novel single-photon counting technique applied to highly sensitive measurement of [Ca2+]i transient in human aortic smooth muscle cells

This study demonstrates that aequorin, a luminescent natural dye, is useful for vascular cell intracellular Ca2+ concentration ([Ca2+]i) determination. A new single-photon counting technique was developed to resolve the effects of fluid flow shear stress on [Ca2+]i in human aortic smooth muscle cells (HASMCs). Confluent HASMCs were grown on petri dishes loaded with aequorin. Then the dishes were placed in a luminometer chamber after the physiological level of shear stress was applied to the HASMC surfaces. The chamber was housed inside a highly sensitive photomultiplier tube. It detected ultraweak photon emission in response to the [Ca2+]i transient. In the presence of 2.0 mM extracellular Ca2+, a shear stress of 12 dyn cm2, applied for 60 s to the top surface of the HASMC monolayer, elicited a sharp increase in [Ca2+]i.