A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Differential integration of Ca2+-calmodulin signal in intact ventricular myocytes at low and high affinity Ca2+-calmodulin targets

Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.     

Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization.

The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, indicating that InsP3 binding did not cause desensitization. When Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP3 caused rapid, partial decreases in fluorescence ratios. Subsequent addition of a maximal InsP3 concentration caused the fluorescence to fall to within 5% of that recorded after ionomycin. Addition of all but the lowest concentrations of InsP3 to stores loaded with the lower affinity indicator, Calcium Green-5N, caused almost complete emptying of the stores at rates that increased with InsP3 concentration. The lowest concentration of InsP3 (10 nM) slowly emptied approximately 80% of the stores, but within 3 min the rate of Ca2+ release slowed leaving approximately 7 microM Ca2+ within the stores, which was then rapidly released by a maximal InsP3 concentration. In stores co-loaded with both indicators, InsP3-evoked Ca2+ release appeared quantal with Fura 2 and largely non-quantal with Calcium Green-5N; the discrepancy is not, therefore, a direct effect of the indicators. The fall in luminal [Ca2+] after activation of InsP3 receptors may, therefore, cause their inactivation, but only after the Ca2+ content of the stores has fallen by approximately 95% to < or = 10 microM.     

Subplasma membrane Ca2+ signals

Ca(2+) may selectively activate various processes in part by the cell's ability to localize changes in the concentration of the ion to specific subcellular sites. Interestingly, these Ca(2+) signals begin most often at the plasma membrane space so that understanding subplasma membrane signals is central to an appreciation of local signaling. Several experimental procedures have been developed to study Ca(2+) signals near the plasma membrane, but probably the most prevalent involve the use of fluorescent Ca(2+) indicators and fall into two general approaches. In the first, the Ca(2+) indicators themselves are specifically targeted to the subplasma membrane space to measure Ca(2+) only there. Alternatively, the indicators are allowed to be dispersed throughout the cytoplasm, but the fluorescence emanating from the Ca(2+) signals at the subplasma membrane space is selectively measured using high resolution imaging procedures. Although the targeted indicators offer an immediate appeal because of selectivity and ease of use, their limited dynamic range and slow response to changes in Ca(2+) are a shortcoming. Use of targeted indicators is also largely restricted to cultured cells. High resolution imaging applied with rapidly responding small molecule Ca(2+) indicators can be used in all cells and offers significant improvements in dynamic range and speed of response of the indicator. The approach is technically difficult, however, and realistic calibration of signals is not possible. In this review, a brief overview of local subplasma membrane Ca(2+) signals and methods for their measurement is provided.     

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.     

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.     

Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.     

A new generation of Ca2+ indicators with greatly improved fluorescence properties

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.     

Spatiotemporal features of Ca2+ buffering and diffusion in atrial cardiac myocytes with inhibited sarcoplasmic reticulum

Ca(2+) signaling in cells is largely governed by Ca(2+) diffusion and Ca(2+) binding to mobile and stationary Ca(2+) buffers, including organelles. To examine Ca(2+) signaling in cardiac atrial myocytes, a mathematical model of Ca(2+) diffusion was developed which represents several subcellular compartments, including a subsarcolemmal space with restricted diffusion, a myofilament space, and the cytosol. The model was used to quantitatively simulate experimental Ca(2+) signals in terms of amplitude, time course, and spatial features. For experimental reference data, L-type Ca(2+) currents were recorded from atrial cells with the whole-cell voltage-clamp technique. Ca(2+) signals were simultaneously imaged with the fluorescent Ca(2+) indicator Fluo-3 and a laser-scanning confocal microscope. The simulations indicate that in atrial myocytes lacking T-tubules, Ca(2+) movement from the cell membrane to the center of the cells relies strongly on the presence of mobile Ca(2+) buffers, particularly when the sarcoplasmic reticulum is inhibited pharmacologically. Furthermore, during the influx of Ca(2+) large and steep concentration gradients are predicted between the cytosol and the submicroscopically narrow subsarcolemmal space. In addition, the computations revealed that, despite its low Ca(2+) affinity, ATP acts as a significant buffer and carrier for Ca(2+), even at the modest elevations of [Ca(2+)](i) reached during influx of Ca(2+).     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside- out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.     

InsP3- and Ca2(+)-induced Ca2+ release in single mouse oocytes

To better understand the mechanism of intracellular Ca2+ mobilization, mouse oocytes were micro-injected with 'caged'-inositol-1,4,5 triphosphate caged-InsP3) together with the Ca2+ indicator Fluo-3 to directly induce and monitor Ca2+ redistribution. Photo-released InsP3 elicits [Ca2+]i changes exhibiting several kinetic phases and threshold behaviour. Often Ca2+ oscillations were induced after a single InsP3 pulse. Autoregenerative Ca2+ transients could also be induced by injections of Ca2+ itself, demonstrating unequivocally the presence of a Ca2(+)-induced Ca2(+)-release mechanism in these cells.     

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.     

The relationship between the free concentrations of Ca2+ and Ca2+-calmodulin in intact cells

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 microM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of approximately 45 nM is produced when the free Ca2+ concentration exceeds 3 microM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1 microM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is approximately 2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (Kd /= 100 nM) occurs only where free Ca2+-calmodulin concentrations can be locally enhanced.     

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers.

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

Iminocoumarin-based low affinity fluorescent Ca2+ indicators excited with visible light.

A series of iminocoumarin-based fluorescent Ca2+ indicators were synthesized and the spectral profiles of their free and Ca2+ bound forms were studied. The newly-synthesized compounds incorporate the Ca2+ chelating structure of BAPTA. The chromophore moieties are iminocoumarins substituted at the 3-position with benzothiazolyl, benzoxazolyl and benzimidazolyl groups. These compounds are excited with visible light and their Ca2+ dissociation constants range from 5.4 to 27.5 microM. Fluorescence spectra studies of these probes indicated a clear shift in their excitation wavelength maxima upon Ca2+ binding along with changes in fluorescence intensity that enable the compounds to be used as low Ca2+ affinity, visible excitable probes.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Interaction of alkali metal ions with Ca2+-binding center of Ca2+- ATPase of the sarcoplasmic reticulum in skeletal muscles

Sodium ion interaction with sarcoplasmic reticulum (SR) membranes leads to considerable alterations of the [23Na]NMR lineshape. Na+ binding to SR in the presence of Ca2+ and H+ is well described by a model which postulates a competitive ion binding to high and low affinity sites of Ca2+-ATPase. The dissociation constant, Kd, for high and low affinity sites is 5 and 10 mM, respectively, for Na+ and (3-5).10(-8) and 1.5.10(-3) M, respectively, for Ca2+. The pK value for high and low affinity sites is 7.3 and 6.1, respectively. Other alkaline metal ions compete with Na+ for the low affinity sites of Ca2+-ATPase; their affinities decrease in the following order: Na+ = K+ greater than Rb+ greater than Cs greater than Li+. Some of the Na+ binding sites (approximately 10%) do not interact with Ca2+.