Murexide-derived in vitro electrochemical sensor for the simultaneous determination of neurochemicals.

This work highlights the protocol employed for the simultaneous electroanalysis of tryptamine, serotonin and dopamine using a conducting poly-murexide-based electrode. To date, this is the first-of-its-kind report of simultaneous electrochemical determination of these three targets. Features of the developed electrode were identified by employing FE-SEM analysis. Under optimized conditions, the analytes underwent an irreversible electro-oxidation at the modified electrode surface, with a linear range of 0.5-40 µΜ, 0.4-40.4 µΜ and 0.5-40 µΜ for dopamine, serotonin and tryptamine, respectively. The electrolytic medium employed for the sensing was a phosphate-buffered solution with pH 7. The specificity of the developed electrode was also satisfactory in the presence of other biomolecules including L-phenylalanine, L-serine, glucose and ascorbic acid. Thus, the developed murexide-derived conducting-polymer-based electrode was used for the simultaneous sensing of the neurochemicals dopamine, serotonin and tryptamine. Electroanalysis was also demonstrated for these targets in human serum.

A visual sensor array based on an indicator displacement assay for the detection of carboxylic acids.

Carboxylic acids (CAs) have been reported as potential biomarkers of specific diseases or human body odors. A visual sensor array is described here that is based on indicator displacement assays (IDAs). The arrays were prepared by spotting solutions of the following metal complexes: Murexide-Ni(II), murexide-Cu(II), zincon-Zn(II) and xylenol orange-Cu(II), with the capability of discrimination of 15 carboxylic acids (CAs) and the quantitation of pyruvic acid (PA). Clear differences can be observed through distinctive difference maps obtained within 5 min by subtraction of red, green and blue (RGB) values of digital images after and before exposure to analytes. After an analysis of multidimensional data by pattern recognition algorithms including HCA, PCA and LDA, excellent classification specificity, and accuracy of >96% were obtained for all samples. The IDA array exhibited a linear range from 10 to 1500 µM with a theoretical detection limit of 3.5 µM towards PA. Recoveries of real samples varied from 84.8% to 114.3%. As-fabricated IDA sensor array showed an excellent selectivity among other organic interfering substances and a good batch to batch reproducibility, demonstrating its robustness. All these observations suggested that the IDA sensor array is one of the most promising paths for the discrimination of CAs. Graphical abstract Schematic diagram of indicator displacement assay (a), the procedure for acquisition of difference maps (b), and pattern recognitions for CAs (c). The method uses hierarchical cluster analysis (HCA), principal component analysis (PCA) and linear discriminant analysis (LDA).

Electrochemical study of a glassy carbon electrode modified by poly-4-nitroaniline-reduced/murexide and its sensitivity for metal ions.

The electrochemical modification of a glassy carbon electrode using reduced poly-4 nitroaniline (P-4NA) and it's applicability for determination of metallic ions was performed in this study. The electrode modification was performed by cyclic voltammetry in the potential range between 0.9 V and 1.4 V vs Ag/Ag+ (in 10 mM AgNO3) at the scan rate of 100 mV/s by 50 cycles in non-aqueous media. The reduction of nitro groups on the P-4NA modified glassy carbon electrode surface was performed in the potential range between -0.1 V and -0.8 V vs Ag/AgCl(Sat. KCl) at a scan rate of 100 mV/s in 100 mM aqueous HCl solution. The reduced P-4NA glassy carbon surface was modified with the murexide. The affinity of the modified glassy carbon electrode with some metallic ions was investigated by electrochemical impedance spectroscopy method in phosphate buffer solution (pH = 5).

Spectrofluorimetric determination of nickel (ΙΙ) with murexide.

A very sensitive and selective spectrofluorimetric method has been developed for nickel (ΙΙ) determination in environmental samples. The method is based on measuring the decrease in fluorescence intensity of murexide after nickel (ΙΙ) binding. The intensity of the fluorescence emission peak was measured at ex/em 345/431 nm in several solutions with pH interval 3.0-7.0. The fluorescence intensity decrease was found to be linear in the concentration range of 0.007 mg.L(-1) to 0.1 mg.L(-1) and 0.1 mg.L(-1) to 20 mg.L(-1) of nickel (ΙΙ) by using 10(-4) M murexide at pH 3. The detection limit was found 0.004 mg.L(-1). Relatively large excesses of over 20 cations and anions do not interfere. The method was successfully applied to the analysis of nickel (ΙΙ) in sea, rain and ground water. This method is very precise and accurate (R.S.D. = 0.42% for the determination of 0.05 mg.L-(1) nickel in 10 replicates).

An indicator-displacement assay for naked-eye detection and quantification of histidine in human urine.

A simple and efficient colorimetric method for the naked-eye detection and quantification of histidine in biological fluids was developed based on an indicator-displacement assay (IDA) and the Ni(2+)-histidine affinity pair. In this IDA approach, a commercially available dye, murexide, was used as the indicator and the selective detection of histidine was achieved based on the competition between indicator and histidine for the binding with Ni(2+). The competition of histidine with murexide for Ni(2+) resulted in an obvious color change of the solution from yellow to purple, and the permitted naked-eye detection of trace histidine. The developed bioassay allows the rapid, sensitive and selective detection of histidine in urine samples, and does not need complicated sample pretreatment. The detection limit was 0.4 µM with a linear range from 2 to 30 µM. The relative standard deviation for 11 replicate detections of 8 µM histidine was 2.0%. The developed sensor was successfully applied to the determination of histidine in human urine samples with recoveries from 97 to 105%.

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide.

One aim of this article was to determine the resting concentration of free Ca(2+) in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca(2+)](SR,R)) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca(2+)](SR,R) to TMX's apparent dissociation constant for Ca(2+) (K(app)) in order to establish the capability of monitoring [Ca(2+)](SR)(t) during SR Ca(2+) release - a signal needed to determine the Ca(2+) permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K(app) as well as a dependence of K(app) on TMX concentration, the results indicate an average [Ca(2+)](SR,R) ranging from 0.43 to 1.70mM. The ratio [Ca(2+)](SR,R)/K(app) averaged 0.256, a relatively low value which should not depend on factors influencing K(app). As a result, the time course of [Ca(2+)](SR)(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.

Synthesis and characterization of cadmium selenide nanoparticles loaded on activated carbon and its efficient application for removal of muroxide from aqueous solution.

In the first, Cadmium selenide Nanoparticle loaded on activated carbon (CdSe-NP-AC) has been synthesized and characterized by different techniques including XRD and SEM. Then, this new adsorbent successfully has been applied for the removal of muroxide (MO) from aqueous solution in batch studies, while the effect of various experimental parameters like initial pH (pH(0)), contact time, amount of (CdSe-NP-AC) and initial MO concentration (C(0)) on its removal percentage was examined by one at a time optimization method. It was found following optimization of variable, the adsorption of MO onto (CdSe-NP-AC) followed pseudo-second-order kinetics and show Tempkin and Langmuir models for interpretation of experimental data. It was observed that by increasing the temperature the removal percentage was improved and the positive change in entropy (ΔS°) and heat of adsorption (ΔH°) show the endothermic nature of process, while the high negative value in Gibbs free energy change (ΔG°) indicates the feasible nature of adsorption process.

Evaluation of proton activity in microemulsions by a kinetic probe.

The decomposition reaction of the purple dye murexide in acidic media is used as a probe indicator for protons in nonionic microemulsions. The reaction kinetics primarily rely on the proton concentration and permit assessment of the proton activity in the nonionic microemulsions of water/cyclohexane/Igepal and water/heptane/Igepal. The experiments performed in the two microemulsions covered a wide range of water-to-oil mass fraction for the two systems. The kinetic runs were monitored under pseudo-first order conditions by the stopped-flow technique. The equilibrium constants for the formation of purpuric acid and the kinetic constants for the ensuing decomposition reaction fulfill a trend consistent with the micro compartmentalized nature of the multicomponent medium, and support the use of murexide as an indicator of the proton activity in microemulsions.

Limitations of the tetramethylmurexide assay for investigating the Fe(II) chelation activity of phenolic compounds.

Limitations of the colorimetric assay involving tetramethylmurexide (TMM) to determine the extent of complex formation between metal ions and phenolic compounds have been studied. Older literature reports using this method to determine bound Fe(II). Our study shows the TMM assay is inadequate when determining the Fe(II) chelation activity of phenolic preparations rich in tannin constituents on account of the high absorbance values derived by control samples (i.e., those that do not contain the TMM reagent). Phenolic test samples comprising the TMM reagent, iron ions, and tannins could not yield meaningful absorbance data on Fe(II) chelation activity. In our study, we investigated commercially available compounds, namely, sinapic acid, catechin, rutin, tannic acid, procyanidin B(2), as well as crude acetonic extracts of almonds, red lentil, buckwheat, and their low-molecular-weight and tannin fractions separated from the crude extracts by Sephadex LH-20 column chromatography. Even as little as 0.5 mg of tannins added per control sample resulted in high absorbance values to the extent of 0.4 for red lentil and almonds, and 1.3 for buckwheat. A strong correlation (r(2) = 0.98) between the content of condensed tannins, as determined by the vanillin reaction, and absorbance of control samples by the TMM assay was found for the plant extracts and their fractions. A more useful colorimetric assay to investigate the Fe(II) chelating ability of tannin-rich preparations may be the method that uses ferrozine.

Solid phase extraction using silica gel modified with murexide for preconcentration of uranium (VI) ions from water samples.

Murexide was chemically bonded to silica gel surface immobilized 3-aminopropyl trimethoxysilane (APMS) to produce the new sorbent. A solid phase extraction method using the new sorbent has been developed to separate and concentrate trace amount of uranium (VI) from aqueous samples for the measurement by spectrophotometry method using Arsenazo III reagent. The influences of some analytical parameters on the quantitative recoveries of the analyte were investigated both in batch and column methods. Quantitative recovery of U(VI) was achieved by stripping with 0.1 mol L(-1) HCl. The maximum sorption capacity of the modified silica gel was 1.13 mmol g(-1) U(VI). A high preconcentration factor value of 400 with a lower limit of detection of 1 microg L(-1) was obtained for U(VI). The practical applicability of the developed sorbent was examined using synthetic and real samples such as sea/ground water samples.

Metal binding to ligands: cadmium complexes with glutathione revisited.

We studied the interaction of gamma-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) with cadmium ions (Cd(2+)) by first performing classical potentiometric pH titration measurements and then turning to additional spectroscopic methods. To estimate the residual concentrations of free cadmium, we studied the competition of glutathione with a Cd(2+)-sensitive dye, either an absorbing dye (murexide) or a fluorescent one (FluoZin-1), and consistent results were obtained with the two dyes. In KCl-containing Tes, Mops, or Tris buffer at pH 7.0 to 7.1 and 37 degrees C (and at a total Cd(2+) concentration of 0.01 mM), results suggest that free cadmium concentration is halved when the concentration of glutathione is approximately 0.05 mM; this mainly reflects the combined apparent dissociation constant for the Cd(glutathione) 1:1 complex under these conditions. To identify the other complexes formed, we used far-UV spectroscopy of the ligand-to-metal charge transfer absorption bands. The Cd(glutathione)(2) 1:2 complex predominated over the 1:1 complex only at high millimolar concentrations of total glutathione and not at low submillimolar concentrations of total glutathione. The apparent conditional constants derived from these spectroscopy results made it possible to discriminate between sets of absolute constants that would otherwise have simulated the pH titration data similarly well in this complicated system. Related experiments showed that although the Cl(-) ions in our media competed (modestly) with glutathione for binding to Cd(2+), the buffers we had chosen did not bind Cd(2+) significantly under our conditions. Our experiments also revealed that Cd(2+) may be adsorbed onto quartz or glass vessel walls, reducing the accuracy of theoretical predictions of the concentrations of species in solution. Lastly, the experiments confirmed the rapid kinetics of formation and dissociation of the UV-absorbing Cd(glutathione)(2) 1:2 complexes. The methods described here may be useful for biochemists needing to determine conditional binding constants for charge transfer metal-ligand complexes under their own conditions.

A simple flow-injection spectrofluorimetric method for the determination of mercury.

A highly sensitive flow-injection spectrofluorimetric method is presented for the rapid and simple determination of Hg (II) in environmental and pharmaceutical samples. Murexide (ammonium purpurate) was used as the fluorescence reagent in the carrier stream. An emission peak of murexide, which is decreased linearly by addition of Hg (II), occurs at 435 nm in aqueous solution with excitation at 335 nm. A linear calibration was obtained for 5-200 ng ml(-1) Hg (II) with the relative standard deviation 2.5% (n = 5) for a 20 microl injection volume Hg (II). The limit of the detection was 1 ng ml(-1) and the sampling rate was 80 h(-1). No significant interference was found by the ions commonly found in the most environmental samples. The proposed method was successfully applied for the determination of trace mercury in real samples and the validation of the proposed methodology is provided.

Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres.

Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+](SR)) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrin's properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One - the EGTA-phenol red method - monitored the content of total Ca in the SR ([Ca(T)](SR)) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mM), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [Ca(T)](SR) versus [Ca2+](SR) as [Ca2+](SR) varied from its resting level ([Ca2+](SR,R)) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca-calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only approximately 25% of released Ca2+ comes from calsequestrin and approximately 75% comes from the free pool. The value of [Ca2+](SR,R) was close to the K(D) for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+](SR,R) is near 1 mM in cut fibres. These results along with the known rapid kinetics of the Ca-calsequestrin binding reaction indicate that calsequestrin's properties are optimized to buffer [Ca2+](SR) during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation-contraction coupling process, they do indicate that passive buffering of [Ca2+](SR) is a very important function of calsequestrin.

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.

Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle.

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca-induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle.

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of thyroid hormones and their analogues on the mitochondrial calcium transport activity.

In this paper the authors studied the effects of thyroid hormones and their structural analogues on the mitochondrial calcium transport activities. The thyroid hormones, 3,5,3' L-triiodothyronine (LT3) and 3,5,3'5' L-tetraiodothyronine (LT4) at physiological intracellular concentrations between 7.2 and 9 nM, decouple total Ca++ transport, as well as inhibit the passive transport of Ca++, either due to oxidation of pyruvate, malate or succinate or after inhibition with rotenone. The optical isomers 3,5,3' D-triiodothyronine (DT3) and 3,5,3',5' D-tetraiodothyronine (DT4) are less effective at all the used concentrations. Furthermore the structural analogues 3,3',5' L-triiodothyronine (LrT3), 3,5-dicloro, 3',5' L-diiodothyronine (LDiClT2) and 3,5 L-diiodothyronine (LT2) furnished even less effects on the same activities. The effect of the thyroid hormones and of their structural analogues has revealed that the mitochondrial calcium transport may be influenced both by a stereospecific interaction between hormones and protein ligands and by a lipophilic chaotropic action on the mitochondrial membranes lipids. In this context it is interesting to consider that both thyroid hormones and Ca++ transport activity are interacting with the energetic metabolism by means of phosphorylation and substrate oxidation mechanism.

Calcium binding to metallochromic dyes and calmodulin in the presence of combined, AC-DC magnetic fields.

The possibility that weak, ac and dc magnetic fields in combination may affect binding equilibria of calcium-ions (Ca2+) was investigated with two metallochromic dyes as calcium-binding molecules: murexide and arsenazo III. Calcium-dye equilibria were followed by measuring solution absorbances with a fiber-optic spectrophotometer. A Ca(2+)-arsenazo solution was also used indirectly to monitor the binding of Ca2+ to calmodulin. Parallel, ac and dc magnetic fields were applied to each preparation. The ac magnetic field was held constant during each of a series of experiments at a frequency in the range between 50 and 120 Hz (sine wave) or at 50 pps (square wave) and at an rms flux density in the range between 65 and 156 microT. The dc magnetic field was then varied from 0 to 299 microT at 1.3 microT increments. The magnetic fields did not measurably affect equilibria in the binding of metallochromic dyes or calmodulin to Ca2+.

Intravesicular calcium transient during calcium release from sarcoplasmic reticulum.

The time course of changes in the intravesicular Ca2+ concentration ([Ca2+]i) in terminal cisternal sarcoplasmic reticulum vesicles upon the induction of Ca2+ release was investigated by using tetramethylmurexide (TMX) as an intravesicular Ca2+ probe. Upon the addition of polylysine at the concentration that led to the maximum rate of Ca2+ release, [Ca2+]i decreased monotonically in parallel with Ca2+ release. Upon induction of Ca2+ release by lower concentrations of polylysine, [Ca2+]i first increased above the resting level, followed by a decrease well below it. The release triggers polylysine, and caffeine brought about dissociation of calcium that bound to a nonvesicular membrane segment consisting of the junctional face membrane and calsequestrin bound to it, as monitored with TMX. No Ca2+ dissociation from calsequestrin-free junctional face membranes or from the dissociated calsequestrin was produced by release triggers, but upon reassociation of the dissociated calsequestrin and the junctional face membrane, Ca2+ dissociation by triggers was restored. On the basis of these results, we propose that the release triggers elicit a signal in the junctional face membrane, presumably in the foot protein moiety, which is then transmitted to calsequestrin, leading to the dissociation of the bound calcium; and in SR vesicles, to the transient increase of [Ca2+]i, and subsequently release across the membrane.