Mag-indo1 Affinity for Ca, Compartmentalization and Binding to Proteins: The Challenge of Measuring Mg Concentrations in Living Cells

A physicochemical study of the Mag-indo1 binding to Ca²⁺ in solution showed that: (i) the characteristic fluorescence spectra of Ca²⁺-bound and Mg²⁺-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca²⁺ concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC₆ loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca²⁺ and concluded that the Mag-indo1 binding to Ca²⁺ is likely to affect drastically the Mg²⁺ concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg²⁺ measurements when using the two-wavelength ratio method.     

Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator of calcium channels

CaBP1 (calcium-binding protein 1) is a 19.4-kDa protein of the EF-hand superfamily that modulates the activity of Ca(2+) channels in the brain and retina. Here we present data from NMR, microcalorimetry, and other biophysical studies that characterize Ca(2+) binding, Mg(2+) binding, and structural properties of recombinant CaBP1 purified from Escherichia coli. Mg(2+) binds constitutively to CaBP1 at EF-1 with an apparent dissociation constant (K(d)) of 300 microm. Mg(2+) binding to CaBP1 is enthalpic (DeltaH = -3.725 kcal/mol) and promotes NMR spectral changes, indicative of a concerted Mg(2+)-induced conformational change. Ca(2+) binding to CaBP1 induces NMR spectral changes assigned to residues in EF-3 and EF-4, indicating localized Ca(2+)-induced conformational changes at these sites. Ca(2+) binds cooperatively to CaBP1 at EF-3 and EF-4 with an apparent K(d) of 2.5 microM and a Hill coefficient of 1.3. Ca(2+) binds to EF-1 with low affinity (K(d) >100 microM), and no Ca(2+) binding was detected at EF-2. In the absence of Mg(2+) and Ca(2+), CaBP1 forms a flexible molten globule-like structure. Mg(2+) and Ca(2+) induce distinct conformational changes resulting in protein dimerization and markedly increased folding stability. The unfolding temperatures are 53, 74, and 76 degrees C for apo-, Mg(2+)-bound, and Ca(2+)-bound CaBP1, respectively. Together, our results suggest that CaBP1 switches between structurally distinct Mg(2+)-bound and Ca(2+)-bound states in response to Ca(2+) signaling. Both conformational states may serve to modulate the activity of Ca(2+) channel targets.     

Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].     

Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin alphaIIb cytoplasmic domain

Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the alphaIIb cytoplasmic domain of integrinalphaIIbbeta(3). We have previously demonstrated that CIB lacks structural stability in the absence of divalent metal ions but that it acquires a well-folded conformation upon addition of Ca(2+) or Mg(2+). Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca(2+)-bound CIB (Ca(2+)-CIB) and the Mg(2+)-bound protein (Mg(2+)-CIB) bind with high affinity and through a similar mechanism to alphaIIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca(2+)-CIB and Mg(2+)-CIB, but involve hydrophobic interactions in each case. Since the Mg(2+) concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the alphaIIb cytoplasmic domain independent of an intracellular Ca(2+) stimulus in vivo. This raises the question of whether CIB can act as a Ca(2+) sensor in alphaIIbbeta(3) signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in alphaIIbbeta(3), post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and alphaIIbbeta(3). Differences in NMR spectra do suggest, however, that Ca(2+)-binding to the Mg(2+)- CIB-alphaIIb complex induces subtle structural changes that could further modulate the activity of alphaIIbbeta(3).     

Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1)

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.     

Characterization of a range of fura dyes with two-photon excitation

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.     

The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

Identification of the Ca(2+)-dependent calmodulin-binding region of chromogranin A.

Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region.     

Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae

The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.     

Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.     

Immunosuppressants inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

Immunosuppressants such as cyclosporinA and FK506 (tacrolimus) are widely prescribed to treat numerous disorders and to treat organ transplant recipients. However, cyclosporine A and FK506 are both known to produce hypomagnesaemia. The mechanism of this effect is still unclear. The present study determined the effects of immunosuppressant treatment on the parathyroid hormone (PTH) mediated Mg(2+) uptake and the mitogen-activated protein kinase (MAPK) activation in mouse distal convoluted tubule (MDCT) cells. The intracellular Ca(2+) and Mg(2+) concentrations in a single MDCT cell were measured by using the fluorescentdye Fura-2 AM and Mag-fura-2 AM, respectively. Cyclosporine A and FK506 illicited a transient increase of intracellular Ca(2+) from a basal level of 99 +/- 16 nM to 685 +/- 105 and 608 +/- 96 nM, respectively. In order to determine the Mg(2+) transport, the MDCT cells were Mg(2+)-depleted by culturing them in Mg(2+)-free media for 16 h, and the Mg(2+) uptake was measured by microfluorescence after placing the depleted cells in 1.5mM MgCl(2). The mean rate of Mg(2+) uptake, d([Mg(2+)](i))/dt, was 140 +/- 16 nM/s in the control MDCT cells. PTH increased the Mg(2+) uptake more than 2 times in this cell. Cyclosporine A (10 microM) and FK506 (0.1 microM) did not affect the basal Mg(2+)uptake (140 +/- 16 and 142 +/- 14 nM/s, respectively), but they inhibited the PTH-stimulated Mg(2+) entry, decreasing it from 248+/-12 to 147 +/- 7 and 148 +/- 14 nM/s, respectively. These effects were inhibited by L685818, which is a potent competitive antagonist of FK506. PTH stimulated the extracellular signal-regulated kinase1/2 (ERK1/2) protein synthesis. This PTH-stimulated ERK1/2 activation was inhibited by cyclosporine A and FK506. In the present study, the role of ERK1/2 activation on the PTH-dependent magnesium uptake was examined in MDCT cells, and we showed that immunosuppressants inhibit the hormone-stimulated Mg(2+) uptake into the MDCT cells by inhibiting the MAPK pathway.     

Magnesium binding to DM-nitrophen and its effect on the photorelease of calcium

The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.     

A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.     

Crystal structure of the Ca(2+)-form and Ca(2+)-binding kinetics of metastasis-associated protein, S100A4

S100A4 takes part in control of tumour cell migration and contributes to metastatic spread in in vivo models. In the active dimeric Ca(2+)-bound state it interacts with multiple intracellular targets. Conversely, oligomeric forms of S100A4 are linked with the extracellular function of this protein. We report the 1.5A X-ray crystal structure of Ca(2+)-bound S100A4 and use it to identify the residues involved in target recognition and to derive a model of the oligomeric state. We applied stopped-flow analysis of tyrosine fluorescence to derive kinetics of S100A4 activation by Ca(2+) (k(on)=3.5 microM(-1)s(-1), k(off)=20s(-1)).