nM For Ca2 Research Articles

Ca2+-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins

Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50-100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.

A novel dual-channel Schiff base fluorescent chemo-sensor for Zn2+ and Ca2+ recognition: Synthesis, mechanism and application

A novel multifunctional chemo-sensor (RHCS-NH2), incorporating Schiff base, rhodamine B hydrazide, hydrazine hydrate group into triazine, was successfully designed and synthesized. It was found that the RHCS-NH2 sensor displays immediate multi-response toward Ca2+ in CH3CN-H2O (v/v = 9/1, Tris-HCl pH 7.0) and Zn2+ in DMF-H2O (95/5, v/v). Under the optimized conditions, the linear range of identifing Ca2+ ions are from 0.01 to 10.0 μM in CH3CN-H2O (v/v = 9/1, Tris-HCl pH 7.0), 0.03–10.0 μM for Zn2+ ions in DMF-H2O (95/5, v/v), and the detection limit were measured to be 0.10 nM for Ca2+ and 0.14 nM for Zn2+, respectively. The reaction mechanism between RHCS-NH2 and metal ions was investigated in detail based on fluorescence titration, Job's plots and 1H NMR titration curve, ESI-MS measurement and theoretical calculation respectively. Simultaneously, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays and inverted fluorescence microscopy imaging also demonstrated that the probe has well cell membrane permeability and hypotoxicity, indicating its potential applicability in vitro.

A highly selective fluorescent sensor for Ca2+ and Sr2+ based on diarylethene with a furan-carbohydrazide unit

A fluorescent sensor based on diarylethene has been designed and synthesized. The sensor not only exhibited excellent photochromic properties, but also has distinguishing ability for Ca2+ and Sr2+ from other metal ions. Upon addition of Ca2+ and Sr2+, its emission intensity enhanced 27-fold and 24-fold respectively, accompanied by the emission peak shifted from 439 nm to 484 nm for Ca2+, and to 479 nm for Sr2+. The 1:1 stoichiometry between the sensor and the two ions was confirmed by Job's plot and HRMS. The LOD for Ca2+ and Sr2+ was determined to be 9.4 × 10−8 mol L−1 and 7.2 × 10−8 mol L−1, respectively. Furthermore, the sensor was applied in the detection of Ca2+ and Sr2+ in practical samples successfully.

Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry

Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase–activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+ levels.

Observing Effects of Calcium/Magnesium Ions and pH Value on the Self-Assembly of Extracted Swine Tendon Collagen by Atomic Force Microscopy

Self-assembly of extracted collagen from swine trotter tendon under different conditions was firstly observed using atomic force microscopy; then the effects of collagen concentration, pH value, and metal ions to the topography of the collagen assembly were analyzed with the height images and section analysis data. Collagen assembly under 0.1 M, 0.2 M, 0.3 M CaCl2, and MgCl2 solutions in different pH values showed significant differences (P < 0.05) in the topographical properties including height, width, and roughness. With the concentration being increased, the width of collagen decreased significantly (P < 0.05). The width of collagen fibers was first increased significantly (P < 0.05) and then decreased with the increasing of pH. The collagen was assembled with network structure on the mica in solution with Ca2+ ions. However, it had shown uniformed fibrous structure with Mg2+ ions on the new cleaved mica sheet. In addition, the width of collagen fibrous was 31~58 nm in solution with Mg2+ but 21~50 nm in Ca2+ solution. The self-assembly collagen displayed various potential abilities to construct fibers or membrane on mica surfaces with Ca2+ ions and Mg2+ irons. Besides, the result of collagen self-assembly had shown more relations among solution pH value, metal ions, and collagen molecular concentration, which will provide useful information on the control of collagen self-assembly in tissue engineering and food packaging engineering.

Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

A novel fluorescent nano-scale sensor for detection of trace amounts of Ca (II) ions

A photo-induced electron transfer (PET) based sensing approach for the direct determination of trace amounts of calcium ions is presented. The Ca2+ selective fluoroionophore Bis, 2,2'-{1,2 phenylenebis [nitrilomethylylidene]} diphenol (DMK) was encapsulated in polymeric ethyl cellulose. The sensing membranes were fabricated in form of nanofibers, exploiting the prepared polymer. When embedded in nanomaterials, the DMK dye yielded strong absorbance, large Stoke's shift, high fluorescence quantum yield, and excellent short and long-term photostability. The sensing ability of the nanofibers was tested by steady state and time resolved fluorescence spectroscopy. To our knowledge, this is the first attempt using the DMK-doped electrospun nanofibrous materials for calcium sensing. The offered nanosensor displays a sensitive response with a detection limit of 0.016nM for Ca2+ ions over a wide concentration range, 1.0×10−10–1.0×10−4M, and exhibits high selectivity over Mg 2+ and other cations. Accuracy of the sensing system was proven by recovery tests.

Double Electron–Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²⁺-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²⁺-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²⁺-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²⁺-free recoverin and 3.7 ± 0.1 nm for Ca²⁺-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²⁺-bound recoverin may represent an intermolecular distance between C39 and N120. ¹⁵N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²⁺-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca²⁺-free recoverin is monomeric. We propose that Ca²⁺-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²⁺-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²⁺-induced dimerization is functionally important but not well understood.

The Discovery and Characterization of ML218: A Novel, Centrally Active T-Type Calcium Channel Inhibitor with Robust Effects in STN Neurons and in a Rodent Model of Parkinson’s Disease

T-type Ca(2+) channel inhibitors hold tremendous therapeutic potential for the treatment of pain, epilepsy, sleep disorders, essential tremor and other neurological disorders; however, a lack of truly selective tools has hindered basic research, and selective tools from the pharmaceutical industry are potentially burdened with intellectual property (IP) constraints. Thus, an MLPCN high-throughput screen (HTS) was conducted to identify novel T-type Ca(2+) channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca(2+) channels. While the HTS provided numerous hits, these compounds could not be optimized to the required level of potency to be appropriate tool compounds. Therefore, a scaffold hopping approach, guided by SurflexSim, ultimately afforded ML218 (CID 45115620) a selective T-Type Ca(2+) (Ca(v)3.1, Ca(v)3.2, Ca(v)3.3) inhibitor (Ca(v)3.2, IC(50) = 150 nM in Ca(2+) flux; Ca(v)3.2 IC(50) = 310 nM and Ca(v)3.3 IC(50) = 270 nM, respectively in patch clamp electrophysiology) with good DMPK properties, acceptable in vivo rat PK and excellent brain levels. Electrophysiology studies in subthalamic nucleus (STN) neurons demonstrated robust effects of ML218 on the inhibition of T-Type calcium current, inhibition of low threshold spike and rebound burst activity. Based on the basal ganglia circuitry in Parkinson's disease (PD), the effects of ML218 in STN neurons suggest a therapeutic role for T-type Ca(2+) channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A(2A) antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-Type Ca(2+) function in vitro and in vivo, and freely available.

Development of an innovative method of fluoro-electrode for simultaneous detection of electrical activity and fluorescence spectroscopy

We report investigations of resonance energy transfer in the green fluorescent protein and calmodulin-based fluorescent indicator constructs for Ca2+ called cameleons using steady-state and time-resolved spectroscopy of the full construct and of the component green fluorescent protein mutants, namely ECFP (donor) and EYFP (acceptor). EYFP displays a complicated photophysical behavior including protonated and deprotonated species involved in an excited-state proton transfer. When EYFP is excited in the absorption band of the protonated species, a fast nonradiative deactivation occurs involving almost 97% of the excited protonated population and leading to a low efficiency of excited-state proton transfer to the deprotonated species. ECFP displays a multiexponential fluorescence decay with a major contributing component of 3.2 ns. The time-resolved fluorescence data obtained upon excitation at 420 nm of Ca2+-free and Ca2+-bound YC3.1 cameleon constructs point to the existence of different conformations of calmodulin dependent on Ca2+ binding. Whereas steady-state data show only an increase in the efficiency of energy transfer upon Ca2+ binding, the time-resolved data demonstrate the existence of three distinct conformations/populations within the investigated sample. Although the mechanism of the interconversion between the different conformations and the extent of interconversion are still unclear, the time-resolved fluorescence data offer an estimation of the rate constants, of the efficiency of the energy transfer, and of the donor-acceptor distances in the Ca2+-free and Ca2+-bound YC3.1 samples.

Mitochondrial ND5 Gene Variation Associated with Encephalomyopathy and Mitochondrial ATP Consumption

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A-->G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (Deltapsim) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.

NADPH OXIDASE‐MEDIATED O 2 −· PRODUCTION AMPLIFIES VASOCONSTRICTOR RESPONSE OF SMALL CORONARY ARTERIES TO M 1 ‐MUSCARINIC RECEPTOR ACTIVATION

The present study test the hypothesis that NADPH oxidase (Nox) mediated O2−· production amplifies coronary arterial constrictor response to M1-receptor (M1R) agonists. Fluorescence imaging analysis showed that M1R agonist, acetylcholine (Ach), simultaneously increased intracellular Ca2+ and O2−· levels in bovine coronary arterial smooth muscle cells (CASMCs) with a maximal increase of 821 + 67 nM in Ca2+ levels accompanied by 75% in O2−· production. However, when cells were transfected with siRNA of p47phox(a key subunit for Nox activation), Ach-induced increases in Ca2+ and O2−· were significantly attenuated. Similarly, p47phoxsiRNA significantly reduced oxotremorine (OXO)-induced O2−· production in CASMCs by 67%. Functionally, OXO-induced constrictor response of endothelium-denuded small bovine coronary arteries was significantly reduced by Nox inhibitor, DPI or O2−· scavenger cocktail (tempol + catalase). Moreover, introduction of siRNA for p47phox, NOX1 and NOX4 into arterial smooth muscle by ultrasound microbubble technology significantly blocked this OXO-induced contraction. We concluded that Nox-derived O2−· is involved in M1R-mediated intracellular Ca2+ release in CASMCs and thereby importantly contributes to amplification of vasoconstrictor response of coronary arteries to M1R activation (Supported by NIH grants HL057244, HL075316 and AHA predoctoral fellowship 0410061Z).

Field-Flow Fractionation−Inductively Coupled Plasma Mass Spectrometry: An Alternative Approach to Investigate Metal−Humic Substances Interaction

Interaction between metal ions and humic matter was investigated using a hyphenated technique, field-flow fractionation-inductively coupled plasma mass spectrometry (FFF-ICP-MS). Aggregation of a metal-spiked commercial Aldrich humic acid in an aqueous solution of calcium ion or in seawater was examined over time intervals of 0-4320 min. The aggregation was demonstrated by shifts in peak maximum of humic matter from smaller size (2.9 nm) to larger size (5.1 or 5.8 nm in Ca2+ solution or in seawater, respectively) and also by the broadening of size distribution profiles. With FFF, size distribution of humic aggregate was characterized. Further, dominant particle size (2.9 nm), mean particle size (3.8 nm), and diffusion coefficient (1.51 x 10(-6) cm2/s) of humic acid solution were determined. With FFF-ICP-MS, associations of Cd, Cu, and Pb with humic aggregates were examined. The mean diameters of Cd-, Cu-, and Pb-bound humic aggregates in the metal-spiked humic acid were 4.1, 4.5, and 5.8 nm, respectively. These diameters were shifted to 6.0, 6.0, and 6.9 nm, respectively, in the humic acid incubated with calcium solution, whereas they were shifted to 6.5, 5.7, and 7.4 nm, respectively, in the humic acid incubated with seawater for three days. Humic aggregate of small size showed more affinity for Cu than Cd and Pb, whereas the large aggregate showed more affinity for Pb than Cd and Cu, respectively.

3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)- 6-methylpyrimidin-2,4-dione (NBI 42902) as a Potent and Orally Active Antagonist of the Human Gonadotropin-Releasing Hormone Receptor. Design, Synthesis, and in Vitro and in Vivo Characterization

Further structure-activity relationship studies of a series of substituted uracils at the 1, 3, and 5 positions resulted in the discovery of several potent antagonists of the human gonadotropin-releasing hormone receptor. Uracils bearing a side chain derived from phenylglycinol at the 3-position were shown to be orally bioavailable in monkeys. 3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidin-2,4-dione (R-13b, NBI 42902) displayed subnanomolar binding affinity (K(i) = 0.56 nM) and was a potent functional antagonist (IC(50) = 3.0 nM in Ca(2+) flux assay) at the human GnRH receptor. It also bound to the monkey GnRH receptor with high affinity (K(i) = 3.9 nM). In addition, R-13bhad good plasma exposure in cynomolgus monkeys after oral administration, with a C(max) of 737 ng/mL and an AUC of 2392 ng/mL.h at a 10 mg/kg dose. Moreover, oral administration of R-13b to castrated male cynomolgus monkeys resulted in a significant decrease in serum levels of luteinizing hormone. These results demonstrate that compounds from this series of uracils are potent GnRH antagonists with good oral bioavailability and efficacy in nonhuman primates.

Photochemical and complexation studies for new fluorescent and colored chelator

The photo-induced intramolecular proton (or hydrogen atom) transfer (ESIPT) and metal binding properties of Oxa-ester were studied in methanol solution. The dissociation constant between Oxa-ester and the metal ion was highly dependent with the metal ion and was determined by Hill plots or iterative least squares fitting to be 447 mM, 14.9 mM, and 290 nM for Ca2+, Zn2+, and Cu2+, respectively, in methanol. The fluorescence intensity of Oxa-ester greatly increased with increasing concentration of Zn2+, while the fluorescence intensity decreased with the addition of Ca2C and Cu2+. Transient absorption spectroscopy revealed that Oxa-ester underwent ESIPT to give Z-NH isomer in the excited state in benzene, while Oxa did not undergo ESIPT probably due to the hydrogen bonding with solvent water.

Sorption and Redox Activity of Cobalt Corrinoids on Hectorite

AbstractHalogenated synthetic organic compounds are widespread contaminants of the environment; however, little is known concerning their potential dehalogenation by extracellular corrinoids. Our primary objective was to determine if corrinoids sorbed to soil colloids are redox active and thus viable electron transfer mediators of contaminant dehalogenation in reduced soils and sediments. Dicyanocobínamide, cyanocobalamin (vitamin B12), and aquocobalamin were sorbed onto Ca2+‐, K+‐, and Na+‐hectorite and sorption isotherms determined. Additional assessment of sorption reactions was performed using x‐ray diffraction (XRD) and infrared (IR) spectroscopy. Redox states of the sorbed corrinoids in hectorite suspensions were monitored using ultraviolet and visible (UV‐VIS) spectrophotometry. Corrinoid molecular size and charge, as well as the saturating cation, controlled corrinoid sorption. The XRD results indicated that Co corrinoids intercalated hectorite, expanding the basal spacing from 1.45 to 2.21 nm for Ca2+‐, 1.17 to 2.70 nm for K+‐, and 1.31 to 2.81 nm for Na+‐hectorite. The IR spectra of bound corrinoids yielded little information on binding mechanisms, but indicated that axial ligands remained intact. The central metal of bound corrinoids was reduced to Co(I) by Ti(III) and reoxidized to Co(III) by introduction of O2, thus demonstrating that redox activity of sorbed corrinoids was maintained and reversible. Microbially produced extracellular corrinoids may act as potential electron transfer mediators when in association with clays, possibly participating in the reductive dehalogenation of organic contaminants.

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40-50% of total Ca2+ uptake was inhibited by TG up to 10 nM (apparent Ki approximately 4.2 nM, Ca2+ pool I). An additional increase of inhibition up to 85-90% of total Ca2+ uptake could be achieved at 15 to 20 nM of TG (apparent Ki approximately 12.1 nM, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki approximately 10 microM). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30-40% of total Ca2+ uptake was inhibited by 100 microM of vanadate (apparent Ki approximately 18 microM, Ca2+ pool II). The remaining 60-70% could be inhibited either by vanadate at concentrations up to 1 mM (apparent Ki approximately 300 microM) or by TG up to 10 nM (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at approximately 25% over a wide range of Ca2+ filling. About 10-20% remained unreleasable by IP3. Reduction of IP3-releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki approximately 3.0 nM for IP3-induced Ca2+ release as compared to approximately 4.2 nM for Ca2+ uptake at TG up to 10 nM) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations > 10 nM which blocked Ca2+ pool II the apparent Ki values were approximately 11.3 and approximately 12.1 nM, respectively. For inhibition by vanadate up to 100 microM the apparent Ki values were approximately 18 microM for Ca2+ uptake and approximately 7 microM for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mM the apparent Ki values were approximately 300 and approximately 200 microM, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 microM. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Estimates of beat to beat handling of activator calcium using measurements of [Ca2+]i in aequorin loaded ferret cardiac muscle.

The aims were (1) to measure simultaneously and on a beat to beat basis intracellular calcium concentration ([Ca2+]i) transients and force transients in isolated ferret cardiac trabeculae; (2) to obtain and compare independent estimates of the recirculating fraction of Ca2+ using the [Ca2+]i data and the force data (recirculating fraction is the fraction of activator Ca2+ taken up by the sarcoplasmic reticulum in each beat and, in the steady state twitch, the fraction of activator Ca2+ released by the sarcoplasmic reticulum); and (3) to estimate the amount of Ca2+ that returns to the sarcoplasmic reticulum and the amount that, during steady state contractions, enters the cytosol, presumably from the extracellular compartment, with each beat. Eight trabeculae were mounted in the myograph. The servo-controlled muscle length was 98% of the length at which developed force was maximal. A modified technique was used for chemical loading of aequorin, and a new method for computer controlled low level photon counting, storage, calibration, and analysis. [Ca2+]i transients and force transients were simultaneously recorded during potentiated beats, together with their respective decays toward control steady state [Ca2+]i transients and force transients. A modified test of postextrasystolic potentiation achieved with a brief train of rapid pacing followed by a pause was used to evoke the potentiated beats. At 2.0 mM extracellular Ca2+ ([Ca2+]o), resting [Ca2+]i was 283(SD 77) nM. The resting tension was 1.6(0.3) g.mm-2. The steady state [Ca2+]i transient and the peak potentiated [Ca2+]i transient averaged 992(165) and 1290(154) nM respectively. The corresponding tensions were 4.0(1.9) and 8.7(3.1) g.mm-2 respectively. The recirculating fraction of Ca2+ calculated from the dissipation of the potentiated [Ca2+]i transient averaged 45(4)%. This recirculating fraction was indistinguishable from the one calculated with another method from the decay of the force potentiation. This is the first study to estimate the recirculating fraction of activator Ca2+ using measurements of [Ca2+]i. The results indicate that over a wide range of [Ca2+]i and tensions the Ca(2+)-force relationship is well approximated by a straight line. At 2.0 mM [Ca2+]o it appears that some 450 nM of Ca2+ recirculates and that a similar amount per steady state beat enters the cytosol, probably from the extracellular compartment.

Measurement of free Zn2+ ion concentration with the fluorescent probe mag-fura-2 (furaptra)

The fluorescent probe mag-fura-2, previously used to measure [Mg2+], can also be used to measure [Zn2+]. The peak in the excitation spectrum occurs at 323 nm for Zn2+, compared with 335 nm for Ca2+ and Mg2+. This allows simultaneous measurements of [Zn2+] and either [Ca2+] or [Mg2+], by using 3 excitations wavelengths. The dissociation constant for Zn2+ is 20 nM at pH 7.0–7.8, ionic strength 0.15 and 37°C. This allows [Zn2+] to be measured in the range from 0.5 nM to 1 μM. Mag-fura-2 was used to measure [Zn2+] in Zn2+/albumin and Zn2+/histidine mixtures in a physiological buffer at 37°C and pH 7.4. The data obtained enable one to formulate Zn2+-buffers for the 1 to 100 nM Zn2+ range.

Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin.

The binding of cations to ATP-G-actin has been assessed by measuring the kinetics of the increase in fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Ca2+ and Mg2+ compete for a single high-affinity site on ATP-G-actin with KD values of 1.5-15 nM for Ca2+ and 0.1-1 microM for Mg2+, i.e. with affinities 3-4 orders of magnitude higher than previously reported (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). As proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886), the Mg-actin complex undergoes a slow isomerization (Kis = 0.03-0.1) to a higher affinity state (K'D = 4-40 nM). The replacement of Ca2+ by Mg2+ at this high-affinity site causes a slow 10% increase in fluorescence that is 90% complete in about 200 s at saturating concentrations of Mg2+. Independently, Ca2+, Mg2+, and K+ bind to low-affinity sites (KD values of 0.15 mM for Ca2+ and Mg2+ and 10 mM for K+) which causes a rapid 6-8% increase in fluorescence (complete in less than 5 s). We propose that the activation step that converts Ca-G-actin to a polymerizable species upon addition of Mg2+ is the binding of Mg2+ to the low-affinity sites and not the replacement of Ca2+ by Mg2+ at the high-affinity site.