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1.
In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC20 values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca2+-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl2 by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca2+-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca2+ influx through L-type Ca2+ channels and intracellular Ca2+ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.  相似文献   

2.
High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC50’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.  相似文献   

3.
The PPARγ agonist Rosiglitazone exerts anti-hyperglycaemic effects by regulating the long-term expression of genes involved in metabolism, differentiation and inflammation. In the present study, Rosiglitazone treatment rapidly inhibited (5-30 min) the ER Ca2+ ATPase SERCA2b in monocytic cells (IC50 = 1.88 μM; p < 0.05), thereby disrupting short-term Ca2+ homeostasis (resting [Ca2+]cyto = 121.2 ± 2.9% basal within 1 h; p < 0.05). However, extended Rosiglitazone treatment (72 h) induced dose-dependent SERCA2b up-regulation, and restored calcium homeostasis, in monocytic cells (SERCA2b mRNA: 138.7 ± 5.7% basal (1 μM)/215.0 ± 30.9% basal (10 μM); resting [Ca2+]cyto = 97.3 ± 8.3% basal (10 μM)). As unfavourable cardiovascular outcomes, possibly related to disrupted cellular Ca2+ homeostasis, have been linked to Rosiglitazone, this effect may be of clinical interest. In contrast, in PPRE-luciferase reporter-gene assays, Rosiglitazone induced non-dose-dependent PPARγ-dependent effects (1 μM: 152.5 ± 4.9% basal; 10 μM: 136.1 ± 5.1% basal (p < 0.05 for 1 μM vs. 10 μM)). Thus, we conclude that Rosiglitazone can exert PPARγ-independent non-genomic effects, such as the SERCA2b inhibition seen here, but that long-term Rosiglitazone treatment did not perturb resting [Ca]cyto in this study.  相似文献   

4.
Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O2 g−1 DW h−1) than at high alkalinity (50 ± 27 μmol O2 g−1 DW h−1), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O2 g−1 DW h−1), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O2 g−1 DW h−1). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O2 mequiv. L−1 g−1 DW h−1) and L. major (244 ± 29 and 82 ± 24 μmol O2 mequiv. L−1 g−1 DW h−1). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.  相似文献   

5.
Leishmaniasis’ treatment is based mostly on pentavalent antimonials or amphotericin B long-term administration, expensive drugs associated with severe side effects. Considering these aforementioned, the search for alternative effective and safe leishmaniasis treatments is a necessity. This work evaluated a neolignan, licarin A anti-leishmanial activity chemically synthesized by our study group. It was observed that licarin A effectively inhibited Leishmania (Leishmania) major promastigotes (IC50 of 9.59 ± 0.94 μg/mL) growth, by inducing in these parasites genomic DNA fragmentation in a typical death pattern by apoptosis. Additionally, the neolignan proved to be even more active against intracellular amastigotes of the parasite (EC50 of 4.71 ± 0.29 μg/mL), and significantly more effective than meglumine antimoniate (EC50 of 216.2 ± 76.7 μg/mL) used as reference drug. The antiamastigote activity is associated with an immunomodulatory activity, since treatment with licarin A of the infected macrophages induced a decrease in the interleukin (IL)-6 and IL-10 production. This study demonstrates for the first time the antileishmanial activity of licarin A and suggests that the compound may be a promising in the development of a new leishmanicidal agent.  相似文献   

6.
We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg−1 H2O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg−1 H2O). Hemolymph [Na+] (323.0 ± 2.5 mmol L−1) and [Mg2+] (34.6 ± 1.0 mmol L−1) are hypo-regulated while [Ca2+] (22.5 ± 0.7 mmol L−1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L−1) but hypo-regulated (6.2 ± 0.7 mmol L−1) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg−1; K0.5 = 7.07 ± 0.01 μmol L−1) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg−1; K0.5 = 0.11 ± 0.3 mmol L−1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site-site interactions, but modulation by Na+ shows Michaelis-Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited ≈80% of total ATPase activity (KI = 464.7 ± 23.2 μmol L−1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min−1 mg−1) and 45‰-acclimated crabs (around 154 nmol Pi min−1 mg−1), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2-3-fold in 45‰-acclimated crabs.  相似文献   

7.
Voltage-gated Ca2+ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca2+ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca2+ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca2+-channel α1 subunits, Cav3.1, Cav3.2 and Cav3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Cav3.1, Cav3.2, and Cav3.3 functionally expressed in Xenopus oocytes. Our data show that all Cav3 channels select Ca2+ over Na+ by affinity. Cav3.1 and Cav3.2 discriminate Ca2+, Sr2+ and Ba2+ based on the ion's effects on the open channel probability, whilst Cav3.3 discriminates based on the ion's intrapore binding affinity. All Cav3s were characterized by much smaller difference in the KD values for Na+ current blockade by Ca2+ (KD1 ∼ 6 μM) and for Ca2+ current saturation (KD2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na+/Ca2+ current at close to physiological Ca2+ concentrations, which was the strongest for Cav3.3, smaller for Cav3.2 and the smallest for Cav3.1. In addition to intrapore Ca2+ binding site(s) Cav3.2, but not Cav3.1 and Cav3.3, is likely to possess an extracellular Ca2+ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca2+ current in native cells.  相似文献   

8.
Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na+-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na+-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na+-ATPase activity with IC50 = 18 ± 5 μg mL−1. This effect was shown to be reversible, dependent on the pH and Ca2+. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na+-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na+-ATPase and PKC activities.  相似文献   

9.
The larvicidal activity of essential oils of four species of Piper from the Amazon Forest was tested using third-instar larvae of Aedes aegypti. The oils were extracted by steam distillation and analyzed by GC and GC–MS. The main components isolated from each Piper species were as follows: viridiflorol (27.50%), aromadendrene (15.55%) and β-selinene (10.50%) from Piper gaudichaudianum; β-selinene (15.77%) and caryophyllene oxide (16.63%) from Piper humaytanum; dillapiol (54.70%) and myristicin (25.61%) from Piper permucronatum; and asaricin (27.37%) and myristicin (20.26%) from Piper hostmanianum. Amongst all essential oils tested, the most active against larvae of A. aegypti was the oil extracted from P. permucronatum, with a LC50 = 36 μg/ml (LC90 = 47 μg/ml), followed by the essential oil of P. hostmanianum, with a LC50 = 54 μg/ml (LC90 = 72 μg/ml). The oils with higher content of arylpropanoids were more active against larvae of A. aegypti.  相似文献   

10.
In the present study, we have examined any possible involvement of L-type Ca2+ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca2+ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca2+ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca2+]i changes. These GTS-mediated [Ca2+]i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg3 are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca2+ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.  相似文献   

11.
N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A2 (PLA2) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca2+-activated chloride current (ICl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited ICl(Ca) in a concentration-dependent manner (IC50 = 4.2 μM, nHill = 1.1), without affecting the L-type Ca2+ current. Unlike ACA, the non-selective PLA2 inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on ICl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on ICl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides ICl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on ICl(Ca) are PLA2-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.  相似文献   

12.
Fractionation of dichloromethane and acetone fractions obtained by serial extraction from the leaf powder of Dodonaea viscosa Jacq. var. angustifolia (Sapindaceae) resulted in the isolation of four kaempferol methyl ethers. The compounds were identified by spectral data (1H NMR, 13C NMR and MS) as: 3, 5, 7-trihydroxy-4'-methoxyflavone (1); 5, 7, 4'-trihydroxy-3, 6-dimethoxyflavone (2); 5, 7-dihydroxy-3, 6, 4'-trimethoxyflavone (santin) (3); and 5-hydroxy -3, 7, 4'-trimethoxyflavone (4) together with 3,4',5,7-tetrahydroxy flavone (kaempferol) (5). Antioxidant potential of the compounds was evaluated using a DPPH spectrophotometric assay, while antibacterial activity was determined using a serial dilution microplate technique. The isolates demonstrated varying degrees of antioxidant and antibacterial activities. Of all the compounds investigated, compounds 1 and 5 demonstrated some antioxidant activity (EC50 = 75.49 ± 1.76 µM and 35.06 ± 0.85 respectively) but lower than l-ascorbic acid (EC50 = 13.55 ± 0.28 µM) used as a standard antioxidant agent. The minimum inhibitory concentration (MIC) of isolated compounds against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa varied from 16 µg/ml to more than 250 µg/ml. Some structure activity relationships could be established for these compounds.  相似文献   

13.

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O2 and 5% CO2 under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (Emax = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (Emax = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca2+-free solution, ADIS inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca2+-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl2, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca2 + channel blocker through both intracellular and extracellular Ca2 + release.  相似文献   

14.
The effects of aluminium (Al) on thyroid function were evaluated in adult Wistar rats intraperitoneally (i.p) injected with 7 mg Al (as lactate)/kg body weight (b.w) per day during a six week period. The time-course kinetics of Na125I (3 μCi per 100 g b.w, i.p) was analysed by measuring gamma-radioactivity of thyroid, serum, serum protein precipitate and bile, at times ranging from 2 to 96 h post-dosing. In Al-treated group the 125I thyroid uptake at 24 h (15,840 ± 570 vs. 18,030 ± 630 dpm/mg, P < 0.05) as well as the rate of 125I release from the gland, calculated as the slope of the plot between 24 and 96 h (84 ± 8 vs. 129 ± 11 dpm/mg/h, P < 0.05) were significantly reduced as compared to control. The biliary 125I excretion was not modified at all studied times. The Al content and lipid peroxidation (69.1 ± 8.5 vs. 53.2 ± 7.0 nmol MDA/g wet weight, P < 0.05) of thyroid tissue were increased in Al-treated rats. The serum concentrations of total thyroxine (T4, 3.78 ± 0.14 vs. 4.68 ± 0.12 μg/dL, P < 0.05) and total triiodothyronine (T3, 47 ± 4 vs. 66 ± 5 ng/dL, P < 0.05) were decreased by effect of Al, but free-T4 (1.05 ± 0.05 vs. 1.04 ± 0.04 ng/dL, NS) and thyrotropin (TSH, 2.7 ± 0.4 vs. 2.6 ± 0.5 ng/ml, NS) remain unchanged. In spite of the Al could indirectly affect thyroid iodide uptake and hormones secretion by a mechanism involving the induction of an oxidative stress state, however, these changes could be managed by the hypothalamus-pituitary-thyroid endocrine axis. We can conclude that in adult rats the Al would not act as a thyroid disruptor.  相似文献   

15.

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe2+ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s20,w = 17.0 S and D20,w = 3.21 × 107 cm2/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s20,w = 18.3 S and D20,w = 3.18 × 107 cm2/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.  相似文献   

16.
Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca2+ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by + 40-200% in a concentration-dependent manner (EC50 ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca2+ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca2+ sensitivity and sarcoplasmic reticulum (SR) Ca2+ cycling. Importantly, NCA did not affect diastolic Ca2+ or SR Ca2+ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.  相似文献   

17.
18.
In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)3], with 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between mS = ± 1/2 (geff ~ 9) or mS = ± 3/2 (geff ~ 4.3) states. Mössbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H37Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the “second-line” therapeutic drugs.  相似文献   

19.
The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca2+ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 106 L mol–1 and (5.26 ± 0.71) × 106 L mol–1, respectively. In addition, in the presence of calcium (10 μmol L–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 107 L mol–1. In addition, the binding constant of KChIP4a with Ca2+ was (7.1 ± 1.5) × 107 L mol–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca2+, and the integrity of the molecular structure of KChIP4a was important for Ca2+ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.  相似文献   

20.
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