共查询到19条相似文献,搜索用时 140 毫秒
1.
目的和方法:采用双微电极电压钳(TEV)法研究细胞外Mn^2+对非洲爪蟾卵母细胞表达的内向整流钾通道(IRK1)的阻断作用。结果:细胞外Mn^2+浓度分别为1、1.25、2.5、5、10和20mmol/L,K^+浓度为90mmol/L,可见Mn^2+对IRK1的瞬间电流(旋加电压后2ms)具有Mn^2+浓度依赖性和电压依赖性阻断作用;细胞外加Mn^2+浓度较高时强;细胞外K^+浓度为90mmol/ 相似文献
2.
两类Ca^2+通道拮抗剂对脑缺血时Ca^2+/CaM PKhⅡ活性抑制的保护 … 总被引:2,自引:1,他引:1
本文以蒙古沙土鼠双颈总动脉结扎(BCAO)前脑缺血模型Ca^2+/CaM PKⅡ活性变化为指标,研究了以氯胺酮(KT)、右美沙芬(DM)、苄丙咯(BP)及硝苯吡啶(ND)为代表的配体门控Ca^2+通道(LGCC)及电压门控Ca^2+通道(VGCC)两类Ca^2+通道拮抗剂对缺血性脑损伤的保护作用。结果如下:(1)脑缺血后,胞浆型及颗粒型Ca^2+/CaM PKⅡ活性均明显下降;(2)缺血前单独用药 相似文献
3.
用膜片钳技术中的细胞贴附方式和内面向外方式,首次在新生大鼠大脑皮层星形神经元胞体膜上记录到一类电压依赖性钾通道。此通道可被20mmol/L TEA,5mmol/L Ba^2+,140mmol/L Cs^+阻断,不受20mmol/L4-AP影响,其激活不依赖Ca^2+。膜外钾离子浓度对通道的特性有显著的影响,逆转电位随K^+的增大而增大,并表现出一定的饱和现象,两者的对数呈线性关系;同一驱动电位下, 相似文献
4.
两类Ca~(2 )通道拮抗剂对脑缺血时Ca~(2 )/CaM PK Ⅱ活性抑制的保护作用 总被引:1,自引:0,他引:1
本文以蒙古沙土鼠双颈总动脉结扎(BCAO)前脑缺血模型Ca2+/CaMPKⅡ活性变化为指标,研究了以氯胺酮(KT)、右美沙芬(DM)、苄丙咯(BP)及硝苯吡啶(ND)为代表的配体门控Ca2+通道(LGCC)及电压门控Ca2+通道(VGCC)两类Ca2+通道拮抗剂对缺血性脑损伤的保护作用。结果如下:(1)脑缺血后,胞浆型及颗粒型Ca2+/CaMPKⅡ活性均明显下降;(2)缺血前单独用药,KT、DM、BP及ND对酶活性均具有剂量依赖性的保护作用;(3)与单独用药相比,联用异类药,KT+BP、KT+ND及DM+BP的保护作用显著增高,而联用同类药,BP+ND及KT+DM的保护作用无明显增高。结果提示:脑缺血中,LGCC及VGCC两类Ca2+通道均参与了缺血性脑损伤过程;两类Ca2+通道的单一拮抗对缺血性脑损伤均有保护作用,而联合拮抗效果更佳。 相似文献
5.
实验用Fluo-3负载细胞,在激光扫描共聚焦显微镜下直接监测缺氧后分散培养的大鼠海马CA1区神经元内游离Ca^2+浓度([Ca^2+]i)的变化,观察腺苷对这种变化的影响并初步探讨其作用机制。结果发现,急性缺氧使海马神经元[Ca^2+]i显著升高;腺苷(100μmol/L)明显抑制缺氧引起的[Ca^2+]i增高,腺苷A1受体拮抗剂CPT以及K^+通道阻断剂4-AP和ATP敏感性K^+通道阻断剂gl 相似文献
6.
LHRH—A2及无机离子促进草鱼脑垂体离体分泌GH的初步研究 总被引:1,自引:0,他引:1
用培养瓶(皿)培养法研究LHRH-A2、Ca^2+、K^+对草鱼脑垂体器官分泌GH的影响。结果表明,1nmol/L、10nmol/L、100nmol/L和1000nmol/L的LHRH-A2都能显著促进GH的分泌;随着Ca^2+浓度(0.1、1、3mmol/L)的增高GH的分泌增强,在1mol/L和3mmol/L Ca^2+条件下的GH分泌水平显著高于在0.1mmol/L Ca^2+条件下的GH分 相似文献
7.
本实验观察了正常及高血压大鼠红细胞膜Ca^2+,Mg^2+-ATP酶对不同浓度Cu^2+Mg^2+的反应。结果表明:(1)在自发性高血压大鼠(SHR),此酶最适反应的Ca^2+浓度为10^-6mol/L;WKY大鼠为10^-4mol/L;两肾一环型肾性高血压大鼠(RHR)为10^-7molg/L,Wistar大鼠为10^-4mol/L;Ca^2+高于以上各相应浓度时该酶活性受到抑制;(2)作为该酶 相似文献
8.
T—2毒素对心肌细胞三型钙通道的阻滞作用 总被引:2,自引:0,他引:2
用膜片钳连细胞电压钳法,在培养的Wistar大鼠单个心肌细胞上记录了T-2毒素对B,L和T三型Ca^2+通道单通道电活动的影响,结果表明,T-2毒素浓度为10mg/L时,心肌细胞B,L和T三型Ca^2+通道均受到有显的阻滞,其阻滞作用表现为Ca^2+通道的开放概率减小,开放时间缩短,关闭时间延长,而对流过Ca^2|通道的Ba^2+流幅值无影响。 相似文献
9.
G蛋白在亮啡肽诱导心肌细胞内钙释放中的作用 总被引:1,自引:0,他引:1
本实验采用分离的SD大鼠心室肌细胞,以Fura-2AM荧光指示剂负载,检测心肌细胞内游离钙浓度(Ca^2+)变化。探讨亮啡肽(LEK)对(Ca^2+)的作用及其机制。实验结果:LEK(60μmol/L)能升高(Ca^2+)移去细胞外液钙此效应仍能出现,用caffeine (5mmol/L)耗竭细胞内钙池的钙,该效应消失,纳洛酮(100μmol/L),百日咳毒素(200ng/L)处理8-10h及pr 相似文献
10.
cGMP对原代培养猪冠状动脉平滑肌细胞钙激活钾通道的作用 总被引:11,自引:1,他引:10
3′,5′-环-磷酸鸟苷(cGMP)具有激活血管平滑肌细胞膜上钙激活钾通道(KCa通道)的作用,从而引起血管平滑肌细胞的舒张。但cGMP激活KCa物机制存在争论。本工作应用膜片箝技术以原代培养猪冠状动脉平滑肌细胞为对象研究了cGMP影响KCa通道的机制。实验结果显示:(1)在cell-attached膜片方式下,当溶液内游离Ca^2+浓度为10^-7mol/L,膜电位为+70mV时,不同浓度的cG 相似文献
11.
Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage. 相似文献
12.
Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants. 总被引:1,自引:0,他引:1
Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system. 相似文献
13.
Monteilh-Zoller MK Hermosura MC Nadler MJ Scharenberg AM Penner R Fleig A 《The Journal of general physiology》2003,121(1):49-60
Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells. 相似文献
14.
Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+
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We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+). 相似文献
15.
Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change. 相似文献
16.
Hayley M Sun M Merschrod EF Davis PJ Robinson JJ 《Journal of cellular biochemistry》2008,103(5):1464-1471
We have investigated the biochemical and functional properties of toposome, a major protein component of sea urchin eggs and embryos. Atomic force microscopy was utilized to demonstrate that a Ca(2+)-driven change in secondary structure facilitated toposome binding to a lipid bilayer. Thermal denaturation studies showed that toposome was dependent upon calcium in a manner paralleling the effect of this cation on secondary and tertiary structure. The calcium-induced, secondary, and tertiary structural changes had no effect on the chymotryptic cleavage pattern. However, the digestion pattern of toposome bound to phosphatidyl serine liposomes did vary as a function of calcium concentration. We also investigated the interaction of this protein with various metal ions. Calcium, Mg(2+), Ba(2+), Cd(2+), Mn(2+), and Fe(3+) all bound to toposome. In addition, Cd(2+) and Mn(2+) displaced Ca(2+), prebound to toposome, while Mg(2+), Ba(2+), and Fe(3+) had no effect. Collectively, these results further enhance our understanding of the role of Ca(2+) in modulating the biological activity of toposome. 相似文献
17.
The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0. 相似文献
18.
Pyruvate dehydrogenase phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by Ca(2+)-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca(2+) was evaluated in activity and L2 binding studies. An increase in the Mg(2+) concentration decreased the Ca(2+) concentration required for half-maximal activation of PDP1 from 3 to 1 microM, but this parameter was unchanged at 3 microM with PDP1c. Near 1 microM Ca(2+), tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg(2+). With just Ca(2+) included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c.Ca(2+).L2 complex is supported by micromolar Ca(2+) concentrations and becomes sensitive to the Mg(2+) level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 +/- 2.5 microM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K(d) of approximately 0.3 microM. Isothermal titration calorimetry and (45)Ca(2+) binding studies failed to detect binding of Ca(2+) (<100 microM) to L2 or PDP1c, alone, but readily detected binding to L2 and PDP1c. Therefore, both proteins are required for formation of a complex with tightly held Ca(2+), and complex formation hinders the tendency of PDP1c to form a dimer. 相似文献
19.
Inward rectification of the IRK1 K+ channel reconstituted in lipid bilayers. 总被引:1,自引:0,他引:1
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Inwardly rectifying potassium (K+) channels (IRK1) were incorporated into lipid bilayers to test the relative contributions of various mechanisms to inward rectification. IRK1 channels were expressed in Xenopus laevis oocytes and oocyte membrane vesicles containing the channels were fused with lipid bilayers. The major properties of the IRK1 channel were similar whether measured in the oocyte membrane or lipid bilayer; the single channel conductance was 21 pS in 140 mM symmetrical [K+] and varied as a square root of external [K+]. Importantly, IRK1 channels display voltage-dependent inward rectification in the absence of divalent ions or charged regulators such as spermine, indicating that they possess an intrinsic rectification mechanism. Although rectification was significantly increased by either Mg2+ or spermine added to the cytoplasmic face of the channel, their effects could not be explained by simple block of the open pore. The Hille and Schwartz (1978) model, originally proposed to explain inward rectification by singly charged blocking particles, cannot be used to explain rectification by multiply charged blocking particles. As an alternative, we propose that in addition to a slow gating mechanism producing long lasting open and closed states, there is a distinct, intrinsic fast gating process amplified by cytoplasmic Mg2+ and/or polyamine binding to the channel. 相似文献