Ca2+-binding Motif of βγ-Crystallins

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca²⁺ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca²⁺-binding sites. βγ-Crystallins make a separate class of Ca²⁺-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca²⁺ binding to βγ-crystallins in mediating biological processes are yet to be elucidated.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

植物细胞Ca2+的微调系统——Ca2+_ATPase

本文对植物体细胞Ca２＋-ATPase的类型、亚细胞定位、生化特性、分子量差异、基因克隆、酶活性调节剂以及生理功能等方面的研究进展进行综述和讨论。     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca2 泵(Ca2 -ATPase)是调节细胞内Ca2 浓度的重要蛋白质之一.Ca2 泵在转运Ca2 的过程中经历一系列构象变化.其中,E1状态为外向的Ca2 高亲和状态,E2状态则为内向的Ca2 低亲和状态.目前,骨骼肌内质网Ca2 泵转运Ca2 过程中的几个中间状态,包括E1-2Ca2 ,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析.介绍这几种状态的晶体结构,并分析Ca2 泵在执行功能过程中结构与功能的关系.     

Is Ca2+ antagonists binding protein from cytosolic fraction the precursor of alpha 1-subunit of Ca2+ channel?

The binding of Ca2+ antagonists to soluble proteins obtained by ammonium sulphate precipitation from cytosol fraction of rabbit skeletal muscles was studied. The KD values for 3H D-888 and 3H PN 200-110 binding to soluble proteins were 21.3 +/- 3.1 nmol.l-1 and 28.8 +/- 8.9 nmol.l-1 respectively. Photoaffinity labelling of the soluble proteins with the arylazide 1,4-dihydropyridine probe 3H azidopine resulted in labelling of the 85-95 K protein band as determined by SDS polyacrylamide gel electrophoresis. Partial purification of prelabelled soluble sample by gel filtration on Sephadex G-150 gave a more precise molecular weight of 90 +/- 2.5K. Polyclonal antibodies prepared against Ca2+ channel complex from rabbit muscle T-tubules inhibited the 3H PN 200-110 binding. Our results suggest that the soluble protein with Mr = 90K +/- 2.5K may be a precursor of the large subunit of the membrane bound L-type Ca2+ channel in rabbit skeletal muscle.     

Are Ca2+ channels targets of praziquantel action?

Praziquantel is the current drug of choice for the control of schistosomiasis. It is highly effective against all species of schistosomes and shows minimal adverse effects. Though introduced for the treatment of schistosomiasis more than 20 years ago, the mode of action of praziquantel remains to be elucidated. This review will focus on advances in defining the molecular target of praziquantel action, with particular emphasis on recent work indicating an important role for voltage-gated calcium channels.     

Ca2+ Channels as Targets of Neurological Disease: Lambert–Eaton Syndrome and Other Ca2+ Channelopathies

In the nervous system, voltage-gated Ca2+ channels regulate numerous processes critical to neuronal function including secretion of neurotransmitters, initiation of action potentials in dendritic regions of some neurons, growth cone elongation, and gene expression. Because of the critical role which Ca2+ channels play in signaling processes within the nervous system, disruption of their function will lead to profound disturbances in neuronal function. Voltage-gated Ca2+ channels are the targets of several relatively rare neurological or neuromuscular diseases resulting from spontaneously-occurring mutations in genes encoding for parts of the channel proteins, or from autoimmune attack on the channel protein responses. Mutations in CACNAIA, which encodes for the alpha1A subunit of P/Q-type Ca2+ channels, lead to symptoms seen in familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6. Conversely, autoimmune attack on Ca2+ channels at motor axon terminals causes peripheral cholinergic nerve dysfunction observed in Lambert-Eaton Myasthenic Syndrome (LEMS), the best studied of the disorders targeting voltage-gated Ca2+ channels. LEMS is characterized by decreased evoked quantal release of acetylcholine (ACh) and disruption of the presynaptic active zones, the sites at which ACh is thought to be released. LEMS is generally believed to be due to circulating antibodies directed specifically at the Ca2+ channels located at or near the active zone of motor nerve terminals (P/Q-type) and hence involved in the release of ACh. However, other presynaptic proteins have also been postulated to be targets of the autoantibodies. LEMS has a high degree of coincidence (approximately 60%) with small cell lung cancer; the remaining 40% of patients with LEMS have no detectable tumor. Diagnosis of LEMS relies on characteristic patterns of electromyographic changes; these changes are observable at neuromuscular junctions of muscle biopsies from patients with LEMS. In the majority of LEMS patients, those having detectable tumor, the disease is thought to occur as a result of immune response directed initially against voltage-gated Ca2+ channels found on the lung tumor cells. In these patients, effective treatment of the underlying tumor generally causes marked improvement of the symptoms of LEMS as well. Animal models of LEMS can be generated by chronic administration of plasma, serum or immunoglobulin G to mice. These models have helped dramatically in our understanding of the pathogenesis of LEMS. This "passive transfer" model mimics the electrophysiological and ultrastructural findings seen in muscle biopsies of patients with LEMS. In this model, we have shown that the reduction in amplitude of Ca2+ currents through P/Q-type channels is followed by "unmasking" of an L-type Ca2+ current not normally found at the motor nerve terminal which participates in release of ACh from terminals of mice treated with plasma from patients with LEMS. It is unclear what mechanisms underlie the development of this novel L-type Ca2+ current involved in release of ACh at motor nerve terminals during passive transfer of LEMS.     

Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

苹果果肉质膜微囊主动运输Ca2+的Ca2+-ATP酶特性

应用４５Ｃａ２ ＋ 示踪法研究了苹果果肉质膜微囊依赖于Ｃａ２＋ 的ＡＴＰ 酶（Ｃａ２＋ＡＴＰ酶）活性与Ｃａ２＋ 运输之间的关系及激素对该酶活性的影响。结果表明：Ｃａ２ ＋ＡＴＰ 酶存在于质膜上并受载体Ａ２３１８７ 刺激而活性增加,该酶活性与依赖于ＡＴＰ 的Ｃａ２ ＋ 运输依抑制剂ＥＢ、游离Ｃａ２＋ 和ＡＴＰ浓度的变化并呈极为相似的饱和动力学特征;而其ＥＢ 半抑制浓度,Ｃａ２＋ 和ＡＴＰ 半饱和浓度分别为０ ．１ ,０ ．１ 和５０ μｍｏｌ／Ｌ,从而证实了正是Ｃａ２＋ＡＴＰ酶推动苹果果肉质膜微囊的Ｃａ２＋ 的主动运输。生长素与萘乙酸均可促进苹果果肉质膜微囊Ｃａ２＋ＡＴＰ酶活性和Ｃａ２＋ 吸收,而赤霉素则无此作用。     

Ca2+和突触细胞融合

神经突触传递对于神经系统功能的实现具有十分重要的意义,而神经突触传递涉及到突触囊泡膜和突触前膜的融合,3种膜蛋白SNARE特异性识别并形成复合物,从而介导了神经递质的释放。Ca^2 通过其感受器突触结合蛋白而调节了突触细胞的融合过程,也最终影响了神经元的胞吐作用。     

Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca2+-binding mode of this C2-domain as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential.     

Ca2+依赖和Ca2+不依赖的囊泡分泌研究进展

Ca2 是促发囊泡胞吐的关键调节因子.最近的研究表明,分泌囊泡和通道之间的空间距离调节囊泡分泌的过程和性质.Ca2 通道开口附近形成的Ca2 微区和Ca2 钠区和囊泡快速递质释放有非常紧密的联系.SNARE蛋白和钙离子传感器synaptotagmins等在触发分泌中起调控作用.同时另有一类不依赖于Ca2 的囊泡分泌存在.Latrotoxin和mastoparan等可以激活这一类不依赖于Ca2 的信号通路,从而触发囊泡释放.本文主要从ca2 对囊泡胞吐的调控作用着手,综述了Ca2 依赖和Ca2 不依赖的囊泡分泌过程和可能的调控机制.     

Annexins—New family of Ca2+-regulated-phospholipid binding protein

Calcium and phospholipid binding proteins have been identified and localized by immunocytochemistry in a wide range of cells and tissues. Two of these proteins (calpactins) also bind F-actin and are substrates for tyrosine kinases. The similar membrane-binding properties of these molecules arise from conserved amino acid sequences and a model is proposed for the tertiary structure of a common calcium and phospholipid binding domain.     

43Ca nuclear magnetic resonance of Ca2+–calmodulin solutions: Effects of trifluoperazine and peptides

By using a conventional FT NMR spectrometer and probe, we first detected ⁴³Ca NMR spectra of the Ca²⁺ (2.9 mM): calmodulin (0.725 mM) (1:1 per binding site) complex in 0.15 M N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid (HEPES)–K⁺ buffer (pH 7.2). The half-band width of the complex was nearly 160 Hz and the signal of the complex was located at the 2.13 ppm (43 Hz) lower field from that of the free Ca²⁺ ion. By adding trifluoperazine, melittin, substance P or glucagon, the half-band widths of the Ca²⁺–calmodulin complex (1:1 per binding site) were remarkably reduced and the chemical shifts of the complex moved back to the upper field. It is suggested that the Ca²⁺ ion may bind to Ca²⁺ low-affinity sites more tightly in the presence of those effectors than in their absence.     

Is a Ca2+ -ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea-pig spermatozoa?

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

川楝素对K+、Ca2+通道活动及细胞内Ca2+浓度的调控

川楝素是我国学者从驱蛔中药中分离、鉴定的一个三萜化合物,已证明具选择地影响神经递质释放,有效地对抗肉毒中毒,促进细胞分化、凋亡,抑制肿瘤增殖,抑制昆虫发育和取食,影响K 、Ca2 通道活动等多种生物效应.综述了证明川楝素抑制多种K 通道,选择地易化L型Ca2 通道和进而升高胞内Ca 浓度的研究资料,并对川楝素产生这些生物效应的机制进行了讨论.