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.     

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。     

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+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.     

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.     

Ca2+ Influx through Store-operated Ca2+ Channels Reduces Alzheimer Disease β-Amyloid Peptide Secretion

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca²⁺ homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca²⁺ homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca²⁺ homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca²⁺ homeostasis. Here, we developed a system in which cellular Ca²⁺ homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca²⁺ entry pathway, to generate cells with constitutive and store depletion-induced Ca²⁺ entry. We found striking effects of Ca²⁺ entry induced by overexpression of the constitutively active STIM1_D76A mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca²⁺ entry by expression of STIM1_D76A significantly reduced Aβ secretion. Our results suggest that disruptions in Ca²⁺ homeostasis may influence AD pathogenesis directly through the modulation of Aβ production.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

How does regulatory Ca2+ regulate the Na+-Ca2+ exchanger?

Spatial and temporal regulation of intracellular Ca2+ concentrations is a fundamental requirement for life. The mammalian cardiac Na+-Ca2+ exchanger serves as the main mechanism for Ca2+ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca2+, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca2+. Together with mutational analysis of the Ca2+ binding sites, this study reveals the crucial role of one of the two bound Ca2+ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.     

Ca2+预处理对热胁迫下辣椒叶肉细胞中Ca2+-ATP酶活性的影响

在常温下生长的辣椒(Capsicum annum L.)叶肉细胞中Ca2+-ATP酶主要分布于质膜、液泡膜上,叶绿体的基质和基粒片层上也有少量分布;在40℃下热胁迫不同的时间,酶活性逐渐下降,直至叶绿体超微结构解体.同样条件下,经过Ca2+预处理后,分布在上述细胞器膜或片层上的酶活性大大提高,表明Ca2+预处理对该酶活性具有激活作用;Ca2+预处理对热胁迫下的超微结构的完整性具有一定的保护作用,并且能使Ca2+-ATP酶在热胁迫下维持较高活性.结果表明,Ca2+预处理增强辣椒幼苗的抗热性,可能与其稳定细胞膜、从而使Ca2+-ATP酶在热胁迫下保持较高活性有一定关系.     

Ca2+对钠通道作用新发现

钠通道是参与心肌兴奋传导和维持心脏节律的重要物质。ca2+在心肌细胞的兴奋-收缩偶联中起重要     

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.     

Ca2+与植物抗旱性的关系

干旱是制约植物生长发育的主要逆境因素，并抑制根系对钙的吸收。近年来研究表明，外源钙能提高植株的抗旱性，抑制干旱胁迫下活性氧物质的生成，保护细胞质膜的结构，维持正常的光合作用，以及调节激素和一些重要的生化物质代谢；此外，细胞内Ca2+可作为第二信使传递干旱信号，调节干旱胁迫导致的生理反应。     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

NaHCO3胁迫下星星草根中Ca2+与Ca2+-ATPase 的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO₃胁迫条件下星星草(Puccinellia tenuiflora)根中Ca²⁺和Ca²⁺-ATPase 进行超微细胞化学定位研究, 旨在进一步探讨Ca²⁺在NaHCO₃胁迫诱导胞内信号转导过程中的作用, 以及Ca²⁺-ATPase活性定位变化与NaHCO₃胁迫下星星草抗盐碱能力的关系。结果表明: 在正常状态下, 根毛区细胞质内Ca²⁺较少, 主要位于质膜附近和液泡中, Ca²⁺-ATPase主要定位于质膜和液泡膜, 有一定活性。在0.448%NaHCO₃胁迫下, 根毛区细胞质中Ca²⁺增多, 液泡中Ca²⁺减少, 且主要集中于液泡膜附近, 质膜和液泡膜Ca²⁺-ATPase活性明显升高。在1.054%NaHCO₃胁迫下,细胞质中分布的Ca²⁺增多, 而液泡中Ca²⁺极少, Ca²⁺-ATPase活性也降低。以上结果表明, Ca²⁺亚细胞定位和Ca²⁺-ATPase活性变化在星星草响应NaHCO₃胁迫的信号传递过程中具有重要作用。     

毛白杨幼苗低温锻炼过程中Ca2+ 的作用及细胞Ca2+-ATP酶活性的变化

观察了低温锻炼以及随后的常温生长中毛白杨幼苗质膜及线粒体Ca2 ATP酶活性、CaM含量和抗冻性的变化。结果表明 ,单纯低温锻炼在一定程度上提高了毛白杨幼苗质膜及线粒体Ca2 ATP酶活性、CaM含量和抗冻性 ,减小了低温胁迫所引起的质膜及线粒体Ca2 ATP酶活性和CaM含量的下降程度 ,促进了胁迫后恢复过程中质膜及线粒体Ca2 ATP酶活性和CaM水平的迅速回升。在低温锻炼的同时 ,用CaCl2 处理能加强低温锻炼的效果 ,但这种效应可被EGTA、LaCl3和CPZ处理所抑制     

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.     

ELKS/Voltage-Dependent Ca2+ Channel-β Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic β Cells

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