Ca2+在植物细胞对逆境反应和适应中的调节作用

钙离子(Ca2+ )信号在植物的生长发育及其对环境的反应和适应中起着十分重要的作用。本文对Ca2+在植物细胞对低温、干旱和盐渍化逆境的反应和适应中的调节功能作一概述, 论述的主要问题包括: (1)Ca2+的亚细胞定位与分布, 细胞内Ca2+相对低水平的稳态平衡是Ca2+信号发生的基础; (2)Ca2+信号的优越性及其发生与传递; (3)Ca2+充当低温信号的传递者诱导抗寒锻炼和基因表达; (4)细胞内高水平Ca2+持久性调控越冬木本植物的生理休眠; (5)Ca2+对干旱、盐渍化及其渗透胁迫的调节作用; (6)Ca2+参与气孔开关运动的调节; (7)Ca2+参与逆境中细胞壁加厚和加固的调节。     

保卫细胞钙信号的研究进展

钙(Ca2+)是多种信号途径的第二信使。Ca2+成像技术的成熟和发展为显示保卫细胞胞 质Ca2+浓度（［Ca2+］cyt）的分布及外界刺激引起［Ca2+］cyt的变化模式提供了很好的研究工具,关于细胞内外Ca2+库释放Ca2+的机制也有了较清楚的认识。拟南芥突变体的研究使Ca2+ 信号上游分子及其排序更加明确,［Ca2+］cyt增加下游的磷酸化和去磷酸化 过程也是气孔关闭必需的生理过程。     

细菌中钙信号的作用

摘要：越来越多的实验证明二价钙离子（Ca2+）在细菌中有重要调控作用。本文从Ca2+ 信号对细菌生理的影响、细胞内Ca2+ 浓度及测定方法、细菌中Ca2+ 的运输和Ca2+ 结合蛋白四个方面综述了目前细菌中钙信号的研究进展。     

钠钙交换体的生理和病理生理功能研究进展

钠钙交换体(sodium calcium exchanger,NCX)是一种广泛分布于膜性结构(细胞质膜、线粒体膜、内质网、分泌小泡膜等)上的阳离子转运蛋白,具有两种转运Ca2+和Na+的模式:介导Na+内流、Ca2+外排的前向模式(forward mode)和作用相反的反向模式(reverse mode)。通过这种双向模式,NCX可以对胞质内Ca2+浓度进行快速精确的调节,继而影响细胞内信号转导、细胞生长发育、可兴奋细胞的兴奋及兴奋耦联的相关功能等一系列生理活动,如心肌和骨骼肌细胞的收缩、神经递质的释放、神经胶质细胞的迁移分化、免疫细胞的活化以及细胞因子与激素的分泌等。此外,在病理情况下,NCX反向模式的异常激活,被认为是NCX参与心血管系统、中枢神经系统、内分泌系统等多个系统病理生理过程的关键。在此,本文对NCX分子及其参与的生理、病理生理过程的研究进展作一综述,以提供关于NCX较为全面的认识。     

Ca~(2+)转运通路对金雀异黄酮舒张大鼠脑血管作用的影响

目的:研究Ca2+转运通路对金雀异黄酮舒张大鼠脑血管作用的影响。方法:75只大鼠被随机分为3组,分别经由二甲亚砜、金雀异黄酮和酪氨酸磷酸化抑制剂A47处理基底动脉及Willis环血管。每组大鼠进一步划分成5个亚组,每个亚组用不同浓度的细胞外Ca2+处理,分为:0、0.6、1.2、1.8和3.6 m M Ca2+组。5-羟色胺诱导血管收缩。测定大鼠基底动脉管壁厚度与官腔周长的比值;荧光成像分析法测定血管平滑肌细胞细胞内Ca2+浓度;免疫印迹分析检测肌球蛋白轻链激酶(MLCK),蛋白质磷酸酶催化亚基1(PP1),肌凝蛋白磷酸酶目标亚基1(MYPT1)的表达来测定血管平滑肌细胞Ca2+敏感性。结果:金雀异黄酮和酪氨酸磷酸化抑制剂A47显著降低大鼠基底动脉管壁厚度与官腔周长的比值(P0.01),Ca2+内流(P0.01,P0.05)及MLCK的表达(P0.01);增加PP1和MYPT1的表达(P0.01)。细胞外Ca2+与金雀异黄酮及酪氨酸磷酸化抑制剂A47有协同效应。硝苯地平和毒胡萝卜素可废除该效应。结论:低细胞外Ca2+水平增强了金雀异黄酮和酪氨酸磷酸化抑制剂A47的血管舒张作用。L型电压门控Ca2+通道(L-VGCC)和肌浆网Ca2+库(SR)参与交互效应。     

钙调神经磷酸酶信号通路参与钙激动剂介导的心肌细胞肥大

目的在于探讨不同来源的细胞内游离钙(［Ca2+］i）对钙调神经磷酸酶（calcineurin,CaN）依赖信号通路和心肌细胞肥大的影响。方法以原代培养的乳鼠心肌细胞为模型,血管紧张素Ⅱ（AngⅡ）、雷尼丁（RY）和三磷酸肌醇（IP3）（浓度均为10-7mol／L）激活心肌细胞［Ca2+］i,应用钙荧光指剂Fura-2／AM动态观测心肌细胞［Ca2+］i浓度,同时检测心肌细胞CaN活性及蛋白表达。用氚-亮氨酸（3H-Leu）掺入量测定心肌细胞蛋白质合成速率。结果发现AngⅡ、RY和IP3明显增加心肌细胞［Ca2+］i浓度、CaN活性及蛋白表达并提高3H-Leu的掺入量,与对照组心肌细胞相比差异显著（P＜0．01）。结论激活心肌细胞［Ca2+］i可明显提高心肌细胞蛋白合成速率,心肌细胞CaN活性及蛋白表达似与细胞［Ca2+］i变化有明显关系而与其来源无关,表明CaN信号通路在心肌细胞生长中发挥重要作用。     

温度逆境交叉适应对葡萄叶片膜脂过氧化和细胞钙分布的影响

 研究了高温锻炼对低温胁迫下和低温锻炼对高温胁迫下葡萄(Vitis vinifera)叶片中丙二醛（MDA）、谷胱甘肽(GSH)和抗坏血酸(AsA)含量变化以及细胞中Ca2+分布的影响。结果表明: 高（低）温胁迫使正常生长的叶片丙二醛含量升高, GSH和AsA含量下降,低（高）温锻炼预处理能减少MDA含量,提高GSH和AsA含量,抑制了由于温度胁迫引起MDA含量升高和GSH和AsA下降趋势。常温下葡萄叶肉细胞的Ca2+主要分布于液泡、细胞间隙中;高温胁迫和低温胁迫后,细胞质中聚集大量Ca2+沉淀颗粒,液泡中和细胞间隙Ca2+沉淀颗粒减少,叶绿体超微结构被破坏,Ca2+稳态平衡遭到破坏。高温锻炼后细胞质出现大量的Ca2+沉淀颗粒,主要来源于细胞间隙,低温锻炼后细胞质也出现大量的Ca2+沉淀颗粒,主要来源于液泡,两者的叶绿体超微结构都完整;高温锻炼的叶片经过低温胁迫和低温锻炼的叶片经过高温胁迫后,细胞间隙和液泡内Ca2+沉淀颗粒增加,细胞质中Ca2+沉淀颗粒很少,叶绿体较完整,Ca2+稳态平衡得以维持。推测高低温锻炼能够通过Ca2+启动抗逆基因表达和维持细胞中Ca2+稳态平衡来交叉适应低高温的胁迫。     

小脑症蛋白ASPM和钙调蛋白的复合物的纯化和表征

常染色体隐性小脑症(Autosomal recessive primary microcephaly, MCPH)是一种与大脑缩小和智力缺陷有关的神经发育障碍。 ASPM(abnormal spindle-like microcephaly-associated)是最常见的MCPH的致病基因,但其潜在机制尚不清楚。我们发现钙调蛋白(calmodulin, CaM)通过与ASPM的IQ区域相互作用而对ASPM的功能有重要的调控作用。我们纯化了ASPM IQ区域和CaM的复合物,并通过分子排阻色谱结合多角度静态光散射（SEC-MALS）和圆二光谱（CD）实验发现了ASPM和apo_CaM的结合比例为1：8。有趣的是,在Ca2+存在时,ASPM的IQ区域与Ca2+_CaM的结合比例变为了1：7。此外,通过比较不同条件下（Ca2+存在与否）的CD光谱,ASPM-CaM复合物显示出Ca2+依赖性的热稳定性变化。综上所述,我们的研究揭示了Ca2+诱导的ASPM-CaM相互作用的调节机制。     

植物体内的钙信使系绕

Ca对植物不仅仅是一种大量营养元素,更重要的是作为偶连胞外信号与胞内生理生化反应的第二信使,作为植物代谢和发育的主要调控者。本文介绍了Ca在植物细胞中的分布及其体内平衡机制,以及Ca2+信使系统调控的植物生理生化过程,讨论了外界信号通过Ca2+信使系统的传递和表达过程,Ca2+信使系统对基因表达的可能影响,以及Ca2+信使系统的作用机制,并提出了今后的研究方向。     

Zn~(2+)对PTP开放和细胞色素c释放的浓度依赖性双向调节

研究Zn2+对Ca2+介导线粒体通透过渡孔道(PTP)开放和线粒体细胞色素c释放的影响,及其与线粒体膜电位(ΔΨm)和Ca2+介导的线粒体Ca2+释放(mCICR)之间的关系.提取大鼠肝线粒体,通过紫外分光光度仪检测不同浓度Zn2+作用下Ca2+介导的PTP开放状态;采用荧光分光光度仪测定不同浓度Zn2+作用下线粒体膜电位的变化;采用双波长双光束紫外分光光度仪检测不同浓度Zn2+作用下测试体系内Ca2+浓度的变化,以反映线粒体Ca2+的转运情况(即mCICR);通过免疫印迹法检测不同浓度Zn2+作用下Ca2+介导的线粒体细胞色素c的释放.高浓度Zn2+完全抑制Ca2+介导的PTP开放和细胞色素c释放.一定浓度的Zn2+部分抑制Ca2+介导的PTP开放和细胞色素c释放.适当浓度Zn2+自身介导PTP开放和细胞色素c释放.低浓度Zn2+加速Ca2+介导PTP开放和Ca2+释放;高浓度和一定浓度Zn2+分别完全或部分破坏ΔΨm;高浓度Zn2+完全抑制mCICR.当抑制mCICR时,Ca2+和Zn2+对PTP开放和细胞色素c释放的作用完全抑制.结果表明,Zn2+以浓度依赖方式双向调节PTP开放和细胞色素c释放.Zn2+的作用可能与Zn2+破坏ΔΨm和影响mCICR相关.     

NAADP receptors

Of the established Ca(2+) mobilizing messengers, NAADP is arguably the most tantalizing. It is the most potent, often efficacious at low nanomolar concentrations. Recent studies have identified a new class of calcium release channel, the two-pore channels (TPCs), as the likely targets for NAADP. These channels are endolysosomal in localization where they mediate local Ca(2+) release, and have highlighted a new role of acidic organelles as targets for messenger-evoked Ca(2+) mobilization. Three distinct roles of TPCs have been identified. The first is to effect local Ca(2+) release that may play a role in endolysosomal function including vesicular fusion and trafficking. The second is to trigger global calcium release by recruiting Ca(2+)-induced Ca(2+) release (CICR) channels at lysosomal-ER junctions. The third is to regulate plasma membrane excitability by the targeting of Ca(2+) release from appropriately positioned subplasma membrane stores to regulate plasma membrane Ca(2+)-activated channels. In this review, I discuss the role of NAADP-mediated Ca(2+) release from endolysosomal stores as a widespread trigger for intracellular calcium signaling mechanisms, and how studies of TPCs are beginning to enhance our understanding of the central role of lysosomes in Ca(2+) signaling.     

Triggering of Ca2+ signals by NAADP-gated two-pore channels: a role for membrane contact sites?

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a potent Ca2+-mobilizing messenger implicated in many Ca2+-dependent cellular processes. It is highly unusual in that it appears to trigger Ca2+ release from acidic organelles such as lysosomes. These signals are often amplified by archetypal Ca2+ channels located in the endoplasmic reticulum. Recent studies have converged on the TPCs (two-pore channels) which localize to the endolysosomal system as the likely primary targets through which NAADP mediates its effects. 'Chatter' between TPCs and endoplasmic reticulum Ca2+ channels is disrupted when TPCs are directed away from the endolysosomal system. This suggests that intracellular Ca2+ release channels may be closely apposed, possibly at specific membrane contact sites between acidic organelles and the endoplasmic reticulum.     

NAADP as an intracellular messenger regulating lysosomal calcium-release channels

Recent studies into the mechanisms of action of the Ca(2+)-mobilizing messenger NAADP (nicotinic acid-adenine dinucleotide phosphate) have demonstrated that a novel family of intracellular Ca(2+)-release channels termed TPCs (two-pore channels) are components of the NAADP receptor. TPCs appear to be exclusively localized to the endolysosomal system. These findings confirm previous pharmacological and biochemical studies suggesting that NAADP targets acidic Ca(2+) stores rather than the endoplasmic reticulum, the major site of action of the other two principal Ca(2+)-mobilizing messengers, InsP(3) and cADPR (cADP-ribose). Studies of the messenger roles of NAADP and the function of TPCs highlight the novel role of lysosomes and other organelles of the endocytic pathway as messenger-regulated Ca(2+) stores which also affects the regulation of the endolysosomal system.     

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.     

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.     

Two-pore channels form homo- and heterodimers

Two-pore channels (TPCs) have been recently identified as NAADP-regulated Ca(2+) release channels, which are localized on the endolysosomal system. TPCs have a 12-transmembrane domain (TMD) structure and are evolutionary intermediates between the 24-TMD α-subunits of Na(+) or Ca(2+) channels and the transient receptor potential channel superfamily, which have six TMDs in a single subunit and form tetramers with 24 TMDs as active channels. Based on this relationship, it is predicted that TPCs dimerize to form functional channels, but the dimerization of human TPCs has so far not been studied. Using co-immunoprecipitation studies and a mass spectroscopic analysis of the immunocomplex, we show the presence of homo- and heteromeric complexes for human TPC1 and TPC2. Despite their largely distinct localization, we identified a discrete number of endosomes that coexpressed TPC1 and TPC2. Homo- and heteromerization were confirmed by a FRET study, showing that both proteins interacted in a rotational (N- to C-terminal/head-to-tail) symmetry. This is the first report describing the presence of homomultimeric TPC1 channels and the first study showing that TPCs are capable of forming heteromers.     

Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.     

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.     

Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease

Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling.