质膜Ca2+-ATPase的结构与功能

质膜Ca2+-ATPase (PMCA)是P型ATPase家族的一员,在真核细胞中主要负责信号刺激后胞内高浓度Ca2+的清除扫尾工作,并对维持静息状态下较低Ca2+浓度起着重要的调节作用.PMCA的一级结构已被确定,拓扑学结构显示,它有10个跨膜区和3个胞浆功能区.它的4个编码基因可产生4种亚型(PMCA 1~4),这些亚型在功能与分布上存在差异.PMCA的活性可被钙调蛋白等多种因素调节,这与其结构特征息息相关.近年来,PMCA已被证实与脂筏结构有一定关联,它在信号传导和细胞凋亡中的作用也成为目前科学研究的焦点.本文主要对PMCA的结构、亚型和功能的研究现状进行综述.     

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。     

低温胁迫下拟南芥CBF1 超表达突变体胞质中Ca2+ 浓度的变化

低温严重影响植物的生长, 低温刺激可引起植物细胞中Ca²⁺浓度迅速升高。以拟南芥(Arabidopsis thaliana) CBF1 超表达突变体为材料, 研究了低温处理时CBF1基因的表达情况及胞质Ca²⁺的浓度变化。结果表明, CBF1本身可受低温诱导。同时将水母发光蛋白基因转入该拟南芥突变体中并检测Ca²⁺的浓度变化, 发现低温刺激时突变体细胞质中Ca²⁺的浓度变化幅度明显高于野生型, 但液泡的胞质面两侧Ca²⁺的浓度变化相似。用EGTA和LaCl₃处理拟南芥后, 胞质Ca²⁺的浓度升高被抑制, 并且CBF1突变体及对照胞质中的Ca²⁺浓度下降到同一水平。上述结果表明, Ca²⁺参与了CBF1应答低温信号的转导过程, 并且CBF1超表达突变体可能是通过提高胞质Ca²⁺浓度来提高植物的抗低温胁迫能力。     

马蔺体内主要阳离子含量月变化及分布

在自然盐碱生境下,通过测定不同月份土壤和马蔺体内主要阳离子Na⁺、K⁺、Ca²⁺、Mg²⁺的含量,研究了主要阳离子的吸收、转运变化及其在马蔺体内的分布.结果表明: 不同月份马蔺体内阳离子含量变动很大.在6月以后,随着马蔺的生长, Na⁺、K⁺、Ca²⁺和Mg²⁺4种离子在植物体内累积量逐渐增加.其中,根中Ca²⁺、Na⁺含量峰值出现在7月,分别为2.30%和0.51%,K⁺、Mg²⁺的含量峰值分别出现在9、10月,分别为0.27%和0.28%;叶片中Na⁺含量在7月达到最大值(0.57%);K⁺、Ca²⁺和Mg²⁺在8月分别达到1.30%、2.69%和0.47%.与Na⁺相比, 7、8月时马蔺对K⁺的选择吸收能力较低,但转运能力较强.马蔺对所测离子有很强的富集能力,各种离子在植物体内的含量都明显高于土壤背景值,且不同部位对离子的利用和累积能力不同,马蔺对各阳离子的累积主要集中在地上30 cm到地下40 cm范围内.马蔺地上部分平均单株K⁺、Na⁺、Ca²⁺和Mg²⁺含量分别是地下部分的9.11、4.07、0.98和2.27倍.     

重组PICK1蛋白的多聚形式及其与Ca2+和Mg2+的结合

蛋白激酶Cα相互作用蛋白1(PICK1) 是从线虫到人的所有生物中非常保守的一类存在于细胞质中的膜结合蛋白,在蛋白质转运,以及细胞内信号转导过程中发挥重要作用.通过基因重组技术获得PICK1及其截短的 N-PDZ(1～110 残基)和 BAR-C(128～416残基)重组蛋白,结合变性与非变性聚丙烯酰胺凝胶电泳,以及分子排阻层析,表明溶液中的PICK1主要以二聚体形式存在.利用荧光光谱分析PICK1与金属离子Ca²⁺和Mg²⁺的结合情况.结果表明,在0.02 mol/L Hepes, pH 7.2,随着2种金属离子的不断滴加,PICK1在338 nm 处的最大荧光强度逐渐降低,PICK1与Ca²⁺结合常数为Ka1=（2.34±0.20）×10.6 L/mol^-1,Ka2=(7.75±0.62)×10.5 L/mol^-1,而Mg²⁺结合常数为Ka=（5.00±0.40）×10.6 L/mol^-1.另外,对PICK1的N端区域N-PDZ和C端区域BAR-C的重组片段与金属离子Ca²⁺和Mg²⁺结合情况进一步分析表明,Ca²⁺既能与PICK1的N 端N-PDZ结合,又可与C端BARC结合,而Mg²⁺只结合在PICK1的N-PDZ区域.比较Ca²⁺或Mg²⁺对PICK1结合脂质的影响,显示Ca²⁺能明显增强蛋白和脂质的结合.     

盐胁迫下构树幼苗各器官中K+、Ca2+、Na+和Cl-含量分布及吸收特征

将当年生构树幼苗置于含有不同浓度（04、1、2、3、4 g·kg^-1）NaCl的土壤中,研究其生物量积累、叶片细胞质膜透性和K⁺、Ca²⁺、Na⁺、Cl^-等离子的吸收、分布及运输,并观察盐害症状.结果表明：构树幼苗的叶片质膜透性随着NaCl浓度的增加和胁迫时间的延长而升高,根冠比随NaCl浓度的升高而增加,大于3 g·kg^-1的土壤盐胁迫对构树叶片的质膜透性及植株的生物量积累影响显著.构树幼苗各器官中Na⁺和Cl^-含量随土壤NaCl浓度升高而显著增加,K⁺和Ca²⁺则随之降低,叶片各离子含量均明显高于根和茎.说明盐胁迫影响根系对K⁺和Ca²⁺的吸收,并抑制了它们向地上部分的选择性运输,使叶和茎的K⁺和Ca²⁺含量下降.构树通过吸收积累Na⁺和Cl^-抵御土壤盐分带来的渗透胁迫,但过量的Na⁺和Cl^-积累会造成单盐毒害.作为抗盐性较高的非盐生植物,构树地上部分的拒盐作用不显著.     

金属离子和脲对白蜡虫碱性磷酸酶的影响

各种金属离子及脲对白蜡虫Ericerus pela (Chavannes)碱性磷酸酶的活性有不同的影响。从白蜡虫雌成虫中分离纯化得到碱性磷酸酶,加入各种不同浓度的金属离子及脲测定酶的活力。一价金属离子Na⁺、K⁺、Li⁺对酶活力没有影响。碱土金属离子Ca²⁺、Mg²⁺、Ba²⁺对酶有激活作用,激活作用的大小顺序依次为Ca²⁺、Ba²⁺、Mg²⁺。第一过渡金属离子中,Mn²⁺、Co²⁺、Ni²⁺对酶有激活作用,而Zn²⁺、Cu²⁺有抑制作用。重金属离子Cd²⁺、Pb²⁺对酶有抑制作用。Ca²⁺激活作用表现为非竞争性激活效应。Cu²⁺抑制作用表现为非竞争性抑制效应。脲对碱性磷酸酶有变性失活作用,按脲浓度可分为低于3 mol/L和高于3 mol/L两种类型。低浓度的脲对白蜡虫碱性磷酸酶的活性抑制的动力学表现为混合型效应。     

不同森林群落类型溪流水化学特征的季节动态

以小兴安岭凉水自然保护区内的阔叶红松林、云冷杉林和落叶松人工林为研究对象, 于2006年3—10月, 分析了其溪流水化学特征的动态变化. 结果表明: 不同月份3种森林群落溪流水的主要阳离子含量均表现为 Ca²⁺>Na⁺>K⁺>Mg²⁺, 主要阴离子含量均为HCO₃^->SO₄^2->NO₃^->Cl^-;不同群落类型的主要离子含量影响显著, 3种森林群落溪流水中Na⁺、Ca²⁺、Mg²⁺、Fe²⁺和Fe³⁺平均含量为云冷杉林>落叶松人工林>阔叶红松林, 而K⁺为落叶松人工林>云冷杉林>阔叶红松林; 主要阴离子平均含量均以落叶松人工林溪流水中为最高.     

番茄calnexin基因的克隆及胁迫表达分析

Calnexin是内质网中重要的类凝集素分子伴侣,其主要作用是辅助糖基化蛋白的折叠和装配,调节内质网中的Ca²⁺稳态平衡和Ca²⁺信号传导过程。从番茄(Lycopersicon esculentum)的cDNA文库中克隆到calnexincDNA全序列,将其命名为Lecnx61.0,并以其3’端DNA片段为探针对番茄基因组进行Southern分析,结果表明Lecnx61.0在该基因组中仅有一个拷贝;Northern和W estern分析表明,Lecnx61.0的表达还受热激、冷害、盐害和内质网应激诱导剂衣霉素的诱导,但对干旱胁迫没有明显的反应。LeCNX 61.0蛋白对Ca²⁺亏缺胁迫的响应呈现组织特异性,但高浓度Ca²⁺并不影响各组织中LeCNX 61.0蛋白的含量,实验结果表明LeCNX 61.0蛋白可能在植物抵抗特定的环境胁迫中发挥作用。     

盐胁迫对桑树幼苗生长、叶片水分状况和离子分布的影响

以黑龙江省两个桑树品种（秋雨桑和泰来桑）为试验材料,研究了不同盐浓度下桑树幼苗生长、叶片水分关系和不同器官中离子的分布.结果表明：盐胁迫明显降低了桑树幼苗的植株高度和每株干物质量,且对新生叶片干质量的影响大于老叶片.随着盐胁迫的加重,两个品种桑树的叶片水势、渗透势、压力势和相对含水量明显下降,根、茎中Na⁺浓度明显增加,当外界NaCl浓度达到或超过150 mmol·L^-1时,各器官中Na⁺浓度达到饱和.盐胁迫明显降低了两个品种桑树根、茎和叶片中K⁺ 和 Ca²⁺浓度,以及茎和叶片中Mg²⁺浓度,而对根中Mg²⁺浓度影响不大.Na⁺在根、茎和老叶中的区域化分布是两个品种桑树生长过程中表现出耐盐性的机理之一,而盐胁迫使叶片中的Ca²⁺、K⁺和Mg²⁺浓度降低,导致植株体内的离子亏缺,从而限制了植株的生长.     

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Plasma membrane Ca²⁺ pumps (PMCA) that expel Ca²⁺ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca²⁺-Mg²⁺-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K_i value of 46 ± 5 µM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca²⁺-Mg²⁺-ATPase in human embryonic kidney-293 cells with a higher K_i value (105 ± 11 µM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 µM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca²⁺] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium. coronary artery; rat aorta; smooth muscle; endothelium     

嗜热子囊菌光孢变种Thermoascus aurantiacus var. levisporus内切β-葡聚糖酶的分离纯化及特性研究

采用培养分离、室内测定等方法,对嗜热子囊菌光孢变种Thermoascus aurantiacus var. levisporus产生的内切β-葡聚糖酶进行了分离纯化及特性研究.粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等步骤获得了凝胶电泳均一的内切β-葡聚糖酶.结果表明,经12% SDS-PAGE测得酶的单亚基分子量约为31.5 kD,凝胶过滤层析测得酶的分子量约为34.5 kD.该酶反应的最适温度为55 ℃,最适pＨ为2.5～3.0该酶在pH3.0条件下60 ℃较为稳定;80 ℃保温30 min有20%原酶活性.金属离子对内切β-葡聚糖酶活性影响较大, 其中K⁺、 Ca²⁺、Mn²⁺对酶有激活作用;Al⁺、Cu²⁺ 、Al³⁺对酶有显著抑制作用.该酶对羧甲基纤维素具有很强的底物特异性.     

Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice

We investigated the roles and relationships of plasma membrane Ca²⁺-ATPase (PMCA), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2, and Na⁺/Ca²⁺ exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4^–/–) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1^+/–Pmca4^–/–) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca²⁺ transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca²⁺-free physiological salt solution containing EGTA. We measured the contributions of Ca²⁺ clearance components by inhibiting SERCA2 (with 10 µM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 µM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be 25–30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca²⁺ extrusion-relaxation relationship, i.e., Ca²⁺ homeostasis, of bladder smooth muscle. ATP2B; sarco(endo)plasmic reticulum Ca²⁺-ATPase 2; Na⁺/Ca²⁺ exchanger; homeostasis     

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca clearance from MCF-7 breast cancer cells

The expression of the plasma membrane Ca²⁺ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca²⁺ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression – either by treatment or overexpression – led to enhanced Ca²⁺ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca²⁺ homeostasis. These results suggest that modulation of Ca²⁺ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.     

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

We previously showed that plasma membrane Ca²⁺-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca²⁺ ([Ca²⁺]_i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1^+/–, Pmca4^+/–, Pmca4^–/–, and Pmca1^+/–Pmca4^–/– mice. There were no differences in basal [Ca²⁺]_i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca²⁺]_i (130–180%) in smooth muscle from Pmca1^+/– and Pmca1^+/–Pmca4^–/– bladders than those in WT or Pmca4^–/–. The responses to carbachol (CCh: 10 µM) were also greater in Pmca1^+/– (120–150%) than in WT bladders. In contrast, the responses in Pmca4^–/– and Pmca1^+/–Pmca4^–/– bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca²⁺]_i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4^–/– (130–190%) and Pmca1^+/–Pmca4^–/– (120–250%) bladders, but not in Pmca1^+/– bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca²⁺ clearance, while PMCA4 is essential for the [Ca²⁺]_i increase and contractile response to the CCh receptor-mediated signal transduction pathway. PMCA; bladder smooth muscle; gene-altered mice     

Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle

Calcineurin mediates repression of plasma membrane Ca²⁺-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which ⁴⁵Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca²⁺ concentrations ([Ca²⁺]_i) increased twofold, and real-time RT-PCR and Western blot revealed a 40% decrease in PMCA4 expression levels from G₀ to G₁/S in the cell cycle, where PMCA4 constituted 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G₀ to G₁/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G₁/S. By contrast, Ca²⁺-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G₀ to G₁/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G₁/S-associated repression of PMCA4. These data show that G₁/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca²⁺]_i through alternate targets. calcineurin; c-Myb; plasma membrane Ca²⁺-ATPase-4; cell cycle     

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.