细胞钙信号转导-WLL-2014

Intracellular Ca2+ Signaling,Wang Linlin Ph.DDepartment of Physiology Zhejiang University School of Medicine,Physiological Function of Ca2+,Physiological Function of Ca2+,参与神经肌肉的反应过程镇静作用：缺钙可引起神经的兴奋性增高 调节细胞功能，激活蛋白激酶，促进蛋白质磷酸化促进内、外分泌腺的分泌，神经介质的分泌参与血液的凝固维持细胞膜的通透性及完整性是十分必要的参与免疫反应其他,Ca2+参与的递质的释放,Ca2+参与的肌肉收缩,Ca2+参与的细胞连接,Ca2+的测定方法,（1）血清钙的测定 5种方法：即滴定法、比色法、火焰法、离子选择电极法、活化分析法。 （2）细胞内钙的测定： 光谱荧光法（fluoromotric） 利用单波长或双波长荧光分光光度计，应用荧光指示剂Quin-2AM或Fura-2AM测定细胞内Ca2+浓度，这是目前研究Ca2+在生命科学中的生理生化功能的有力武器。,血Ca2+浓度与Ca2+的转运,血浆Ca2+浓度正常维持在8.5 - 11.1 mg 之间，其中46 与血浆蛋白特别是白蛋白结合，47.5 为游离钙(Ca2+)，仅有6.5 的钙以磷酸钙、柠檬酸钙、重碳酸钙、酒石酸钙、棕榈酸钙等复合物形式存在。只有血浆离子钙才具有生理活性。正常人的血浆Ca2+不是固定的，每日可有 0.1 mgdl的变化范围。,影响血Ca2+的因素,来源：食物三个主要靶器官：肾、肠、骨 其他影响因素,激素对Ca2+转运的作用,3种激素：PTH, l，25-(OH)2-D3 , CT此外生长激素、甲状腺素、性激素、前列腺素等也有调节血浆Ca2+的作用,细胞内Ca2+的分布,胞内钙的分布极不均匀 细胞内总钙浓度约为l mmolL 50 存在于细胞核 线粒体占30 ，浓度为0.6 mmolL 内质网占14 ，约0.28 mmoIL 质膜(外层)占5 ，约0.1 mmolL 而细胞溶质中仅占总钙的0.5 (结合态)或0.005 (离子态),储存Ca2+的肌浆网/内质网,Berridge MJ et al., Nature. 1998; 395: 645-648.,Ca2+ a life and death signal,Calcium is Spark of Life, Kiss of Death for Nerve Cells,静息状态下的Ca2+梯度,1. Sarcolemma impermeable to Ca2+ Ca2+ pump (Ca2+-ATPase)Na+-Ca2+ exchanger2. Endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+-ATPase3. Mitochondria,Initiation of intracellular Ca2+ signal,Two primary sources:1. External Extracellular Ca2+2. InternalER/SR,1. External胞外Ca2+内流途径 :(1) VOC: 电压门控 Ca2+ 通道(2) ROC: 受体操作 Ca2+ 通道(3) SOC: store-operated Ca2+ channels(4) Leak system,Voltage-Gated Calcium Channels,SIGMA-ALDRICH,电压依赖性钙通道类型: (1) L-type (long-lasting) (Nowycky, 1985) (2) T-type (transient) (Nowycky, 1985) (3) N-type (neither L-type nor T-type) (Nowycky, 1985) (4) P-type (Purkinje) (Llinas, 1992) (5) F-type (fetal) (Tohse, 1992),In muscle cells, including skeletal muscle, cardiac muscle and most types of smooth muscle, the major Ca2+ inward current is that through the L-type Ca2+ channels.,L-type Ca2+ channels 的调节:,Increases in:numberprobabilityopen time,ROC: A receptor protein which has an ion channel, as an integral part of its structure that is gated, when the normal ligand binds to the receptor.,(2) ROC (1) VOC,2. InternalCa2+ release from ER/SRRyanodine receptor (2) IP3 receptor,Ca2+ waves in hepatocytes,(1) Ryanodine receptor (RyR): mediates the efflux of Ca2+ from the SR and plays a central role in excitation-contraction (E-C) coupling.,Mechanisms of E-C coupling兴奋-收缩耦联机制,RyR异构体,RyR1: Skeletal muscleRyR2: Cardiac muscleRyR3: Brain,(Coronado, 1994),SR,Regulation of RyR,Activation: Caffeine 咖啡因ryanodine (nM) CICR钙诱导钙释放Phosphorylation磷酸化Inhibition:ryanodine (M)ruthenium red钌红,钙火花（calcium sparks）,进入胞内的少量钙离子激活RyRs，导致大量的钙离子涌出SR的过程被称为钙诱导性钙释放(CICR, calcium induced calcium release)。肌浆内的钙离子浓度瞬时增高，这种现象被称为钙火花(calcium sparks)。钙火花第一次被人们发现便是在静息心肌细胞中。这项工作于1993年由 Mark B. Cannell和 Peace Cheng（程和平）揭示的。,心肌细胞钙火花是肌浆网上一个或几个成簇状分布的RyR所产生的局部钙释放,可通过单个L 型钙通道开放所触发或自发发放钙火花呈随机发放,出现在Z线附近,其动力学特征受多种因素的调节T 型钙电流、Na+ /Ca2+交换、ICa(TTX)和IP3 等也是钙火花触发的来源,(2) IP3 receptor (IP3R):,IP3R1: brain, nonexcitable cells (ubiquitous)IP3R2: cardiac muscle, nonexcitable cells, liverIP3R3: brain, nonexcitable cells,胞内 Ca2+回复到静息水平,1. Sarcolemma Ca2+-ATPase Na+-Ca2+ exchanger2. Endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+-ATPase3. Mitochondria,*,Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA),SERCA1fast-twitch skeletal muscle (SM)adult (SERCA1a) neonatal (SERCA1b)SERCA2cardiac muscle (SERCA2a) slow-twitch SM (SERCA2b)SERCA3muscle and nonmuscle cells,(Arai, 1994),Regulation of SERCA by phospholamban,Phospholamban:low-molecular-weight protein (52-amino-acid),磷酸化: phospholamban inhibits the SERCAby decreasing its affinity for Ca2+去磷酸化: phospholamban enhances the activity of SERCA by increasing its affinity for Ca2+calcium/ calmodulin-dependent protein kinase and cAMP-dependent protein kinase A,采用转基因动物研究研究phospholamban 对心脏的作用Phospholamban-deficient mice 敲除小鼠Increase in SERCA affinity for Ca2+ with enhanced rates of contraction and relaxation as well as increased pump rate (Luo, 1994, 1996) Phospholamban-overexpressing mice过度表达Depressed SR SERCA affinity for Ca2+ and myocardial dysfunction(Kadambi, 1994, 1996),Ca2+研究背景,蛙心灌流 (Ringer, 1883)跨膜电流 (Heillbriunn&Kamada ,1953) 膜片钳单通道技术 (Neher &Sakmann, 1978)Fura-2荧光探针 (Tsien, 1985),Voltage Clamp,How to study ?,Patch Clamp,激光共聚焦,Calcium spark,Calcium homeostasis,ExternalVOC, ROC, SOC, Na+-Ca2+ exchangerInternal RyRIP3R,SERCASarcolemma Ca2+-ATPase Na+-Ca2+ exchangerMitochondria,*,*,*,Calcium signal pathway,Ca2+ 稳态与病理生理学,Hypertension 原发性高血压hyper/hypo-thyroidism 甲亢/甲低ischemia/reperfusion injury缺血复灌损伤Cardiomyopathy心肌病heart failure心衰其他,Ectopic Calcification,膀胱结石,Apoptosis and Ca2+ abnormity,细胞凋亡（apoptosis）：由体内外因素触发细胞内预存的死亡程序而导致的细胞死亡过程称为细胞凋亡。胞浆内Ca2+池耗竭及胞外Ca2+内流 Ca2+升高 是细胞凋亡的信号胞浆内Ca2+池耗竭,但是胞浆内Ca2+不升高细胞凋亡,Ca2+超载与缺血性损伤,calcium overload：各种原因引起的细胞内钙含量异常增多并导致细胞结构损伤和功能代谢障碍的现象，称为钙超载。,缺血后再灌注时细胞内Ca2+超载的机制尚无定论，但可能与下列因素有关电压依赖性钙通道；质膜钙泵；Na+-Ca2+交换；线粒体；肌浆网；细胞内蛋白或阴离子结合的钙；结合于质膜糖被的钙,细胞内Ca2+ 浓度引起细胞损伤和死亡的可能机制氧自由基的产生NO过度生成Ca2+依赖性蛋白激酶的激活内源性核酸内切酶激活线粒体破坏酸中毒,Ca2+超载对老年