细胞钙信号转导WLL.ppt

Intracellular Ca2+ SignalingWang Linlin Ph.DDepartment of Physiology Zhejiang University School of MedicinePhysiological Function of Ca2+Physiological Function of Ca2+1.参与神经肌肉的反应过程2.镇静作用：缺钙可引起神经的兴奋性增高 3.调节细胞功能，激活蛋白激酶，促进蛋白质磷酸化4.促进内、外分泌腺的分泌，神经介质的分泌5.参与血液的凝固6.维持细胞膜的通透性及完整性是十分必要的7.参与免疫反应8.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-2／AM或Fura-2／AM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l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l正常人的血浆Ca2+不是固定的，每日可有  0.1 mg ／dl的变化范围 影响血Ca2+的因素1.来源：食物2.三个主要靶器官：肾、肠、骨 3.其他影响因素 激素对Ca2+转运的作用l3种激素：PTH, l，25-(OH)2-D3 , CTl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l 胞内钙的分布极不均匀l 细胞内总钙浓度约为l mmol／Ll 50 ％存在于细胞核l 线粒体占30 ％，浓度为0.6 mmol／Ll 内质网占14 ％，约0.28 mmoI／Ll 质膜(外层)占5 ％，约0.1 mmol／Ll 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 signalCalcium is Spark of Life, Kiss of Death for Nerve Cells静息状态下的Ca2+梯度[Ca2+]o 10-3 M[Ca2+]i 10-7 MHOW ?1. Sarcolemma impermeable to Ca2+ Ca2+ pump (Ca2+-ATPase) Na+-Ca2+ exchanger2. Endoplasmic/sarcoplasmicreticulum (ER/SR) Ca2+-ATPase3. MitochondriaInitiation of intracellular Ca2+ signal[Ca2+]o 10-3 M[Ca2+]i 10-7 MHOW ?Two primary sources:1. External Extracellular Ca2+2. Internal ER/SR[Ca2+]i 10-5 M1. 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 systemVoltage-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) Ca2+ channelsDuration of currentlong-lastingtransientActivation kineticsslowerfasterInactivation kineticsslowerfasterThresholdhigh (-35mV)Low (-60mV)cAMP/cGMP-regulatedYesNoPhosphorylation-regulatedYesNoOpenersBay-K-8644-BlockersvarapamilTetramethrinnifedipine, diltiazemNi2+ Inactivation by [Ca2+]iYesslightPatch-clamp recordingrun-downrelatively stableL-type T-typeIn 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+ channelPKAphosphorylationL-type Ca2+ channels 的调节:Increases in:numberprobabilityopen timeROC: 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) VOC2. InternalCa2+ release from ER/SR(1) Ryanodine receptor (2) IP3 receptorCa2+ 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) SRRegulation of RyRActivation: •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l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l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lT 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) SERCA1 fast-twitch skeletal muscle (SM) adult (SERCA1a) neonatal (SERCA1b)SERCA2 cardiac muscle (SERCA2a) slow-twitch SM (SERCA2b)SERCA3 muscle and nonmuscle cells(Arai, 1994) Regulation of SERCA by phospholamban Phospholamban:low-molecular- weight protein(52-amino-acid)SERCAPhospholamban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l蛙心灌流 (Ringer, 1883)l跨膜电流 (Heillbriunn&Kamada ,1953) l膜片钳单通道技术 (Neher &Sakmann, 1978)lFura-2荧光探针 (Tsien, 1985)lVoltage ClampHow to study ?Nobel Prize in Physiology or Medicine 1963“for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and ce。