细胞钙信号转导-WLL-2014

IntracellularCa2+SignalingWangLinlin

Ph.DDepartmentofPhysiologyZhejiangUniversitySchoolofMedicinePhysiologicalFunctionofCa2+PhysiologicalFunctionof

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-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+的转运血浆Ca2+浓度正常维持在8.5-11.1mg％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

0.1mg／dl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lmmol／L50％存在于细胞核线粒体占30％，浓度为0.6mmol／L内质网占14％，约0.28mmoI／L质膜(外层)占5％，约0.1mmol／L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BerridgeMJetal.,Nature.1998;395:645-648.

Ca2+

–alifeanddeathsignalCalciumisSparkofLife,KissofDeathforNerveCells静息状态下的Ca2+梯度[Ca2+]o10-3M[Ca2+]i10-7M

HOW?1.Sarcolemma

impermeabletoCa2+

Ca2+pump(Ca2+-ATPase)Na+-Ca2+exchanger2.Endoplasmic/sarcoplasmicreticulum(ER/SR)

Ca2+-ATPase3.MitochondriaInitiationofintracellularCa2+signal[Ca2+]o10-3M[Ca2+]i10-7M

HOW?Twoprimarysources:1.ExternalExtracellularCa2+2.InternalER/SR[Ca2+]i10-5M1.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-operatedCa2+channels(4)LeaksystemVoltage-GatedCalciumChannels

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(neitherL-typenorT-type)

(Nowycky,1985)

(4)P-type(Purkinje)(Llinas,1992)

(5)F-type(fetal)(Tohse,1992)

Ca2+channelsDurationofcurrent long-lasting transientActivationkinetics slower faster Inactivationkinetics slower fasterThreshold high(-35mV) Low(-60mV)cAMP/cGMP-regulated Yes NoPhosphorylation-regulated Yes NoOpeners Bay-K-8644 -Blockers

varapamil

Tetramethrin

nifedipine,diltiazem

Ni2+

Inactivationby[Ca2+]i

Yes slightPatch-clamprecording run-down relativelystableL-type

T-type

Inmusclecells,includingskeletalmuscle,cardiacmuscleandmosttypesofsmoothmuscle,themajorCa2+

inwardcurrentisthatthroughtheL-typeCa2+channels.

L-typeCa2+

channelPKAphosphorylationL-typeCa2+channels

的调节:Increasesin: number probability opentimeROC:Areceptorproteinwhichhasanionchannel,asanintegralpartofitsstructurethatisgated,whenthenormalligandbindstothereceptor.(2)ROC (1)VOC2.InternalCa2+releasefromER/SRRyanodinereceptor(2)IP3receptorCa2+wavesinhepatocytes

(1)Ryanodinereceptor(RyR): mediatestheeffluxofCa2+

fromtheSRandplaysacentralroleinexcitation-contraction(E-C)coupling.MechanismsofE-Ccoupling兴奋-收缩耦联机制

RyR异构体RyR1:SkeletalmuscleRyR2:CardiacmuscleRyR3:Brain(Coronado,1994)

SRRegulationofRyRActivation:Caffeine咖啡因ryanodine(nM)CICR钙诱导钙释放Phosphorylation磷酸化…Inhibition:ryanodine(M)rutheniumred钌红…钙火花（calciumsparks）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,calciuminducedcalciumrelease)。肌浆内的钙离子浓度瞬时增高，这种现象被称为钙火花(calciumsparks)。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MarkB.Cannell和PeaceCheng（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)IP3receptor(IP3R):IP3R1:brain,nonexcitablecells(ubiquitous)IP3R2:cardiacmuscle,nonexcitablecells,liverIP3R3:brain,nonexcitablecells胞内Ca2+回复到静息水平1.Sarcolemma

Ca2+-ATPaseNa+-Ca2+exchanger2.Endoplasmic/sarcoplasmicreticulum(ER/SR)

Ca2+-ATPase3.Mitochondria*Sarco-endoplasmicreticulumCa2+-ATPase(SERCA)

SERCA1fast-twitchskeletalmuscle(SM) adult(SERCA1a) neonatal(SERCA1b)SERCA2cardiacmuscle(SERCA2a) slow-twitchSM(SERCA2b)SERCA3muscleandnonmusclecells(Arai,1994)

RegulationofSERCAbyphospholamban

Phospholamban:low-molecular-weightprotein(52-amino-acid)SERCAPhospholamban磷酸化:phospholambaninhibitstheSERCAbydecreasingitsaffinityforCa2+

去磷酸化:phospholambanenhancestheactivityofSERCAbyincreasingitsaffinityforCa2+calcium/calmodulin-dependentproteinkinaseandcAMP-dependentproteinkinaseA采用转基因动物研究研究phospholamban

对心脏的作用Phospholamban-deficientmice敲除小鼠IncreaseinSERCAaffinityforCa2+withenhancedratesofcontractionandrelaxationaswellasincreasedpumprate(Luo,1994,1996)

Phospholamban-overexpressingmice过度表达DepressedSRSERCAaffinityforCa2+andmyocardialdysfunction (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)VoltageClampHowtostudy?NobelPrizeinPhysiologyorMedicine1963"fortheirdiscoveriesconcerningtheionicmechanismsinvolvedinexcitationandinhibitionintheperipheralandcentralportionsofthenervecellmembrane"Eccles Hodgkin HuxleyPatchClampNobelPrizeinPhysiologyorMedicine1991"fortheirdiscoveriesconcerningthefunctionofsingleionchannelsincells"ErwinNeher BertSakmann

激光共聚焦CalciumsparkCalciumhomeostasis[Ca2+]i[Ca2+]iExternalVOC,ROC,SOC,Na+-Ca2+exchangerInternalRyRIP3RSERCASarcolemma

Ca2+-ATPaseNa+-Ca2+exchangerMitochondria****CalciumsignalpathwayCa2+

稳态与病理生理学Hypertension原发性高血压hyper/hypo-thyroidism

甲亢/甲低ischemia/reperfusioninjury缺血复灌损伤Cardiomyopathy心肌病heartfailure心衰其他EctopicCalcification膀胱结石ApoptosisandCa2+

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