細胞鈣信號轉導-WLL-2014

IntracellularCa2+SignalingWangLinlin

Ph.DDepartmentofPhysiologyZhejiangUniversitySchoolofMedicinePhysiologicalFunctionofCa2+PhysiologicalFunctionof

Ca2+參與神經肌肉的反應過程鎮(zhèn)靜作用：缺鈣可引起神經的興奮性增高調節(jié)細胞功能，激活蛋白激酶，促進蛋白質磷酸化促進內、外分泌腺的分泌，神經介質的分泌參與血液的凝固維持細胞膜的通透性及完整性是十分必要的參與免疫反應其他Ca2+參與的遞質的釋放Ca2+參與的肌肉收縮Ca2+參與的細胞連接Ca2+的測定方法（1）血清鈣的測定5種方法：即滴定法、比色法、火焰法、離子選擇電極法、活化分析法。

（2）細胞內鈣的測定：光譜熒光法（fluoromotric）利用單波長或雙波長熒光分光光度計，應用熒光指示劑Quin-2／AM或Fura-2／AM測定細胞內Ca2+濃度，這是目前研究Ca2+在生命科學中的生理生化功能的有力武器。血Ca2+濃度與Ca2+的轉運血漿Ca2+濃度正常維持在8.5-11.1mg％之間，其中46％與血漿蛋白特別是白蛋白結合，47.5％為游離鈣(Ca2+)，僅有6.5％的鈣以磷酸鈣、檸檬酸鈣、重碳酸鈣、酒石酸鈣、棕櫚酸鈣等復合物形式存在。只有血漿離子鈣才具有生理活性。正常人的血漿Ca2+不是固定的，每日可有

0.1mg／dl的變化范圍。影響血Ca2+的因素來源：食物三個主要靶器官：腎、腸、骨其他影響因素

激素對Ca2+轉運的作用3種激素：PTH,l，25-(OH)2-D3,CT此外生長激素、甲狀腺素、性激素、前列腺素等也有調節(jié)血漿Ca2+的作用細胞內Ca2+的分布胞內鈣的分布極不均勻細胞內總鈣濃度約為lmmol／L50％存在于細胞核線粒體占30％，濃度為0.6mmol／L內質網占14％，約0.28mmoI／L質膜(外層)占5％，約0.1mmol／L而細胞溶質中僅占總鈣的0.5％(結合態(tài))或0.005％(離子態(tài))

儲存Ca2+的肌漿網/內質網BerridgeMJetal.,Nature.1998;395:645-648.

Ca2+

–alifeanddeathsignalCalciumisSparkofLife,KissofDeathforNerveCells靜息狀態(tài)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+內流途徑

:(1)VOC:電壓門控Ca2+

通道(2)ROC:受體操作Ca2+

通道(3)SOC:store-operatedCa2+channels(4)LeaksystemVoltage-GatedCalciumChannels

SIGMA-ALDRICH電壓依賴性鈣通道類型:

(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

的調節(jié):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興奮-收縮耦聯機制

RyR異構體RyR1:SkeletalmuscleRyR2:CardiacmuscleRyR3:Brain(Coronado,1994)

SRRegulationofRyRActivation:Caffeine咖啡因ryanodine(nM)CICR鈣誘導鈣釋放Phosphorylation磷酸化…Inhibition:ryanodine(M)rutheniumred釕紅…鈣火花（calciumsparks）進入胞內的少量鈣離子激活RyRs，導致大量的鈣離子涌出SR的過程被稱為鈣誘導性鈣釋放(CICR,calciuminducedcalciumrelease)。肌漿內的鈣離子濃度瞬時增高，這種現象被稱為鈣火花(calciumsparks)。鈣火花第一次被人們發(fā)現便是在靜息心肌細胞中。這項工作于1993年由MarkB.Cannell和PeaceCheng（程和平）揭示的。心肌細胞鈣火花是肌漿網上一個或幾個成簇狀分布的RyR所產生的局部鈣釋放,可通過單個L型鈣通道開放所觸發(fā)或自發(fā)發(fā)放鈣火花呈隨機發(fā)放,出現在Z線附近,其動力學特征受多種因素的調節(jié)T型鈣電流、Na+/Ca2+交換、ICa(TTX)和IP3等也是鈣火花觸發(fā)的來源(2)IP3receptor(IP3R):IP3R1:brain,nonexcitablecells(ubiquitous)IP3R2:cardiacmuscle,nonexcitablecells,liverIP3R3:brain,nonexcitablecells胞內Ca2+回復到靜息水平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采用轉基因動物研究研究phospholamban

對心臟的作用Phospholamban-deficientmice敲除小鼠IncreaseinSERCAaffinityforCa2+withenhancedratesofcontractionandrelaxationaswellasincreasedpumprate(Luo,1994,1996)

Phospholamban-overexpressingmice過度表達DepressedSRSERCAaffinityforCa2+andmyocardialdysfunction (Kadambi,1994,1996)

Ca2+研究背景蛙心灌流(Ringer,1883)跨膜電流(Heillbriunn&Kamada,1953)

膜片鉗單通道技術(Neher&Sakmann,1978)Fura-2熒光探針(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+

穩(wěn)態(tài)與病理生理學Hypertension原發(fā)性高血壓hyper/hypo-thyroidism

甲亢/甲低ischemia/reperfusioninjury缺血復灌損傷Cardiomyopathy心肌病heartfailure心衰其他EctopicCalcification膀胱結石ApoptosisandCa2+

abnormity細胞凋亡（apoptosis）：由體內外因素觸發(fā)細胞內預存的死亡程序而導致的細胞死亡過程稱