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神经电活动的分子基础-离子通道厦门大学医学院 戚智21timeVoltage(mV)Stim ElecREC 1REC 2REC 3+First try:a small depolarizing stimulus(-60 mV)-70 -60 -50 -40 -30 -20 -10 -0 -+10 -+20 -+30 -+40 -+22timeVoltage(mV)Stim ElecREC 1REC 2REC 3+Next try:a slightly larger depolarizing stimulus(-55 mV)-70 -60 -50 -40 -30 -20 -10 -0 -+10 -+20 -+30 -+40 -+23timeVoltage(mV)Stim ElecREC 1REC 2REC 3+-70 -60 -50 -40 -30 -20 -10 -0 -+10 -+20 -+30 -+40 -+Next try:a slightly larger depolarizing stimulus(-50 mV)RMPLocal PotentialsAction Potentials23timeVoltage(mV)Stim ElecREC 1REC 2REC 3+-70 -60 -50 -40 -30 -20 -10 -0 -+10 -+20 -+30 -+40 -+RMPAction PotentialsCan we get even larger Action Potentials?25timeVoltage(mV)Stim ElecREC 1REC 2REC 3-70 -60 -50 -40 -30 -20 -10 -0 -+10 -+20 -+30 -+40 -RMPAction PotentialsTry an even larger depolarizing stimulus(-45 mV)No higher,no larger,Identical!Can we get even larger Action Potentials?动作电位及其产生机制电压钳技术电压钳Voltage clamp电流变化阻断剂对电流成分的分离电压钳Voltage clamp电流变化河豚鱼Tetrodotoxin is a lethal toxin found in pufferfish that inhibits the voltage-sensitive sodium channel,halting action potentials.二、动作电位（Action potenial）（一）动作电位的概念及特点去极化-Depolarization；阈值-Threshold value，超射-Overshoot；锋电位-Spike potential；后电位-After potential；后去极化电位-After depolarization（负后电位）；后超极化电位-After hyperpolarization（正后电位）离子通道由三个功能区离子通道由三个功能区构成构成（一）Pore离子通路(包含Selectivity Filter);（二）Gate通道门；（三）Sensor感受器。选择性及通透性机制What determines Ion Selectivity?+-How are Channels Selective?-charge-atomic radius-HydrationNa+K+Cl-Ca2+-Selectivity by chargeNa+K+Cl-+-Na+K+Positive ringof charges will allow(-)ions to conductAnd repel+ionsCa2+Ca2+Ion Selection:Chloride ChannelsBasicResiduesGateRegionModified from Keramidas et al.,Prog.Biophys.Mol.Biol.86:161(2004)+-Na+K+Cl-+-Na+K+-Negative chargesAllow+ionsTo enter the channel-Selectivity by chargeCa2+Ca2+Ion Selection:Cationic ChannelsAcidicResiduesGateRegionModified from Keramidas et al.,Prog.Biophys.Mol.Biol.86:161(2004)(for contrast)Carrier Proteins Transport proteins that can bind to specific substances on one side of the cell membrane,carry the substance across the cell membrane and release it one the other side They do not extend through the membrane.They bond and drag molecules through the bilipid layer and release them on the opposite side.Selectivity of ValinomycinK+Na+Na+channelNa+K+Na+K+-Smaller Na+ionsFit,but not largerK+or Ca+-Selection by atomic radiusCa2+Ca2+Na+K+Na+K+-Selection by Dehydration:-Charges on the channelMust effectively substitute The(-)polar Charges of the O on H20:The hydration shell mustbe stripped before K+can pass through.-OHH+OHH+OHH+H20Hydration Shell around ionsK+Channel钠离子半径0.95埃钙离子半径0.99埃钾离子半径1.33埃Selectivity of K+,Na+and Ca2+channelsK+channel:Pna/Pk1/100;Pca/Pk1/1000Na+channel:Pk/Pna8/100;Pca/Pna10/100Ca+channel(L-type):Pna/Pca=1/1170;Pk/Pca=1/3000 挑战公认的难题挑战公认的难题3 K+Na+Ca+Hard Spheres Born 1956 1978 B.Sc.in Biochemistry Brandeis U.1981 M.D.Tufts U.School of Medicine 1985 Internal Medicine Beth Israel Hospital,Boston 1987 back to science:post-doc Brandeis 1989 Assoc.prof.Harvard U.1996 X-ray crystallography Rockefeller U.1998 K+channel structure resolved at 0.32 nm resolution 2001 0.2 nmRoderick MacKinnonBiological Membrane=Lipid BilayerApproximately 30 thickHydrophobic core+Hydrophilic or charged headgroupsMixture of lipids that vary in type of head groups,lengths of acyl chains,number of double bonds(Some membranes also contain cholesterol)Presence of Hydrophobic TM Domain can result in:Low levels of expressionDifficulties in solubilizationDifficulties in crystallizationAttempting crystallization and structure solution of transmembrane proteins is considered difficult and risky.Roderick MacKinnon et al.1998-Nobel prize in Chemistry 2003试验材料选取的重要性 链霉菌钾离子通道链霉菌钾离子通道(KcsA)WHY？Bacterial K+channelX-ray crystallographySelectivity filterGateInner vestibuleDoyle et al.Science 1998Elementary electrostatic considerations Negative charges raise local K+availability at channel entrance.Hydrophobic residues line pore,allowing water molecules to interact strongly with the K+ion.Structural basis of the K+selectivity filterSelection of ion by selectivity filterSodium ion is too small for carbonyl oxygen interaction and water expelCarbonyl groups serve as“surrogate water”The selectivity filter is occupied by two K+ions alternating between two configurations.Carbonyl rings can be thought of as K+holes.+Na+channelNa+K+Na+K+-Smaller Na+ionsFit,but not largerK+or Ca+-Selection by atomic radiusCa2+Ca2+钠离子半径0.95埃钙离子半径0.99埃钾离子半径1.33埃Selectivity of K+,Na+and Ca2+channelsK+channel:Pna/Pk1/100;Pca/Pk1/1000Na+channel:Pk/Pna8/100;Pca/Pna10/100Ca+channel(L-type):Pna/Pca=1/1170;Pk/Pca=1/3000 3 K+Na+Ca+Hard SpheresSelection of ion by selectivity filterSodium ion is too small for carbonyl oxygen interaction and water expelThe Ca2+current saturates at Ca2+concentration of 14 mM.The Na+current saturates slowly,with a half-saturation point of 240 mM.Corry et al.Biophys.J.2001.Corry et al.Biophys.J.2001.Electrostatic energy profile of an ion traversing the channel.The potential energy of a calcium ion(solid line,a)and a sodium ion(dashed line,b)in the absence of any fixed charges.When the glutamate groups and mouth dipoles are included,the profiles are replotted for calcium(solid curve,d)and sodium ions(dashed line,c).No applied potential is used.Corry et al.Biophys.J.2001.Corry et al.Biophys.J.2001.When a calcium ion is resident in the channel,a sodium ion can enter from the right.However,it cannot push the calcium ion out of the selectivity filter as the calcium ion sees a huge energy barrier to its left.The presence of a calcium ion in the channel prohibits monovalent ions from traversing.Corry et al.Biophys.J.2001.If a sodium ion is resident in the channel,then a calcium ion can enter and the sodium ion sees only a very small energy barrier preventing it from crossing to the intracellular side of the channel.Even if there are two sodium ions in the filter,a calcium ion can easily enter the channel and push them out.Thus,the presence of sodium cannot stop calcium ions from crossing the channel.Dutzler et al.(2002)Nature 415,287-294Structure of a chloride channel at 3.0 Stereo view from the extracellular side(a)and side view with the extracellular solution above(b)Dutzler et al.(2002)Nature 415,287-294Dutzler et al.(2002)Nature 415,287-294Two architectures for ion channel proteins:antiparallel(Cl-)and parallel(K+)Agres WorkAquaporins SelectivityProtons,ions,and charged molecules cannot be transported.Agres Work In 2000 with other research scientists,he was able to show the first three-dimensional of an aquaporin(Aquaporin-1 from human red blood cell).Monomeric poresWater,glycerol,Tetrameric porePerhaps ions?GlpF E.coli glycerol channel(aquaglycerolporin)AQP1 Mammalian aquaporin-1(pure water channel)Agres WorkProton Blocking by a Global Orientation MechanismAquaporinProteins are Dynamic Structures Water traveling through Aquaporin poreControl of the selectivity of the aquaporin water channel family by global orientational tuning.Tajkhorshid E,Nollert P,Jensen M,Miercke LJ,OConnell J,Stroud RM,Schulten K.Science.2002 Apr 19;296(5567):525-30.All voltage-gated ion channels have a similar architecture e.g.sodium channelS5/S6 loop related to ion selectivityS4 related to voltage sensingKv1Kv2Kv3Kv4Kv5+KCNQColor coding:Red=+Green=-Blue=polarGray=nonpolarS4 alignment Ion Channels and Diseases-Channelopathies 高血钾型周期性瘫痪（hyperkalemic periodic paralysis，HyPP） http:/ Twenty years ago,a quarterhorse named“Impressive”won all of the titles of his class.He was the top-winning,top-producing Quarter Horse stallion of all time.As a breeding stallion,he proved himself equally deserving of his name,turning out champion after champion.Many of Impressives offspring bore the same dramatic physical stature as their sire-they too went on to become outstanding and prolific stallions and broodmares.Of the top 15 halter horses in 1992,13 were descendants of Impressive.Even at the age of 23,Impressive himself was fourth on the list.In 1993,it was estimated that more than 55,000 Quarter Horses,Paints,and Appaloosas bore his pedigree.From the American Quarterhorse AssociationHistoryAffects descendents of the Quarter Horse stallion ImpressiveQuarter Horses,Appaloosas,and PaintsResearch project funded by AQHA in 1989.Research results released in 1992Proliferated because he was successful in the show ring and possessed desired physical features.Soon his progeny were seen to be affected by a strange muscular twitching that often left them temporarily unable to move.Usually mis-diagnosed as tying-up syndrom or colic,these episodes varied widely in degree and duration.but all had one factor in common,their pedigree.As a result,many people now know HYPP by its more common name:Impressive Syndrome.This turned out to be a genetic mutation that only recently has been implicated in the rare but burgeoning-and sometimes fatal-muscular disorder known as hyperkalemic periodic Skeletal muscle Na+channel NaV1.4Potassium-aggravated myotonia mutations affect single channel behaviourCannon,1997Computer model of myotonia and paralysisf:Fraction of Na+current that failed to inactivate高血钾型周期性瘫痪临床表现：高血钾型周期性瘫痪临床表现：较少见，有遗传史，童年起病，常因寒冷或服钾盐诱发，白天发病。发作期钾离子自肌肉进入血浆，因而血钾升高，可达5-7mgEq/L。也以下肢近端较重，持续时间较短，不足一小时，一日多次或一年一次。部分病员发作时可有强直体征，累及颜面和手部，因而面部强直，眼半合，手肌僵硬，手指屈曲和外展。进食、一般活动、静注钙剂、胰岛素或肾上腺素均可终止发作。事先给予能增加钾排泄的醋氮酰胺及双氢克尿塞等利尿剂可预防发作医学|教育网搜集整理。发作时心电图心电图改变，初是T波增高，QT间期延长，以后逐渐出现R波降低，S波增深，ST段下降，P-R间期及QRS时间延长。对诊断诊断有困难者，可作诱发试验：口服氯化钾（3-8g）常可诱发或使原有瘫痪症状加重。这是因为高钾型周期性瘫痪，对外源性钾的摄入比本身血清钾含量变化更为敏感。http:/ is caused by a flaw in a sodium channel in the muscle membrane.This flaw makes the person with HyperKPP extremely sensitive to increases in serum potassium that wouldnt bother the average person.Anyone can be made weak by a drastic increase in serum potassium,but the person with HyperKPP gets weak with even a slight elevation in potassium level,and patients with HyperKPP may become profoundly paralyzed while their potassium levels remain well within normal limits,even when their potassium is on the lower end of normal.The human defect is also in Na channelsHyperekplexia(a startle disease)Spasmodic mouse:a point mutation in Gly Rc(an inhibitoryRc in the spinal cord and brain stem)Decreased frequency of openingSpastic mouse:Less number of Gly Rc expressedGlycine vs.GABA(spinal cord vs.brain)Delicate balance of synaptic excitation and inhibition Shunting inhibition in regions close to soma and axon hillockIPSP at restingmemb.potential?Shaft synapseRLLKNDQHKLLRFRRKRRHHKEDEAGEGRFNFSVPAERKLFAYGMGPACQLADGSKGNANKTNPPPSSKTSNPEQRKDKKARG Y YYPS LLVLSS FL E Y AA V NV SNDAAPASS S GT TL TT T VL GV GARSPK V S YVKADLYM F YF NA F LF PRNHVDERRVKQHFRAECLERQNHTFKGTYKTCEKD LRYQEQGQVAEKLFQFLDNTEPDGLT LAVYGFDKLDTCQVESLQPFNC PALTLTRSAFFNNGRSETTHFAHGKEPNDLKSDLMSPLDDYLVSYNEYPDDSLQQNDPRLADY R VNFL RTTASGFEP GK FNPRFNCSV N VPSP S DF LSNAPKRADTYGD KLG RTSASRYASKEAEASSLAFVIIIILIQIIIIIMMMWWMMWMWMMMMWWMMMMW M AV C LL F V FS A LIIIIIIIIIIIIIIIIIIIIIIAGFLR L TNFS L Y(Human)-28QI244NP250TR252HR392HRQ266HR271L/QK276E Y279CGFEIAmino acid positions are quoted for the rat sequenceR252 E/Q and R271 E/Q-Langosch et.al.,1993R271L/Q-Shiang et.al.,1993;Langosch et.al.,1994I244N-Rees et.al.,1994Y279C-Shiang et.al.,1995K276E-Elmslie et.el.,1996;Lewis et.al.,1998Q266H-Milani et.al.,1996;Moorhouse et.al.,1999R271N/K/H-Lynch et.al.,1997;Langosh et.al.,1994P250T-Saul et.al.,1999R252H and R392H-Vergouwe et.al.,1999 N/K/H E/Q E/QRea et al(2002)Compound heterozygosity in hyperekplexiaPHYSIOLOGY OF PAINDefinitionPain is an unpleasant sensory and emotional experience associated with tissue damage.It is a protective mechanism that warns us that there is something wrong and can provoke body response to avoid further injury.What would it be like to never feel the sensation of pain?Bliss?A Life Without Pain is a documentary film by Melody Gilbert about children who cant feel pain.Jamilah(10 year old)Gabby(3 year old)Miriam(7-year-old)When you first see her,you notice that she seems happy.she is blind in one eye,as she has no way to tell that she is hurting herself when she scratches or touches it.Her parents even go as far as to say that when she was teething,she would chew her tongue“like a piece of bubble gum”.She grabs a hot light bulb and blisters her hand without a single tear.She runs smack into a table corner,bouncing off it like a bumper car.Burns,bumps,broken bones and Band-Aids _ these are the constant accessories of the children.What does pain tell us?To avoid harmful situations To get away from noxious stimuli To rest an injured part of our body To remind us not forgetting doctors Helpful to usThe life and death of a young street performer from Pakistan who could walk on hot coals and drive knives through his arms without flinching has led scientists to a genetic discovery that could revolutionise the treatment of pain.Why?unable to feel pain,but otherwise completely healthy.None related to the child had experienced pain at any time in their lives.They all carried an extremely unusual mutation in a single gene,called SCN9A.This paper shows that rare diseases can still be of great importance because of the insights they give into biological and developmental processes.“This gives us an excellent target to develop painkillers”.The boy who could walk on hot coals14 December 2006脊髓小脑共济失调 脊髓小脑共济失调(spinocerebellar ataxias，SCAs)在普通人群发病率为110万510万，是累及人类神经系统的主要遗传疾病之一。遗传性脊髓小脑共济失调是一大类迟发型神经退化性疾病，呈常染色体显性遗传，具有多神经病理症状，脊髓小脑萎缩，神经元丢失等。临床症状为迟发性共济失调。World without pain is hell The inability to feel pain,which at first blush seems like a gift,is,in fact,a devastating disability.How lucky we are,because we live life with the pain we receive.This paper shows that rare diseases can still be of great importance because of the insights they give into biological and developmental processes.”None related to the child had experienced pain at any time in their lives.They all carried an extremely unusual mutation in a single gene,called SCN9A.思考题简述钾离子通道的离子选择机制。
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