1、神經(jīng)電活動的分子基礎(chǔ) -離子通道,廈門大學(xué)醫(yī)學(xué)院 戚智,21,time,Voltage (mV),Stim Elec,REC 1,REC 2,REC 3,+,First try: a small depolarizing stimulus (-60 mV),-70 -,-60 -,-50 -,-40 -,-30 -,-20 -,-10 -,0 -,+10 -,+20 -,+30 -,+40 -,+,+,+,22,time,Voltage (mV),Stim Elec,REC 1,REC 2,REC 3,+,Next try: a slightly larger depolarizing sti

2、mulus (-55 mV),-70 -,-60 -,-50 -,-40 -,-30 -,-20 -,-10 -,0 -,+10 -,+20 -,+30 -,+40 -,+,+,+,23,time,Voltage (mV),Stim Elec,REC 1,REC 2,REC 3,+,-70 -,-60 -,-50 -,-40 -,-30 -,-20 -,-10 -,0 -,+10 -,+20 -,+30 -,+40 -,+,+,+,Next try: a slightly larger depolarizing stimulus (-50 mV),RMP,Local Potentials,Ac

3、tion Potentials,23,time,Voltage (mV),Stim Elec,REC 1,REC 2,REC 3,+,-70 -,-60 -,-50 -,-40 -,-30 -,-20 -,-10 -,0 -,+10 -,+20 -,+30 -,+40 -,+,+,+,RMP,Action Potentials,Can we get even larger Action Potentials?,25,time,Voltage (mV),Stim Elec,REC 1,REC 2,REC 3,-70 -,-60 -,-50 -,-40 -,-30 -,-20 -,-10 -,0

4、-,+10 -,+20 -,+30 -,+40 -,RMP,Action Potentials,Try an even larger depolarizing stimulus (-45 mV),No higher, no larger, Identical!,Can we get even larger Action Potentials?,動作電位及其產(chǎn)生機(jī)制,電壓鉗技術(shù),電壓鉗 Voltage clamp 電流變化,阻斷劑對電流成分的分離,電壓鉗 Voltage clamp 電流變化,河豚魚 Tetrodotoxin is a lethal toxin found in pufferfi

5、sh that inhibits the voltage-sensitive sodium channel, halting action potentials.,二、動作電位 （Action potenial） （一）動作電位的概念及特點,去極化-Depolarization；閾值-Threshold value，超射-Overshoot；鋒電位-Spike potential；后電位-After potential；后去極化電位-After depolarization（負(fù)后電位）； 后超極化電位-After hyperpolarization（正后電位）,離子通道由三個功能區(qū)構(gòu)成,（一）

6、Pore離子通路(包含Selectivity Filter ); （二）Gate通道門； （三）Sensor感受器。,選擇性及通透性機(jī)制,What determines Ion Selectivity?,+,+,-,How are Channels Selective?,-charge -atomic radius -Hydration,Na+,K+,Cl-,+,+,-,-Selectivity by charge,Na+,K+,Cl-,+,+,-,Na+,K+,+,+,+,+,Positive ring of charges will allow (-) ions to conduct,An

7、d repel + ions,Ion Selection: Chloride Channels,Modified from Keramidas et al., Prog. Biophys. Mol. Biol. 86: 161 (2004),+,+,-,Na+,K+,Cl-,+,+,-,Na+,K+,-,-,-,-,Negative charges Allow + ions To enter the channel,-Selectivity by charge,Ion Selection: Cationic Channels,Modified from Keramidas et al., Pr

8、og. 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

9、molecules through the bilipid layer and release them on the opposite side.,Selectivity of Valinomycin,K+,Na+,+,+,Na+ channel,Na+,K+,+,+,Na+,K+,-,-,-,-,Smaller Na+ ions Fit, but not larger K+ or Ca+,-Selection by atomic radius,+,+,Na+,K+,+,+,Na+,K+,-,-,-,-Selection by Dehydration: -Charges on the cha

10、nnel Must effectively substitute The (-) polar Charges of the O on H20 : The hydration shell must be stripped before K+ can pass through.,-,-,-,-,-,-,-,H20,Hydration Shell around ions,K+ Channel,鈉離子半徑0.95埃 鈣離子半徑0.99埃 鉀離子半徑1.33埃,Selectivity of K+, Na+ and Ca2+ channels K+ channel: Pna/Pk1/100; Pca/Pk

11、1/1000 Na+ channel: Pk/Pna8/100; Pca/Pna10/100 Ca+ channel (L-type): Pna/Pca=1/1170; Pk/Pca=1/3000,挑戰(zhàn)公認(rèn)的難題,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. pr

12、of. Harvard U. 1996 X-ray crystallography Rockefeller U. 1998 K+ channel structure resolved at 0.32 nm resolution 2001 0.2 nm,Roderick MacKinnon,Biological Membrane = Lipid Bilayer,Approximately 30 thick Hydrophobic core + Hydrophilic or charged headgroups Mixture of lipids that vary in type of head

13、 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 expression Difficulties in solubilization Difficulties in crystallization Attempting crystallization and structure solution of transmembran

14、e proteins is considered difficult and risky.,鉀離子通道選擇及 通透性機(jī)制,Roderick MacKinnon et al. 1998 - Nobel prize in Chemistry 2003,試驗材料選取的重要性,鏈霉菌鉀離子通道(KcsA) WHY？,Bacterial K+ channel,X-ray crystallography,Selectivity filter,Gate,Inner vestibule,Doyle et al. Science 1998,Elementary electrostatic considerati

15、ons,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 filter,Selection of ion by selectivity filter,Sodium ion is too small for carbonyl oxygen intera

16、ction and water expel,Carbonyl 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.,鈉離子通道選擇及 通透性機(jī)制,+,+,Na+ channel,Na+,K+,+,+,Na+,K+,-,-,-,-,Smaller Na+ ions Fit, but not larger K+ or

17、 Ca+,-Selection by atomic radius,鈉離子半徑0.95埃 鈣離子半徑0.99埃 鉀離子半徑1.33埃,Selectivity of K+, Na+ and Ca2+ channels K+ channel: Pna/Pk1/100; Pca/Pk1/1000 Na+ channel: Pk/Pna8/100; Pca/Pna10/100 Ca+ channel (L-type): Pna/Pca=1/1170; Pk/Pca=1/3000,Selection of ion by selectivity filter,Sodium ion is too small

18、for carbonyl oxygen interaction and water expel,鈣離子通道選擇及 通透性機(jī)制,The 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 tra

19、versing 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 app

20、lied 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.

21、 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 s

22、ide 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.,氯離子通道選擇及 通透性機(jī)制,Dutzler et al. (2002) Nature 415, 287-294,Structure of a chloride channel

23、 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, 287-294,Dutzler et al. (2002) Nature 415, 287-294,Two architectures for ion channel proteins: antiparallel (Cl-) and parallel (K+),Agres Work,Aquaporins Sele

24、ctivity,Protons, 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 pores Water, glycerol, ,Tetrameric pore Perhaps ions?,Aquaporins o

25、f known structure: GlpF E. coli glycerol channel (aquaglycerolporin) AQP1 Mammalian aquaporin-1 (pure water channel),Agres Work,Proton Blocking by a Global Orientation Mechanism,Aquaporin,Proteins are Dynamic Structures,Water traveling through Aquaporin pore,Control of the selectivity of the aquapor

26、in 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.,離子通道門控 （電壓感受）機(jī)制,All voltage-gated ion channels have a similar architecture e.g. sodium channel,S5/S6 loop related to ion s

27、electivity S4 related to voltage sensing,Kv1,Kv2,Kv3,Kv4,Kv5+,KCNQ,Color coding: Red = + Green = - Blue = polar Gray = nonpolar,S4 alignment,Ion Channels and Diseases- Channelopathies,高血鉀型周期性癱瘓（hyperkalemic periodic paralysis，HyPP）,Origins of HYPP,The mutation traces back to the Quarter Horse Stalli

28、on, Impressive It is speculated by some that the mutation actually links back to his dam, Glamour Bars,Today: Who its affecting now,Impressive now has over 100,000 descendants Stock-type breeds: Quarter Horses, Paints, Appaloosas, and Quarter Horse crosses,Why is HYPP so wide spread now?,Halter hors

29、es are judged on their conformation HYPP causes a favorable phenotypic expression of well-developed musculature This results in superior halter horses who win and breed on these genes,Twenty years ago, a quarterhorse named “Impressive” won all of the titles of his class. He was the top-winning, top-

30、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 stalli

31、ons 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 Assoc

32、iation,History,Affects descendents of the Quarter Horse stallion Impressive Quarter Horses, Appaloosas, and Paints Research project funded by AQHA in 1989. Research results released in 1992 Proliferated because he was successful in the show ring and possessed desired physical features.,Soon his prog

33、eny 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 HY

34、PP 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 paralysis.,How HYPP affects horses: Normal K+ Metabolism,Take note of w

35、here on the graph the Na+ channels are open or closed in relation to the polarization of the muscle cell,Skeletal muscle Na+ channel NaV1.4,Potassium-aggravated myotonia mutations affect single channel behaviour,Cannon, 1997,Computer model of myotonia and paralysis,f: Fraction of Na+ current that fa

36、iled to inactivate,高血鉀型周期性癱瘓臨床表現(xiàn)： 較少見，有遺傳史，童年起病，常因寒冷或服鉀鹽誘發(fā)，白天發(fā)病。發(fā)作期鉀離子自肌肉進(jìn)入血漿，因而血鉀升高，可達(dá)5-7mgEq/L。也以下肢近端較重，持續(xù)時間較短，不足一小時，一日多次或一年一次。部分病員發(fā)作時可有強(qiáng)直體征，累及顏面和手部，因而面部強(qiáng)直，眼半合，手肌僵硬，手指屈曲和外展。進(jìn)食、一般活動、靜注鈣劑、胰島素或腎上腺素均可終止發(fā)作。事先給予能增加鉀排泄的醋氮酰胺及雙氫克尿塞等利尿劑可預(yù)防發(fā)作醫(yī)學(xué)|教育網(wǎng)搜集整理。 發(fā)作時心電圖改變，初是T波增高，QT間期延長，以后逐漸出現(xiàn)R波降低，S波增深，ST段下降，P-R間期及QRS時

37、間延長。 對診斷有困難者，可作誘發(fā)試驗：口服氯化鉀（3-8g）?？烧T發(fā)或使原有癱瘓癥狀加重。這是因為高鉀型周期性癱瘓，對外源性鉀的攝入比本身血清鉀含量變化更為敏感。 ,The human defect is also in Na channels,Hyperekplexia (a startle disease) Spasmodic mouse: a point mutation in Gly Rc (an inhibitory Rc in the spinal cord and brain stem) Decreased frequency of opening Spastic mouse:

38、 Less number of Gly Rc expressed Glycine vs. GABA (spinal cord vs. brain) Delicate balance of synaptic excitation and inhibition,Shunting inhibition in regions close to soma and axon hillock IPSP at resting memb. potential? Shaft synapse,Rea et al (2002),Compound heterozygosity in hyperekplexia,PHYS

39、IOLOGY OF PAIN,Definition Pain 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? B

40、liss?,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

41、 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 “l(fā)ike 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 ou