(3.235.139.152) 您好!臺灣時間:2021/05/11 12:04
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:黃仁裕
研究生(外文):Ren-Yu
論文名稱:藉異源性表達系統探討人類 CLCN1 氯離子通道可能磷酸化位點之功能性研究
論文名稱(外文):Functional Study of the Possible Phosphorylation Sites in the Human CLCN1 Cl- Channel via Heterologous Expression System.
指導教授:林明忠林明忠引用關係
指導教授(外文):Min-Jon Lin
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:73
相關次數:
  • 被引用被引用:0
  • 點閱點閱:87
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
CLCN1(Chloride channel 1)電位依賴型離子通道在哺乳類動物骨骼肌細胞的膜電位再極化作用中扮演著很重要的角色。如果人類CLCN1發生變異很有可能會造成先天性人類肌強直症。目前己知蛋白質磷酸化酵素C (Protein kinase C,PKC) 可以調控人類CLCN1氯離子通道,但其磷酸化位點之所在還是未知的。本實驗的目的就是要研究人類CLCN1氯離子通道蛋白中可能的磷酸化位點。首先我們使用單點突變的技術針對可能的磷酸化位點,做出了21個CLCN1的突變種,所有突變點點都位於人類CLCN1氯離子通道蛋白的C端位置。再將從 hCLCN1/pTLN質體反轉錄作用而來的CLCN-1 cRNA以顯微注射的方式打入非洲爪蟾卵母細胞 (Xenopus laevis oocyte) 之中,再利用雙電極電位鉗定法 (Two-electrode voltage clamp, TEVC) 來測量人類CLCN1表達在爪蟾卵母細胞膜的離子電流。
可以以一個量化的數值V1/2 (當離子通道開啟機率為0.5時之膜電位)的變化做為觀察離子通道性質變化的標準。其中,野生型的人類CLCN1氯離子通道會受到PMA的抑制,而使其V1/2偏向較正電方向, (由 -42.9 12.5 偏移到 -13.7 5.0, n = 8)。而其它突變種的CLCN1也有類似於野生型的情況,如S682A、S720A、S740A。但是在這之中,S892P和 雙重點突變的S892A/T893A卻在PMA作用後沒出現明顯的改變,而Ser892所在,即Thr890到Ser903之間的這序列充滿了多個可能的磷酸化位點,所以可能有磷酸化位點存在。
因此我們將892突變為 Aspartic acid,用以模擬受磷酸化後性質偏向酸性的情況。而S892D本身表現出來的V1/2相對於野生型更為偏向於正向,但卻和野生型受到PMA作用後的V1/2極為接近。同時,S892D在受到PMA作用後幾乎沒有任何改變。但是突變為Proline或是進行雙重點突變都有可能改變三級結構而造成重大的改變,所以無法確定Ser892 就是磷酸化位點,而S892D的突變也有可能會干擾到該區域的磷酸化作用,所以推測Thr890到Ser903序列之間有磷酸化位點存在。


CLCN1 voltage-dependent chloride channel play an important role in membrane potential repolarization of mammalian skeletal muscle. Mutations in human CLCN1 lead to the myotonia congenital which is an inherited human disease. It has been known protein kinase C (PKC) can modulate the CLCN1 channel, however, the phosphorylated sites are still unclear. The purpose of this study was to investigate the possible phosphorylation sites in the human CLCN1 Cl- channel. The human CLCN1 cRNA was in vitro transcribed from linearized hCLCN1/pTLN plasmid, and then inject it into the Xenopus laevis oocytes using a microinjector. Two-electrode voltage clamp (TEVC) technique was used for the measurement of ionic current of human CLCN1. In the present study we construct 21 mutant recombinant plasmids by use the technique of site-directed mutagenesis. All mutants were designed according to the putative phosphorylation sites in C-terminus of CLCN1. Human CLCN1 and mutants were transient expressed in Xenopus oocytes by the microinjection of CLCN1 cRNA. To test whether mutant proteins lacking sensitivity to protein kinase C, an activator of PKC phorbol 12-myristate 13-acetate (PMA, 2 μM) was used in this study.
The functional effects of PMA on wild-type and various mutants were determined by using TEVC recording from Xenopus oocytes. The treatment with PMA causes an inhibiting effect of wild-type CLCN1 current and a shift of the half-maximum activation of open probability (V1/2) to more positive membrane potentials (from -42.9 12.5 to -13.7 5, n = 8). These shifted-effects of most CLCN1 mutants by treated with PMA are similar to the wild-type, including S682A (from -32.9 4.2 to -15.9 10.2, n = 4), S720A (from -26.8 5.7 to -5.2 12.1, n = 4), S740A (from-55.7 13.5 to -24.2 9.1, n = 4), T782A (-40.4 10.3 to -8.1 11.5, n = 4), T891A (from -36.5 9.2 to -14.8 6, n = 6), S892A (from -27.5 6.8 to -1.3 10.4, n = 4) and S896R (from -45.4 13.4 to -13.7 12.9, n = 8). As so far only the S892P (from -39.5 11.2 to -35.7 14, n = 6) and a double mutant S892A/T893A (from -45.2 4.4 to -38.4 4.4, n = 4) were not change V1/2 by treated with PMA.
In order to mimic phosphorylation at position 892, we made a mutant where a serine residue at 892 was substituted by aspartic acid. The result showed that the V1/2 of S892D was more positive than the wild-type, but similar to wild-type in the presence of PMA (-10.2 9.7 vs -13.7 5.0). Furthermore, the treatment of S892D with PMA did not change the value of V1/2 (from -10.2 9.7 to -9.6 6.9, n = 4). These results suggest that sequence including serine 892 has functional phosphorylated site for protein kinase, and therefore to modulate the functional properties of human CLCN1 channels.


中文摘要…………………………………………………………………3

Abstract………………………………………………………………….5

序論 (Introduction)……………………………………………………..7

材料與方法 (Materials and Methods)……………………………..…19
人類氯離子通道基因 CLCN1質體之建構………………………………………..19
磷酸化位置之預測…………………………………………………………………..19
定點突變 (Site-Directed Mutagenesis)……………………………………………...20
轉型 (transformation) 製備…………………………………………………………20
轉型 (transformation) 流程........................................................................................22
Plasmid 的抽取...........................................................................................................22
DNA 膠體電泳 (Electropphoresis)............................................................................23
cRNA 的製備 (in vitro transcription)........................................................................24
非洲爪蟾卵母細胞之製備 ( Xenopus laevis oocyte)………………………………25
cRNA顯微注射 ( cRNA microinjection)...................................................................29
電生理實驗紀錄..........................................................................................................30
資料分析 (Data Analysis)…………………………………………………………...33
藥劑 (Chemicals)……………………………………………………………………33

結果 (Results).........................................................................................35
磷酸化位點之預測結果……………………………………………………………..35
CLCN1氯離子通道於非洲瓜蟾卵母細胞內異源性表達氯離子電流的特徵……35
蛋白質磷酸化酵素C (Protein Kinase C) 對 CLCN1 氯離子通道間之關係…….38
蛋白質序列662到720之間可能的磷酸化位點…………………………………..39
蛋白質序列721到820之間可能的磷酸化位點……………………………………40
蛋白質序列872之後可能的磷酸化位點…………………………………………..41
Ser892所在的序列存在蛋白質磷酸化酵素C (Protein Kinase C) 的磷酸化位點……………………………………………………………………………………..42

討論 (Discussion)...................................................................................45
人類 CLCN1 基因在非洲爪蟾卵母細胞上異源性表達人類 CLCN1 氯離子通道………………………………………………………………………………….….45
非洲爪蟾卵母細胞與哺乳類動物的蛋白質磷酸化酵素 C (PKC) 系統…….…...47
CLCN1氯離子通道C端上可能的磷酸化位點……………………………………48

圖表 (Figures and Table)……...............................................................54
圖一:利用非洲爪蟾卵母細胞異源性表達 CLCN1 氯離子通道。……………..54
圖二:比對注射 CLCN1 cRNA、注射 Rnase-free water及無注射之非洲爪蟾卵母細胞的電生理性質。…………………………………………………………………55
圖三:CLCN1 氯離子通道受到PMA影響前與後的差異。……………………..56
圖四:蛋白質序列662到720之間變異點受到PMA作用的影響。……………57
圖五:蛋白質序列721到820之間變異點受到PMA作用的影響。…………….58
圖六:蛋白質序列872後變異點受到PMA作用的影響。………………………59
圖七:T891A、T893A及S892A+T893A雙重點突變受到PMA作用的電位依賴性曲線。……………………………………………………………………………...61
圖八:Ser892變異對CLCN1受磷酸化功能之影響。………………………………62
表一:目前所測量過的所有可能磷酸化位點及受到PMA影響之情況。……………………..…………………………………………………………....63

參考文獻 (References)...........................................................................64

附圖……………………………………………………………………..72
附圖一:人類CLCN1氯離子通道蛋白的結構圖。………………………………..72
附圖二:非洲爪蟾卵母細胞表達載體示意圖。…………………………………..73


Aromataris E. C. & Rychkov G. Y. ClC-1 chloride channel – matching its properties to a role in skeletal muscle. Clin Exp Pharmacol Physiol 2006, 33: 1118-1123.

Aromataris E. C., Astill D. S., Rychkov G. Y., Bryant S. H., Bretag A. H., Roberts M. L. Modulation of the gating of ClC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid. Br. J. Pharmacol. 1999, 126: 1375-1382.

Astill D. S., Rychkov G., Clarke J. D., Hughes B. P., Roberts M. L., Bretag A. H. Characteristics of skeletal muscle chloride channel ClC-1 and point mutant R304E expressed in Sf-9 insect cells. Biochim. Biophys. Acta 1996, 1280: 178-186.

Bennetts B., Roberts M. L., Bretag A. H., Rychkov G. Y. Temperature dependence of human muscle ClC-1 chloride channel. J. Physiol. 2001, 535: 83-93.

Blair E., Redwood C., Ashrafian H., Oliveira M., Btoxholme J., Kerr B. et al. Mutations in the γ2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 2001, 10:1215-1220.

Bowne S. J., Suilivan L. S., Blanton S. H., Cepko C. L., Blackshaw S., Birch D. G. et al. Mutations in the inosine monophoshate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa. Hum Mol Genet 2002, 11:559-568.

Bretag A. Antimyotonic agents and myotonia. Proc. Aust. Physiol. Pharmacol. Soc. 1983, 14: 170-191.

Bretag AH. Muscle chloride channels. Physiol. Rev. 1987, 67: 618-724.

Bryant S. H. Muscle membrane of normal and myotonicgoats in normal and low external chloride. Fed. Proc.1962, 21: 312.

Bryant, S. H. & Conte-Camerino, D. Chloride channel regulation in the skeletal muscle of normal and myotonic goat. Pfl.ugers Archiv 1991, 417: 605-610.

Camerino C. D., De Luca A., Mambrini M., Ferrannini E. et al. :The effects of taurine on pharmacologically induced myotonia. Muscle Nerve 1989, 12: 898-904.

Chen T. Y., Miller C. Nonequilibrium gating and voltage dependence of the ClC-0 Cl− channel. J. Gen. Physiol. 1996, 108: 237-250.

Chiloeches A., Mason C. S., Marais R. S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3. Mo Cell Biol 2001, 21: 2423-2434.

Cleiren E., Benichou O., Van Hul E., Gram J., Bollerslev J., Singer F. R. et al. Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the CLCN7 chloride channel gene. Hum Mol Genet 2001, 10:2861-2867,

Dominguez I., Diaz-meco M. T., Municio M. M., Berra E., Herreros A. G. DE et al. Evidence for a role of protein kinase C ζ subspecies in maturation of Xenopus laevis oocytes. Mol Cell Biol. 1992, 12: 3776–3783.

Duan D., Winter C., Cowley S., Hume J. R., Horowitz B. Molecular identification of a volume-regulated chloride channel. Nature 1997, 390:417-421.

Duffield M., Rychkov G., Bretag A., Roberts M. Involvement of Helices at the Dimer Interface in ClC-1 Common Gating. J. Gen. Physiol. 2003, 121: 149-161.

Dutzler R. Structural basis for ion conduction and gating in ClC chloride channels. FEBS Letters 2004, 564:229 – 233.

Estevez R., Pusch M., Ferrer-Costa C., Orozco M., Jentsch T. J .Functional and structural conservation of CBS domains from CLC chloride channels. J Physiol 2004, 557.2:363-378.

Fahlke C., Rhodes T. H., Desai R. R., George A. L. Jr. Pore stoichiometry of a voltage-gated chloride channel. Nature 1998, 394:687-690.

Fahlke C. & Rudel R. Chloride currents across the membrane of mammalian skeletal muscle fibres. J. Physiol. 1995, 484: 355-368.

Fahlke C., Rudel R., Mitrovic N., Zhou M., George A. L. Jr. An aspartic acid residue important for voltage dependent gating of human muscle chloride channels. Neuron 1995, 15: 463-472.

Gaxiola R. A., Yuan D. S., Klausner R. D., Fink G. R. The yeast CLC chloride channel functions in cation homeostasis. Proc Natl Acad Sci USA 1998, 95:4046-4050.

Grunder S., Thiemann A., Pusch M., Jentsch T. J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. Nature 1992, 360:759-762.

Gurdon J. B, Lane C. D., Woodland H. R. & Marbaix G. Use of frog eggs and oocytes for the study of messenger RNA and it’s translation in living cell. Nature 1971, 233:177-182.

Gurnett C. A., Kahl S. D., Anderson R. D., Campbell K. P. Absence of the skeletal muscle sarcolemma chloride channel ClC-1 in myotonic mice. J. Biol. Chem. 1995, 270: 9035-9038.

Hryciw, D. H., Rychkov, G. Y., Hughes, B. P. and Bretag, A. H.:Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1. J. Biol. Chem. 1998,273: 4304–4307.

Jentsch T. J. Chloride channels: a molecular perspective. Curr Opin Neurobiol 1996, 6:303-310.

Jentsch T. J., Friedrich T., Schriever A., Yamada H. The CLC chloride channel family. Pflugers Arch 1999, 437:783-795.

Jentsch T. J., Steinmeyer K., Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 1990, 348:510-514.

Jentsch T.J., Stein V., Weinreich F., Zdebik A. A. Molecular structure and physiological function of chloride channels. Physiol. Rev. 2002, 82: 503-568.

Jhee K. H., McPhie P. & Miles E. W. Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis andregulation. Biochemistry 2000, 39:10548-10556.

Kennan A., Aherne A., Palfi A., Humphries M., McKee A., Stitt A. et al. identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho (-/-) mice. Hum Mol Genet 2002, 11:547-557.

Klocke R., Steinmeyer K., Jentsch T. J., Jockusch H. Role of innervation, excitability, and myogenic factors in the expression of the muscular chloride channel ClC-1. A study on normal and myotonic muscle. J. Biol. Chem. 1994, 269: 27635-27639.

Koch M. C., Steinmeyer K., Lorenz C., Ricker K et al. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 1992, 257:797-800.

Kornak U., Kasper D., Bosl M. R., Kaiser E., Schweizer M., Schulz A. et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001, 104:205-215.

Kubisch C., Schroeder B. C., Friedrich T., Lutjohann B. et. al. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell 1999, 96:437-446.

Kwiecinski H. Myotonia induced by chemical agents. Crit. Rev. Toxicol. 1981, 8: 279-310.

Ludewig U., Pusch M., Jentsch T. J. Independent gating of single pores in CLC-0 chloride channels. Biophys. J. 1997, 73: 789-797.

Ludewig U., Pusch M., Jentsch T. J. Two physically distinct pores in the dimeric ClC-0 chloride channel. Nature 1996, 383: 340-343.

Maduke M., Miller C., Mindell J. A. A decade of CLC chloride channels: structure, mechanism, and many unsettled questions. Annu Rev Biophys Biomol Struct 2000, 29:411-438.

Maduke M., Williams C., Miller C. Formation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains. Biochemistry 1998, 37:1315-1321.

Matsumura Y., Uchida S., Kondo Y., Miyazaki H. et al. Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel. Nat Genet 1999, 21:95-98.

Middleton R. E., Pheasant D. J., Miller C. Homodimeric architecture of a ClC-type chloride ion channel. Nature 1996, 383:337-340.

Mindell J. A. & Maduke M. ClC chloride channels. Genome Biology 2001, 2(2):reviews3003.1–3003.6.

Mindell J. A., Maduke M., Miller C., Grigorieff N. Projection structure of a ClC-type chloride channel at 6.5 Å resolution. Nature 2001, 409:219-223.

Palade P.T. & Barchi R. L. Characteristics of the chlorideconductance in muscle fibers of the rat diaphragm. J. Gen. Physiol. 1977, 69: 325-342.

Papponen H., Kaisto T., Myllyla V. V., Myllyla R., Metsikko K. Regulated sarcolemmal localization of the muscle-specific ClC-1 chloride channel. Exp. Neurol. 2005, 191: 163-173.

Piwon N., Gunther W., Schwake M., Bosl M. R., Jentsch T. J. ClC-5 Cl-channel disruption impairs endocytosis in a mouse model for Dent’s disease. Nature 2000, 408:369-373.

Purdy M. D., Wiener M. C. Expression, purification, and initial structural characterization of YadQ, a bacterial homolog of mammalian ClC chloride channel proteins. FEBS Lett 2000, 466:26-28.

Pusch M., Ludewig U., Rehfeldt A., Jentsch T. J. Gating of the voltage-dependent chloride channel ClC-0 by the permeant anion. Nature 1995,373: 5275-31.

Pusch M. Myotonia caused by mutations in the muscle chloride channel gene CLCN1. Hum. Mutat. 2002, 19: 423-434.

Pusch M. & Jentsch T. J. Molecular physiology of voltage gated chloride channels. Physiol. Rev. 1994, 74: 8138-27.

Pusch M., Ludewig U., Jentsch T. J. Temperature dependence of fast and slow gating relaxations of ClC-0 chloride channels. J. Gen. Physiol. 1997, 109: 105-116.

Pusch M., Ludewig U., Rehfeldt A., Jentsch T. J. Gating of the voltage-dependent chloride channel ClC-0 by the permeant anion. Nature 1995, 373: 527-531.

Rosenbohm A., Rudel R., Fahlke C. Regulation of the human skeletal muscle chloride channel hClC-1 by protein kinase C. J. Physiol 1999, 514.3:677-685

Rychkov G. Y., Pusch M., Astill D. S., Roberts M. L., Jentsch T. J., Bretag A. H. Concentration and pH dependence of skeletal muscle chloride channel ClC-1. J. Physiol. 1996, 497: 423-435.

Saviane C., Conti F., Pusch M. The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia. J. Gen. Physiol. 1999, 113: 457-468.

Schmidt-Rose T. & Jentsch T. J. Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1. J Biol Chem 1997, 272:20515-20521.

Schmidt-Rose T. & Jentsch T. J. Transmembrane topology of a CLC chloride channel. Proc Natl Acad Sci USA 1997, 94:7633-7638.

Schwappach B., Stobrawa S., Hechenberger M., Steinmeyer K. & Jentsch T. J. Golgi localization and functionally important domains in the NH2 and COOH terminus of yeast CLC putative chloride channel Geflp. J boil Chem 1998, 273:15110-15118.

Shan X., Dunbrack R. I., Christopher S. A. & Kruger W. D. Mutations in the regulatory domain of cystathionine beta-synthase can functionally suppress patient-derived mutations in cis. Hum Mol Genet 2001, 10:635-643.

Simon D. B., Bindra R. S., Mansfield T. A., Nelson-Williams C. et al. Mutations in the chloride channel gene, CLCNKB, cause Bartter’s syndrome type III. Nat Genet 1997, 17:171-8.

Staley K., Smith R., Schaack J., Wilcox C., Jentsch T. J. Alteration of GABAA receptor function following gene transfer of the CLC-2 chloride channel. Neuron 1996, 17:543-551.

Surti T. S., Huang L., Jan Y. N., Jan L. Y. & Cooper E. C. Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels. PNAS 2005, 102:17828-17833.

Tricarico, D., Conte-Camerino, D., Govoni, S., Bryant S. H. Modulation of rat skeletal muscle chloride channels by activators and inhibitors of protein kinase C. Pfl.ugers Archiv 1991, 418:500-503.

Tricarico, D., Wagner, R., Bryant, S. H., Conte-Camerino, D. Regulation of resting ionic conductances in frog skeletal muscle. Pfl.ugers Archiv 1993, 423:189-192.

Waldegger S. & Jentsch T.J. From tonus to tonicity: physiology of CLC chloride channels. J. Am. Soc. Nephrol. 2000, 11: 1331-1339.
Warner A. E. Kinetic properties of the chloride conductance of frog muscle. J. Physiol. 1972, 227: 291-312.

Weiping W. U., Grigori Y., RYCHKOV, Bernard P., Hughes, Allan H. BRETAG. Functional complementation of truncated human skeletal-muscle chloride channel (hClC-1) using carboxyl tail fragments. Biochem. J. 2006, 395:89-97.

Wong Y. H., Lee T. Y, Liang H. K., Huang C. M. et al. KinasePhos 2.0: a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns. Nucleic Acids Research 2007,35:W588-594.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔