臺灣博碩士論文加值系統

ClC-2是屬於ClC 氯離子通道和運輸器家族的成員之一。ClC-2通道主要位在細胞膜上，並廣泛的表現在各個組織及細胞中，在大腦以及上皮組織中表現量更為豐富。先前的研究指出，ClC-2通道在神經細胞中與調節其神經興奮性有關，人類中若在ClC-2基因有突變可能會導致全身性的癲癇發作。目前對於ClC-2通道在生理上所扮演的角色雖然已有初步的了解，但它在細胞中是如何被調控及表現的，至今尚不清楚。因此為了更進一步了解調控ClC-2通道的分子機制，我們使用酵母雙雜交技術尋找與ClC-2通道相互作用之蛋白質。藉由這些蛋白質在細胞中已知參與的機制、訊息傳導路徑，可以進一步推測C lC-2的調控性質。
我們將ClC-2未在細胞質內的C端CBS1到CBS2 domain之前的這段序列，亦即第581-794個胺基酸接在pGilda載體上，找尋與這段序列有相互作用的蛋白質。對於大鼠腦部cDNA library進行篩選後，共發現了124個蛋白質可能與ClC-2有相互作用。在經過進一步的序列分析，扣除胺基酸序列有frame shift，剩下53個可能與ClC-2有相互作用的蛋白質。目前從中挑選10個蛋白質，進行進一步的實驗以確認其與ClC-2通道的相互作用能力，我們使用X-gal測試分析和leucine需求分析實驗，結果顯示出這10個蛋白質都能在進行X-gal分析時使菌落由白變藍，並且在缺乏leucine的培養皿上生長。接下來我們以共同免疫沈澱法設法確認其相互作用關係。雖然結果顯示NSF (N-ethylmaleimide sensitive fusion protein)、SMAD 1及Carhsp1(calcium regulated heat stable phosphoprotein 1)似乎有被共同免疫沈澱的跡象。但由於訊號太微弱，我們仍不能確認其彼此之間的相互作用關係。在GSTpull down的結果中，NSF似乎能被GST-ClC-2給pull-down。在進一步確認這些蛋白質與ClC-2的相互關係後，我們將利用電生理技術研究這些蛋白質是否會改變ClC-2之電生理特性，以釐清兩者之間的相關性。

ClC-2 is a member of the ClC family of chloride channels and transporters. It is mainly located on the plasma membrane and expressed in various kind of tissues, and most abundant in the brain and epithelium. Previous studies have shown that ClC-2 channel expression in neurons may modulate their membrane excitability. A disruption in the ClC-2 gene was reported to correlate with the presence of generalized epilepsy in human. In spite of the recent progress in the understanding of the physiological and pathophysiological significance of ClC-2 channels, their regulatory as well as signaling pathways remain unclear. Therefore, we applied yeast two-hybrid screen to search for ClC-2-interacting proteins, which will provide important insight on the physiological function of ClC-2 channels.
We focused on a sequence between the CBS1-CBS2 region (amino acids 581-794) at the C-terminal tail of ClC-2, which was cloned into the bait vector pGilda. After screening a rat brain cDNA library, 124 prey clones were identified. By eliminating the clones with incorrect reading frames, we have obtained 51 positive clones. 10 potential candidates from 51 positive clones were chosen for further characterization. X-gal assays and Leucine requirement tests were performed to reconfirm the interaction between ClC-2 and potential candidate proteins. All of the 10 candidate proteins showed blue patches on X-gal-containing plates and were grown on leucine-deficient plates, suggesting that these clones may indeed interact with ClC-2 channels. We performed co-immunoprecipitation and GST pull-down assay to verify the interaction between ClC-2 and potential candidates of ClC-2-interacting proteins. Despite of the fact that the signal intensity of co-immunoprecipitation was not optimal, our current data suggest that ClC-2 probably display direct interaction with with NSF (N-ethylmaleimide sensitive fusion protein), SMAD 1, and Carhsp 1 (calcium regulated heat stable phosphoprotein 1). Moreover, our preliminary GST pull-down results also support a direct interaction between NSF and ClC-2. Further experiments, hower, will be required to verify the foregoing observation. In the future, we plan to apply electrophysiological techniques to determine whether these candidate proteins may affect the biophysical properties and/or trafficking process of ClC-2 channels.

口試委員審定書 P1
致謝 P2
圖次 P6
中文摘要 P7
英文摘要 P8
緒論 P10
1. 氯離子通道 P10
2. ClC氯離子通道 P10
3. ClC-2氯離子通道 P12
4. 已知與ClC-2有相互作用之蛋白質相關研究 P15
研究目的 P18
材料與方法 P19
1. Molecular biology P19
1.1 ClC-2載體置備 P19
1.2 ClC-2誘餌載體置備 P20
1.3 GST-ClC-2載體置備 P20
2. Yeast Two-Hybrid system P20
2.1 酵母雙雜交技術之原理及方法 P20
2.2 將載體轉殖入酵母菌中 P21
2.3 X-gal 測試及Leucine 需求分析 P22
2.4 大鼠腦部cDNA library篩選 P22
2.5 萃取酵母菌中pJG4-5 plasmid P22
2.6 將pJG4-5 plasmid轉殖入細菌菌株 P23
2.7 Colony PCR P23
2.8 DNA電泳 P23
3. Protein Biochemustry P24
3.1 將plasmid DNA轉殖至HEK293T cell P24
3.2 共同免疫沉澱分析法(Co-immunoprecipitation) P24
3.3 SDA-PAGE電泳與西方墨點分析 P25
3.4 GST pull down 測定 P26
結果 P28
1. 酵母雙雜交篩選 P28
1.1 誘餌蛋白所包含之ClC-2區域 P86
1.2 Autoactivation test P29
1.3 以西方點墨法確認ClC-2誘餌蛋白的表現 P30
1.4 利用酵母雙雜交系統對大鼠腦部cDNA library進行篩選 P31
2. 進一步驗證所篩選出之蛋白質與ClC-2之相互作用 P32
2.1 以酵母雙雜交確認篩選出的蛋白質與ClC-2相互作用關係 P32
2.2 以免疫螢光染色及西方點墨法確認ClC-2及篩選出的蛋白質在
HEK 293T的相互作用關係 P33
2.3 以共同免疫沈澱法（Co-immunoprecipitation）設法確認ClC-2
和其作用蛋白在HEK293T細胞的相互作用關係 P34
2.4 以GST pull-down assay檢驗和ClC-2有作用的蛋白質 P35
討論 P36
1. 所篩選出的10個蛋白質在細胞中可能參與之功能 P36
2. 所篩選之蛋白質與ClC-2相互作用的結果探討 P38
2.1 酵母雙雜交篩選 P38
2.2 共同免疫沉澱法 P39
2.3 GST pull-down P40
3. ClC-2與NSF、Carhsp 1及SMAD 1作用時可能扮演之角色 P41
4. 待解決之問題及未來目標 P42
參考文獻 P44
圖表 P53

Baolin Li, Yuan Su, John Ryder, Lei Yan, Songqing Na, Binhui Ni (2003) RIFLE: A Novel Ring Zinc Finger-Leucine-Rich Repeat Containing Protein, Regulates Select Cell Adhesion Molecules in PC12 Cells. Journal of Cellular Biochemistry 90:1224–1241
Bateman A. (1997) The structure of a domain common to archaebacteria and the homocystinuria disease protein. Trends Biochem Sci. 22(1):12-3
Blaisdell CJ, Edmonds RD, Wang XT, Guggino S, Zeitlin PL. (2000) pH-regulated Chloride secretion in fetal lung epithelia. Am. J. Physiol. Lung Cell Mol.Physiol. 278:L1248–55
Blanz, J., Schweizer, M., Auberson, M.,Maier, H., Muenscher, A., Hu¨ bner, C.A., Jentsch, T.J. (2007) Leukoencephalopathy upon disruption of the chloride channel ClC-2. J Neurosci 27:6581–6589
Bosl MR, Stein V, Hubner C, Zdebik AA, Jordt SE, Mukhophadhyay AK, Davidoff MS, Holstein AF, Jentsch TJ. (2001) Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl- channel disruption. EMBO J 20: 1289–1299
Carew MA, Thorn P (1996) Identification of ClC-2-like chloride currents in pig pancreatic acinar cells. Pflugers Arch. 433(1-2):84-90
Claus Schafer, Hanna Steffen, Karen J. Krzykowski, Burkhard Goke, Guy E. Groblewski (2003) CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 285: G726–G734
Clayton GH, Staley KJ, Wilcox CL, Owens GC, Smith RL (1998) Developmental expression of C1C-2 in the rat nervous system. Brain Res Dev Brain Res. 108(1-2):307-18
Dhani SU, Mohammad-Panah R, Ahmed N, Ackerley C, Ramjeesingh M, Bear CE. (2003) Evidence for a functional interaction between the ClC-2 chloride channel and the retrograde motor dynein complex. J Biol Chem 278:16262–16270.
Dutzler R, Campbell EB, Cadene M, Chait BT, MacKinnon R (2002) X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature 415(6869):287-94
Estévez R, Boettger T, Stein V, Birkenhäger R, Otto M, Hildebrandt F, Jentsch TJ (2001) Barttin is a Cl–-channel β-subunit crucial for renal Cl– reabsorption and inner ear K+-secretion. Nature 414:558-561.
Estévez R, Jentsch TJ (2002) CLC chloride channels: correlating structure with function. Current Opinion in Structural Biology 2002, 12:531–539
Estévez R, Pusch M, Ferrer-Costa C, Orozco M, Jentsch TJ. (2004) Functional and structural conservation of CBS domains from CLC chloride channels. J Physiol. 557(Pt 2):363-78
Fahlke C, Yu HT, Beck CL, Rhodes TH, George AL Jr (1997) Pore-forming segments in voltage-gated chloride channels. Nature 390(6659):529-32
FurukawaT, OguraT, Zheng YJ, Tsuchiya H, Nakaya H ( 2002) Phosphorylation and functional regulation of ClC-2 chloride channels expressed in Xenopus oocytes byM-cyclin-dependent protein kinase. J. Physiol. 540:883–93
Gulbins, E., A. Jekle, K. Ferlinz, H. Grassme, and F. Lang. (2000) Physiology of apoptosis. Am. J. Physiol. 279:F605–F615.
Jordt SE, Jentsch TJ. (1997) Molecular dissection of gating in the ClC-2 chloride channel. EMBO J. 16:1582–92
Haug K, Warnstedt M, Alekov AK, Sander T, Ramirez A, et al. (2003) Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies. Nat. Genet.33:527–32
Hinzpeter A, Lipecka J, Brouillard F, Baudoin-Legros M, Dadlez M, Edelman A, Fritsch J (2005) Association between Hsp90 and the ClC-2 chloride channel upregulates channel function. Am J Physiol Cell Physiol. 290(1):C42-4
Hinzpeter A, Fritsch J, Borot F, Trudel S, Vieu DL, Brouillard F, Baudouin-Legros M, Clain J, Edelman A, Ollero M (2007) Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress. J Biol Chem. 282(4):2423-32
K. Gyömörey, H. Yeger, C. Ackerly, E. Garami, C.E. Bear (2000) Expression of the chloride channel ClC-2 in the murine small intestine epithelium. Am. J. Physiol. 279 C1787–C1794
Kenyon, E., Maminishkis, A., Joseph,D.P., Miller,S.S. (1997) Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium. Am. J. Physiol., 273, C456-C472.
Kevin Strange, Francesco Emma,Paul S. Jackson (1996) Cellular and molecular physiology of volume-sensitive anion channels. Am J Physiol. 270(3 Pt 1):C711-30
Klein Gunnewiek, J.M.T., Van de Putte, L.B.A. and van Venrooij,W. J. (1997) The U1 snRNP complex: an autoantigen in connective tissue disease. Clin. Exp. Rheumatol. 15: 549–560.
Klocke R, Steinmeyer K, Jentsch TJ, Jockusch H. (1994) 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. 269(44):27635-9
Kornak U, Kasper D, Bösl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ (2001) Cell. 104(2):205-15
Lee S, Wishart MJ, Williams JA (2009) Identification of calcineurin regulated phosphorylation sites on CRHSP-24. Biochem Biophys Res Commun. 385(3):413-7
Li H, Ayer LM, Polyak MJ, Mutch CM, Petrie RJ, Gauthier L, Shariat N, Hendzel MJ, Shaw AR, Patel KD, Deans JP. (2004) The CD20 calcium channel is localized to microvilli and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism. J. Biol Chem. 279(19):19893-901
Lopes MH, Hajj GN, Muras AG, Mancini GL, Castro RM, Ribeiro KC, Brentani RR, Linden R, Martins VR (2005) Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways. Neurosci. 25(49):11330 –11339
Ludewig U, Pusch M, Jentsch TJ. (1997) Independent gating of single pores in CLC-0 chloride channels. Biophys. J. 73:789–97
Makara JK, Rappert A, Matthias K, Steinhäuser C, Spät A, Kettenmann H. (2003) Astrocytes from mouse brain slices express ClC-2-mediated Cl- currents regulated during development and after injury. Mol Cell Neurosci. 23(4):521-30
Malinowska DH, Kupert EY, Bahinski A, Sherry AM, Cuppoletti J. (1995)
Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel. Am. J. Physiol. Cell Physiol.268:C191–200
Matthias Heinze, Michael Kofler, Christian Freund (2007) Investigating the functional role of CD2BP2 in T cells. International Immunology, Vol. 19, No. 11, pp. 1313–1318
Mohammad-Panah R, Harrison R, Dhani S, Ackerley C, Huan LJ (2003) The chloride channel ClC-4 contributes to endosomal acidification and trafficking. J.Biol. Chem. 278:29267–77
Monica P, Michael D, Christoph B, Siegfried W,Florian L (2004) Serum and glucocorticoid inducible kinases functionally regulate ClC-2 channels.
Biochemical and Biophysical Research Communications 321 (2004) 1001–1006
Michael P. Keith, Chantal Moratz, George C. Tsokos (2007) Anti-RNP immunity: Implications for tissue injury and the pathogenesis of connective tissue disease. Autoimmunity Reviews 6 232–236
Najma A, Mohabir R, Simeon W, Alison V, Elizabeth G and Christine E. B (2000) Chloride channel activity of ClC-2 is modified by the actin cytoskeleton. Biochem. J. 352, 789±794
Nehrke K, Arreola J, Nguyen HV, Pilato J, Richardson L, et al. (2002) Loss of hyperpolarization-activated Cl− current in salivary acinar cells from Clcn2 knockout mice. J. Biol. Chem. 26:23604–11
Nishizawa K, Freund C, Li J, Wagner G, Reinherz EL (1998) Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation. Proc. Natl.Acad. Sci. U. S. A. 95, 14897–14902
Picollo A, Pusch M. (2005) Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436(7049):420-3
Pontier, S.M. (2006) Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy. Embo J. 25, 2698–2709.
Prashant K. Nighot, Adam J. Moeser, Kathleen A. Ryan, Troy Ghashghaei, Anthony T. Blikslager (2009) ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum. Experimental Cell Research 315 110-118
Pratt WB. (1997) The role of the hsp90-based chaperone system in signal
transduction by nuclear receptors and receptors signaling via MAP kinase.Annu Rev Pharmacol Toxicol 37: 297–326
Pusch M, Ludewig U, Rehfeldt A,Jentsch TJ.(1995) Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion. Nature 373:527–31
Pusch M, Jordt SE, Stein V, Jentsch TJ.(1999) Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol. 515:341-53
Raimund Dutzler (2007) A structural perspective on ClC channel and transporter function. FEBS Letters 581 2839–2844
Raúl Estévez, Thomas J Jentsch (2002) CLC chloride channels: correlating structure with function. Current Opinion in Structural Biology 12:531–539
Romanenko VG, Fang Y, Byfield F, Travis AJ, Vandenberg CA, Rothblat GH, Levitan I. Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels. (2004) Biophys J. 87(6):3850-61
Rutledge E, Denton J, Strange K. (2002) Cell cycle- and swelling-induced activation of a Caenorhabditis elegans ClC channel is mediated by CeGLC-7alpha/beta phosphatases. J Cell Biol 158:435–444.
Sarah Ross 1, Caroline S. Hill (2008) How the Smads regulate transcription. IJBCB 40 (2008) 383–408
S.-H. Shi, Y. Hayashi, R. S. Petralia, S. H. Zaman, R. J. Wenthold, K. Svoboda, R. Malinow (1999) Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284, 1811–1816
Sander T, Schulz H, Saar K, Gennaro E, Riggio MC, Bianchi A, Zara F, Luna D, Bulteau C, Kaminska A, Ville D, Cieuta C, Picard F, Prud''homme JF, Bate L, Sundquist A, Gardiner RM, Janssen GA, de Haan GJ, Kasteleijn-Nolst-Trenité DG, Bader A, Lindhout D, Riess O, Wienker TF, Janz D, Reis A (2000) Genome search for susceptibility loci of common idiopathic generalised epilepsies. Hum Mol Genet. 9(10):1465-72
Scheel O, Zdebik AA, Lourdel S, Jentsch TJ. (2005) Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature 21;436 (7049):424-7
Schwiebert EM, Cid-Soto LP, Stafford D, Carter M, Blaisdell CJ (1998) Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells. Proc. Natl. Acad.Sci. USA 95:3879–84
Scott JW, Hawley SA, Green KA, Anis M, Stewart G, Scullion GA, Norman DG, Hardie DG. (2004) CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J Clin Invest 113:274–284.
Sík A, Smith RL, Freund TF (2000) Distribution of chloride channel-2-immunoreactive neuronal and astrocytic processes in the hippocampus. Neuroscience. 101(1):51-65
Smith RL, Clayton GH, Wilcox CL, Escudero KW, Staley KJ (1995) Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific modulation of postsynaptic inhibition. J Neurosci 15: 4057–4067
Sollner,T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J.E.(1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324
Sonja U. Dhani, Patrick Kim Chiaw, Ling-Jun Huan, Christine E. Bear. (2007) ATP Depletion Inhibits the Endocytosis of ClC-2. J. Cell. Physiol. 214: 273–280
Thiemann A, Grunder S, PuschM,Jentsch TJ. (1992) A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356:57–60
Thomas J. Jentsch, Valentin Stein, Frank Weinreich and Anselm A. Zdebik. (2002) Molecular Structure and Physiological Function of Chloride Channels. Physiol Rev 82:503-568
Thomas J. Jentsch,Mallorie Poet, Jens C. Fuhrmann, Anselm A. Zdebik (2005) Physiological Functions Of ClC Cl− Channels Gleaned From Human Genetic Disease And Mousemidels. Annu. Rev. Physiol. 67:779–807
Thomas R Soderling (2000) CaM-kinases: modulators of synaptic plasticity
Current Opinion in Neurobiology 10:375–380
Tserentsoodol, N., Shin, B.C., Suzuki, T., Takata,K. (1998) Colocalization of tight junction proteins, occludin and ZO-1 and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye. Histochem. Cell Biol., 110, 543-551.
Vandewalle A, Cluzeaud F, Bens M, Kieferle S, Steinmeyer K, Jentsch TJ. (1997) Localization and induction by dehydration of ClC-K chloride channels in the rat kidney. Am J Physiol. 272(5 Pt 2):F678-88
Victor G. Romanenko, Tetsuji Nakamoto, Marcelo A. Catalan, Mireya Gonzalez-Begne, George J. Schwartz, Yasna Jaramillo, Francisco V. Sepu lveda, Carlos D. Figueroa, James E. Melvin (2008) Am J Physiol Gastrointest Liver Physiol 295: G1058–G1067
Weinreich F, Jentsch TJ (2001) Pores formed by single subunits in mixed dimers of different CLC chloride channels. J Biol Chem. 276(4):2347-53
Wondergem, R., W. Gong, S.H. Monen, S.N. Dooley, J.L. Gonce, T.D. Conner, M.Houser, T.W. Ecay, K.E. Ferslew. (2001) Blocking swelling-activated chloride current inhibits mouse liver cell proliferation. J. Physiol. 532:661–672.
Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE. (2001) Clathrin exchange during clathrin-mediated endocytosis. J Cell Biol 155:291–300.
X. Lu, M.Wyszynski, M. Sheng, M. Baudry (2001) Proteolysis of glutamate receptor-interacting protein by calpain in rat brain: Implications for synaptic plasticity. J. Neurochem. 77, 1553–1560
Xiong H, Li C, Garami E, Wang Y, Ramjeesingh M (1999) ClC-2 activation
modulates regulatory volume decrease. J.Membr. Biol. 167:215–21
Y. Huang, K. K. W. Wang (2001) The calpain family and human disease. Trends Mol. Med. 7, 355–362
Zheng YJ, Furukawa T, Ogura T, Tajimi K, Inagaki N (2002) M phase-specific expression and phosphorylation-dependent ubiquitination of the ClC-2 channel. J Biol Chem 277(35):32268-73