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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20346
標題: 高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T鉀離子通道蛋白 基因選殖、異源表現與特性分析
Cloning, Expression, and Functional Characterization of Potassium Channel from Methanohalophilus portucalensis FDF1T
作者: 王俊文
Wang, Jiun-Wen
關鍵字: 太古生物;Archaea;高鹽甲烷菌;鉀離子通道;膜電壓感應器;Halophilic methanogen;Potassium channel;Voltage sensor
出版社: 生命科學系所
引用: 1. 賴美津。1995。極端嗜鹽菌分離純化與特性分析及探討其在生物科技上發展的潛力。行政院國科會專題研究計畫成果報告。 2. 王家麒。2002。受質專一性和鉀離子濃度對嗜鹽甲烷古生菌相容質甜菜鹼生合成酵素的影響。國立中興大學植物系碩士論文。 3. 丁俊彥。2011。極端高鹽太古生物之分類鑑定、比較基因體分析及其生物科技應用潛力。國立中興大學生命科學系博士論文。 4. 賴姝蓉。2011。高鹽甲烷太古生物之相容質甜菜鹼自體生合成酵素特性分析並探討其應用於模式生物阿拉伯芥以及斑馬魚抗鹽抗旱的可行性。國立中興大學生命科學系博士論文。 5. 吳靜儀。2011。極端高鹽太古生物鉀離子通道蛋白基因的選殖與特性分析。國立中興大學生命科學系碩士論文。 6. 洪娟娟。2012。甲烷太古生物之相容質Nε-acetyl-β-lysine 自體生合成基因以及酵素特性分析。國立中興大學生命科學系博士論文。 7. Alam, A., and Y. Jiang. 2011. Structural studies of ion selectivity in tetrameric cation channels. J. Gen. Physiol. 137:397-403. 8. Amann, E., B. Ochsa and K. J. Abel. Tightly regulated tac promoter vectors useful for the expression of unfused proteins in Escherichia coli. Gene. 69:301-15 9. Asha, H., and J. Gowrishankar. 1993. Regulation of kdp operon expression in Escherichia coli: evidence against turgor as signal for transcriptional control. J. Bacteriol. 175:4528-37. 10. Ashley, M. K., M. Grant, and A. Grabov. 2006. Plant responses to potassium deficiencies: a role for potassium transport proteins. J. Exp. Bot. 57:425-36. 11. Ballal, A., B. Basu, and S. K. Apte. 2007. The Kdp-ATPase system and its regulation. J. Biosci. 32:559-68. 12. Boone, D. R., I. M. Mathrani, M. Y. Liu, J. A. D. F. Menaia, R. A. Mah and J. E. Boone. 1993. Isolation and characterization study of the genus Methanohalophilus. Int. J. Sys. Bact. 43:430-37 13. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49:359-78. 14. Choe, S. 2002. Potassium channel structures. Nat. Rev. Neurosci. 3:115-21. 15. Choe, S., and T. Roosild. 2002. Regulation of the K channels by cytoplasmic domains. Biopolymers. 66:294-9. 16. Corratge-Faillie, C., M. Jabnoune, S. Zimmermann, A. A. Very, C. Fizames, and H. Sentenac. 2010. Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family. Cell Mol. Life. Sci. 67:2511-32. 17. Corvini, P. F., H. Gautier, E. Rondags, H. Vivier, J. L. Goergen, and P. Germain. 2000. Intracellular pH determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis. Microbiology. 146 (Pt 10):2671-8. 18. Cuello, L. G., J. G. Romero, D. M. Cortes, and E. Perozo. 1998. pH-dependent gating in the Streptomyces lividans K+ channel. Biochemistry. 37:3229-36. 19. Dosch, D. C., G. L. Helmer, S. H. Sutton, F. F. Salvacion, and W. Epstein. 1991. Genetic analysis of potassium transport loci in Escherichia coli: evidence for three constitutive systems mediating uptake potassium. J. Bacteriol. 173:687-96. 20. Doyle, D. A., J. Morais Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280:69-77. 21. Durell, S. R., E. P. Bakker, and H. R. Guy. 2000. Does the KdpA subunit from the high affinity K(+)-translocating P-type KDP-ATPase have a structure similar to that of K(+) channels? Biophys. J. 78:188-99. 22. Durell, S. R., Y. Hao, T. Nakamura, E. P. Bakker, and H. R. Guy. 1999. Evolutionary relationship between K(+) channels and symporters. Biophys. J. 77:775-88. 23. Dvir, H., E. Valera, and S. Choe. 2010. Structure of the MthK RCK in complex with cadmium. J. Struct. Biol. 171:231-7. 24. Empadinhas, N., and M. S. da Costa. 2008. Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes. Int. Microbiol. 11:151-61. 25. Epstein, W. 2003. The roles and regulation of potassium in bacteria. Prog. Nucleic Acid Res. Mol. Biol. 75:293-320. 26. Epstein, W., and S. G. Schultz. 1965. Cation Transport in Escherichia coli: V. Regulation of cation content. J. Gen. Physiol. 49:221-34. 27. Gassel, M., T. Mollenkamp, W. Puppe, and K. Altendorf. 1999. The KdpF subunit is part of the K(+)-translocating Kdp complex of Escherichia coli and is responsible for stabilization of the complex in vitro. J. Biol. Chem. 274:37901-7. 28. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-80. 29. Haynes, W. J., K. Y. Ling, Y. Saimi, and C. Kung. 2003. PAK paradox: Paramecium appears to have more K(+)-channel genes than humans. Eukaryot Cell. 2:737-45. 30. Heginbotham, L., T. Abramson, and R. MacKinnon. 1992. A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels. Science. 258:1152-5. 31. Heginbotham, L., Z. Lu, T. Abramson, and R. MacKinnon. 1994. Mutations in the K+ channel signature sequence. Biophys. J. 66:1061-7. 32. Jiang, Y., A. Lee, J. Chen, M. Cadene, B. T. Chait, and R. MacKinnon. 2002. Crystal structure and mechanism of a calcium-gated potassium channel. Nature. 417:515-22. 33. Jiang, Y., A. Lee, J. Chen, V. Ruta, M. Cadene, B. T. Chait, and R. MacKinnon. 2003. X-ray structure of a voltage-dependent K+ channel. Nature.423:33-41. 34. Kroning, N., M. Willenborg, N. Tholema, I. Hanelt, R. Schmid, and E. P. Bakker. 2007. ATP binding to the KTN/RCK subunit KtrA from the K+ -uptake system KtrAB of Vibrio alginolyticus: its role in the formation of the KtrAB complex and its requirement in vivo. J. Biol. Chem. 282:14018-27. 35. Kung, C., and P. Blount. 2004. Channels in microbes: so many holes to fill. Mol. Microbiol. 53:373-80. 36. Kuo, M. M., K. A. Baker, L. Wong, and S. Choe. 2007. Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity. Proc. Natl. Acad. Sci. U S A. 104:2151-6. 37. Kuo, M. M., W. J. Haynes, S. H. Loukin, C. Kung, and Y. Saimi. 2005. Prokaryotic K(+) channels: from crystal structures to diversity. FEMS Microbiol Rev. 29:961-85. 38. Kuo, M. M., Y. Saimi, and C. Kung. 2003. Gain-of-function mutations indicate that Escherichia coli Kch forms a functional K+ conduit in vivo. EMBO J. 22:4049-58. 39. Lai, M. C., and R. P. Gunsalus. 1992. Glycine betaine and potassium ion are the major compatible solutes in the extremely halophilic methanogen Methanohalophilus strain Z7302. J. Bacteriol. 174:7474-7. 40. Lai, M. C., K. R. Sowers, D. E. Robertson, M. F. Roberts, and R. P. Gunsalus. 1991. Distribution of compatible solutes in the halophilic methanogenic archaebacteria. J. Bacteriol. 173:5352-8. 41. Lai, M. C., D. R. Yang and M. J. Chuang. 1999. Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betasine in the Halophilic Methanoarchaeon Methanohalophilus portucalensis. Appl. Envi. Microbiol. 65:828-33 42. Lai, M. C., C. C. Wang, M. J. Chuang, Y. C. Wu, Y. C. Lee. 2006. Effects of substrate and potassium on betaine- synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase from a halophilic methanoarchaeon Methanohalophilus portucalensis. Res. Microbiol. 157:948-55 43. Lai, S. J., and M. C. Lai. 2011. Characterization and regulation of the osmolyte betaine synthesizing enzymes GSMT and SDMT from halophilic methanogen Methanohalophilus portucalensis. PLoS One. 6:e25090. 44. Lee, S. Y., A. Lee, J. Chen, and R. MacKinnon. 2005. Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane. Proc. Natl. Acad. Sci. U S A. 102:15441-6. 45. Li, Y., I. Berke, L. Chen, and Y. Jiang. 2007. Gating and inward rectifying properties of the MthK K+ channel with and without the gating ring. J. Gen. Physiol. 129:109-20. 46. Loukin, S. H., M. M. Kuo, X. L. Zhou, W. J. Haynes, C. Kung, and Y. Saimi. 2005. Microbial K+ channels. J. Gen. Physiol. 125:521-7. 47. Martinac, B., Y. Saimi, and C. Kung. 2008. Ion channels in microbes. Physiol. Rev. 88:1449-90. 48. Mathrani, I. M., and D. R. Boone. 1985. Isolation and characterization of a moderately halophilic methanogen from a solar saltern. Appl. Environ. Microbiol. 50:140-3. 49. Matsuda, N., H. Kobayashi, H. Katoh, T. Ogawa, L. Futatsugi, T. Nakamura, E. P. Bakker, and N. Uozumi. 2004. Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock. J. Biol. Chem. 279:54952-62. 50. Milkman, R. 1994. An Escherichia coli homologue of eukaryotic potassium channel proteins. Proc. Natl. Acad. Sci. U S A. 91:3510-4. 51. Morais-Cabral, J. H., Y. Zhou, and R. MacKinnon. 2001. Energetic optimization of ion conduction rate by the K+ selectivity filter. Nature. 414:37-42. 52. Moss, A. J., P. J. Schwartz, R. S. Crampton, E. Locati, and E. Carleen. 1985. The long QT syndrome: a prospective international study. Circulation. 71:17-21. 53. Munsey, T. S., A. Mohindra, S. P. Yusaf, A. Grainge, M. H. Wang, D. Wray, and A. Sivaprasadarao. 2002. Functional properties of Kch, a prokaryotic homologue of eukaryotic potassium channels. Biochem. Biophys. Res. Commun. 297:10-6. 54. Oren, A. 2002. Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J. Ind. Microbiol Biotechnol. 28:56-63. 55. Oren, A. 2008. Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Systems. 4:2. 56. Parfenova, L. V., B. M. Crane, and B. S. Rothberg. 2006. Modulation of MthK potassium channel activity at the intracellular entrance to the pore. J. Biol. Chem. 281:21131-8. 57. Polarek, J. W., G. Williams, and W. Epstein. 1992. The products of the kdpDE operon are required for expression of the Kdp ATPase of Escherichia coli. J. Bacteriol. 174:2145-51. 58. Prince, W. S., and M. R. Villarejo. 1990. Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex. J. Biol. Chem. 265:17673-9. 59. Rhoads, D. B., F. B. Waters, and W. Epstein. 1976. Cation transport in Escherichia coli. VIII. Potassium transport mutants. J. Gen. Physiol. 67:325-41. 60. Richey, B., D. S. Cayley, M. C. Mossing, C. Kolka, C. F. Anderson, T. C. Farrar, and M. T. Record, Jr. 1987. Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression. J. Biol. Chem. 262:7157-64. 61. Roberts, M. F., M. C. Lai, and R. P. Gunsalus. 1992. Biosynthetic pathways of the osmolytes Nε-acetyl-β-lysine, b-glutamine, and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses. J. Bacteriol. 174:6688-93. 62. Rodriguez-Navarro, A. 2000. Potassium transport in fungi and plants. Biochim Biophys. Acta. 1469:1-30. 63. Roosild, T. P., S. Miller, I. R. Booth, and S. Choe. 2002. A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch. Cell. 109:781-91. 64. Ruta, V., Y. Jiang, A. Lee, J. Chen, and R. MacKinnon. 2003. Functional analysis of an archaebacterial voltage-dependent K+ channel. Nature. 422:180-5. 65. Schlosser, A., S. Kluttig, A. Hamann, and E. P. Bakker. 1991. Subcloning, nucleotide sequence, and expression of trkG, a gene that encodes an integral membrane protein involved in potassium uptake via the Trk system of Escherichia coli. J. Bacteriol. 173:3170-6. 66. Schlosser, A., M. Meldorf, S. Stumpe, E. P. Bakker, and W. Epstein. 1995. TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli. J. Bacteriol. 177:1908-10. 67. Schrempf, H. 2005. Deciphering Streptomyces lividans KcsA as a K Channel Model, p41-67. In Andrzej, K and B. Martinac (ed.), Baterial Ion Channels and their Eukaryotic Homologs. vol. 3. ASM Press, Washington, D.C. 68. Schultz, S. G., and A. K. Solomon. 1961. Cation transport in Escherichia coli. I. Intracellular Na+ and K+ concentrations and net cation movement. J. Gen. Physiol. 45:355-69. 69. Seoh, S. A., D. Sigg, D. M. Papazian, and F. Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron. 16:1159-67. 70. Sesti, F., S. Rajan, R. Gonzalez-Colaso, N. Nikolaeva, and S. A. Goldstein. 2003. Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel. Nat. Neurosci. 6:353-61. 71. Shi, N., W. Zeng, S. Ye, Y. Li, and Y. Jiang. 2011. Crucial points within the pore as determinants of K(+) channel conductance and gating. J. Mol. Biol. 411:27-35. 72. Sleator, R. D., and C. Hill. 2002. Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS Microbiol. Rev. 26:49-71. 73. Stumpe, S., and E. P. Bakker. 1997. Requirement of a large K+-uptake capacity and of extracytoplasmic protease activity for protamine resistance of Escherichia coli. Arch. Microbiol. 167:126-36. 74. Suelter, C. H. 1970. Enzymes activated by monovalent cations. Science. 168:789-95. 75. Tholema, N., E. P. Bakker, A. Suzuki, and T. Nakamura. 1999. Change to alanine of one out of four selectivity filter glycines in KtrB causes a two orders of magnitude decrease in the affinities for both K+ and Na+ of the Na+ dependent K+ uptake system KtrAB from Vibrio alginolyticus. FEBS Lett. 450:217-20. 76. Thompson, A. N., D. J. Posson, P. V. Parsa, and C. M. Nimigean. 2008. Molecular mechanism of pH sensing in KcsA potassium channels. Proc. Natl. Acad. Sci. U S A. 105:6900-5. 77. Trchounian, A., and H. Kobayashi. 1999. Kup is the major K+ uptake system in Escherichia coli upon hyper-osmotic stress at a low pH. FEBS Lett. 447:144-8. 78. Ungar, D., A. Barth, W. Haase, A. Kaunzinger, E. Lewitzki, T. Ruiz, H. Reilander, and H. Michel. 2001. Analysis of a putative voltage-gated prokaryotic potassium channel. Eur. J. Biochem. 268:5386-96. 79. Ye, S., Y. Li, L. Chen, and Y. Jiang. 2006. Crystal structures of a ligand-free MthK gating ring: insights into the ligand gating mechanism of K+ channels. Cell. 126:1161-73. 80. Zhou, Y., J. H. Morais-Cabral, A. Kaufman, and R. MacKinnon. 2001. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature. 414:43-8. 81. Zulkifli, L., M. Akai, A. Yoshikawa, M. Shimojima, H. Ohta, H. R. Guy, and N. Uozumi. 2010. The KtrA and KtrE subunits are required for Na+-dependent K+ uptake by KtrB across the plasma membrane in Synechocystis sp. strain PCC 6803. J Bacteriol 192:5063-70.
摘要: 
鉀離子是細胞內最主要的陽離子，並且在滲透壓調控以及酸鹼平衡中扮演著重要的角色。鉀離子通道可快速運送鉀離子，每秒達107個鉀離子。生長在高達2.9 M鹽濃度環境的高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T可在細胞內累積1.13 M濃度的鉀離子以及低分子量的有機分子作為相容質抵抗外界鹽濃度壓力。本研究利用M. portucalensis FDF1T基因解序所得到的序列獲得完整的鉀離子通道基因(kvmp)全長。TOPCONS分析KvMp二級結構具有六個穿膜片段，多重胺基酸序列比對顯示KvMp具有典型的Voltage sensor(RXXRXXRXXK) 以及selectivity filter(TXGYGD)之保守序列。將高鹽太古生物的kvmp轉殖到鉀離子運輸缺陷的E. coli LB2003，表現KvMp的轉殖株可生長於在低鉀離子KML(添加0, 1 ,10 mM) 培養基，顯示KvMp蛋白具有鉀離子通道的功能。證實甲烷太古生物的鉀離子通道可以互補細菌的鉀離子運送能力。以Escherichia coli BL21(DE3) RIL異源表現膜蛋白KvMp，利用超高速離心以及界面活性劑dodecyl-β-D-maltoside可成功萃取約30 % 膜蛋白KvMp，將來可利用size exclusion chromatography純化KvMp蛋白並構築於脂質體(liposome)，以分析KvMp蛋白in vitro活性。此研究是首次探討高鹽甲烷太古生物鉀離子通道。

Potassium is the major cation in all cells and plays a role in osmotic regulation and pH homeostasis. Potassium channel is a rapid potassium transport system to maintain the intracellular potassium concentration. Halophilic methanoarchaeon Methano- halophilus portucalensis FDF1T could grow at environment with salinity up to 2.9 M NaCl by accumulating 1.13 M of potassium ions and organic solutes as osmoprotectants. In this study, the complete gene of potassium channel (kvmp) was identified by metagenomic pyrosequencing. The secondary structure prediction revealed that KvMp consists of six transmembrane helices (TM) that is surrounded with the selectivity filter. In addition, the conserved positive charge amino acids (RxxRxxRxxRxxK) as voltage sensor in the fourth TM suggested that KvMp is a voltage activated potassium channel. In vivo activity of KvMp was demonstrated by the heterologous expression of kvmp gene in K+ uptake deficient mutant E. coli LB2003, which could not grow at low potassium condition (<10 mM). Campare with potassium transport deficient mutant E. coli LB2003, kvmp containing E. coli LB2003 could grow in low potassium (0, 1, 10 mM) environment. This result confirmed that KvMp is a potassium channel and archaeal potassium channel could complement the K+ transport activity in E. coli. In addition, the recombinant KvMp were heterologously expressed in E. coli BL21 (DE3) RIL. The membrane fraction of recombinant KvMp was obtained through ultracentrifugation and was further extracted by dodecyl-β-D-maltoside. The soluble KvMp were partially purified by Ni2+-NTA affinity chromatography. This is the first study of the voltage potassium channel in halophilic methanoarchaea.
URI: http://hdl.handle.net/11455/20346
其他識別: U0005-2408201211483200
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