臺灣博碩士論文加值系統

電位控制鉀離子通道(voltage-gated K+ channel)的基本架構，是由四個鉀離子通道次單位(subunit)所組成的同質或異質(homotetramer or heterotetramer)四聚體。Eag鉀離子通道屬於電位控制鉀離子通道大家族之一，廣泛表現在整個中樞神經系統中。在大鼠Eag鉀離子通道次家族中包含兩個成員：rEag1與rEag2。根據之前研究指出，rEag1與rEag2在培養的海馬迴神經細胞中之次細胞分佈(subcellular localization)情形不同：rEag2主要分布在細胞本體與近端樹突；rEag1除了出現在細胞本體和近端樹突之外，還廣泛的分布於遠端樹突。此外，rEag1最特別的是在樹突上還具有點狀分布的特性。本篇論文的目的是要了解rEag1和rEag2鉀離子通道蛋白的生物化學特性，我們特別是想探究rEag1和rEag2在大鼠前腦(forebrain)及海馬迴(hippocampus)組織中形成herterotetramer和homotetramer的可能性，以及此現象是否可增加rEag鉀離子通道在次細胞分布情形的多樣性。
我們首先觀察在不同培養天數的海馬迴神經細胞上之rEag鉀離子通道分布狀況：rEag1和rEag2鉀離子通道都分布在神經細胞本體以及近端樹突，且rEag1鉀離子通道還分布到遠端樹突，並呈現斑點狀染色型態，顯示兩個鉀離子通道之次細胞層次分布的確不同。我們的溶解度分析實驗結果顯示rEag1和rEag2兩者對不同的清潔劑的溶解度有所不同：rEag1鉀離子通道在各種清潔劑中的溶解度相對高於rEag2鉀離子通道。進一步利用共同免疫沉澱的實驗，我們證實在HEK293T細胞株，rEag1與rEag2可能可以形成heterotetramer的鉀離子通道；而在大鼠大腦與海馬迴組織，rEag1與rEag2也可能有形成heterotetramer的鉀離子通道。但是進一步的分析發現有相當比例的rEag1是形成homotetramer的鉀離子通道。當我們以RT-PCR與real-time PCR偵測rEag1與rEag2 的mRNA於各腦區間的表現差異，顯示在大腦皮質處的rEag1與rEag2 mRNA含量遠高於其他腦區，且rEag1的含量高於rEag2。
綜合本研究的結果顯示rEag1與rEag2兩者在生化特性上有所差異，且彼此可形成heterotetramer或homotetramer的鉀離子通道。我們推測形成homotetramer和heterotetramer的rEag鉀離子通道的現象，可能是造成rEag1和rEag2在海馬迴神經細胞的次細胞分布不同的主要機制之一。

Functional voltage-gated potassium (K+) channel comprises the assembly of four pore-forming α-subunits in a homo- or hetero-tetrameric configuration. The ether-á-go-go (Eag) K+ channel is a member of the superfamily of voltage-gated K+ channel and broadly expressed over the entire central nervous system. There are two isoforms of Eag K+ channels in rat, rEag1 and rEag2. Previous studies from our lab have shown that rEag1 and rEag2 K+ channels display differential subcellular localization patterns in cultured hippocampal neurons. Both rEag1 and rEag2 K+ channels are expressed at soma and proximal dendrites; whereas only rEag1 channels display punctate staining patterns throughout the dendrites. The purpose of this study is to characterize the biochemical properties of rEag1 and rEag2 K+ channels. In particular, we investigated whether rEag1 and rEag2 subunits co-assemble to form heterotetrameric channels in rat forebrain and hippocampus, the result of which may shed light on the mechanism underlying the differential localization of rEag channels in neurons,
Firstly, we examined the immunoreactivities of rEag K+ channels in cultured hippocampal neurons that were subjected to different days in vitro. All neurons studied revealed a similar pattern of differential localization of rEag1 and rEag2 K+ channels. We then applied a variety of different detergents in rat brain homogenates to examine the relative solubility of rEag1 and rEag2 proteins. For the majority of the detergents used, the solubility of rEag1 subunits was significantly greater than that of rEag2. Furthermore, co-immunoprecipitation experiments showed that rEag1 and rEag2 K+ channels can be co-sedimented in cell lysates prepared from transfected HEK293T cells, forebrain and hippocampus, suggesting the formation of heterotetrameric K+ channels. Nevertheless, a considerable portion of rEag1 subunits were found to be homotetramers. Finally, RT-PCR and real-time PCR analyses of rEag1 and rEag2 mRNA in different brain regions suggested that the expression level of rEag1 subunit was significantly higher than that of rEag2 in most of the brain area.
In conclusion, our results indicate that rEag1 and rEag2 K+ channels exhibit different lipid-solubility, suggesting that they may be preferentially localized at distinct membrane microdomains. Moreover, in neurons rEag1 and rEag2 subunits may exist as either hetero- or homo-tetrameric K+ channels. We therefore propose that the differential tetrameric assembly configuration of rEag isomers may contribute to the subcellular localization pattern of rEag1 and rEag2 K+ channels.

圖次………………………………………………………… 2

中文摘要…………………………………………………… 3

英文摘要…………………………………………………… 5

導論………………………………………………………… 7

材料與方法………………………………………………… 18

結果………………………………………………………… 29

討論………………………………………………………… 37

結論………………………………………………………… 44

圖片與圖片說明…………………………………………… 45

參考文獻…………………………………………………… 57

Armstrong CM, Hille B (1998) Voltage-gated ion channels and electrical excitability. Neuron 20:371-380.
Arnold DB (2007) Polarized targeting of ion channels in neurons. Pflugers Arch 453:763-769.
Bairoch A (1991) PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res 19 Suppl:2241-2245.
Bairoch A (1992) PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res 20 Suppl:2013-2018.
Bauer CK, Schwarz JR (2001) Physiology of EAG K+ channels. J Membr Biol 182:1-15.
Bauer CK, Engeland B, Wulfsen I, Ludwig J, Pongs O, Schwarz JR (1998) RERG is a molecular correlate of the inward-rectifying K current in clonal rat pituitary cells. Receptors Channels 6:19-29.
Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, Steinlein OK (1998) A potassium channel mutation in neonatal human epilepsy. Science 279:403-406.
Broughton SJ, Kitamoto T, Greenspan RJ (2004) Excitatory and inhibitory switches for courtship in the brain of Drosophila melanogaster. Curr Biol 14:538-547.
Camacho J (2006) Ether a go-go potassium channels and cancer. Cancer Lett 233:1-9.
Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD (2004) K+ channels as targets for specific immunomodulation. Trends Pharmacol Sci 25:280-289.
Chung JJ, Li M (2005) Biochemical characterization of the native Kv2.1 potassium channel. Febs J 272:3743-3755.
Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B (1999) Molecular diversity of K+ channels. Ann N Y Acad Sci 868:233-285.
Demo SD, Yellen G (1991) The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker. Neuron 7:743-753.
Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. J Neurosci 8:1454-1468.
Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69-77.
Edward C. Cooper KDA, Aviva Abosch, Nicholas M. Barbaro, Mitchel S. Berger,, Warwick S. Peacock YNJ, and Lily Yeh Jan (2000) Colocalization and coassembly of two human brain M-type potassium channel subunits that are mutated in epilepsy. Proc Natl Acad Sci U S A vol. 97:4914–4919.
Engeland B, Neu A, Ludwig J, Roeper J, Pongs O (1998) Cloning and functional expression of rat ether-a-go-go-like K+ channel genes. J Physiol 513 ( Pt 3):647-654.
Farrant M, Feldmeyer D, Takahashi T, Cull-Candy SG (1994) NMDA-receptor channel diversity in the developing cerebellum. Nature 368:335-339.
Fletcher TL, Banker GA (1989) The establishment of polarity by hippocampal neurons: the relationship between the stage of a cell's development in situ and its subsequent development in culture. Dev Biol 136:446-454.
Fletcher TL, De Camilli P, Banker G (1994) Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development. J Neurosci 14:6695-6706.
Flynn GE, Johnson JP, Jr., Zagotta WN (2001) Cyclic nucleotide-gated channels: shedding light on the opening of a channel pore. Nat Rev Neurosci 2:643-651.
Ganetzky B, Wu CF (1983) Neurogenetic analysis of potassium currents in Drosophila: synergistic effects on neuromuscular transmission in double mutants. J Neurogenet 1:17-28.
Ganetzky B, Robertson GA, Wilson GF, Trudeau MC, Titus SA (1999) The eag family of K+ channels in Drosophila and mammals. Ann N Y Acad Sci 868:356-369.
Gavrilova-Ruch O, Schonherr K, Gessner G, Schonherr R, Klapperstuck T, Wohlrab W, Heinemann SH (2002) Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells. J Membr Biol 188:137-149.
Gilles-Gonzalez MA, Gonzalez G (2004) Signal transduction by heme-containing PAS-domain proteins. J Appl Physiol 96:774-783.
Gu C, Jan YN, Jan LY (2003) A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301:646-649.
Guy HR, Seetharamulu P (1986) Molecular model of the action potential sodium channel. Proc Natl Acad Sci U S A 83:508-512.
Guy HR, Durell SR, Warmke J, Drysdale R, Ganetzky B (1991) Similarities in amino acid sequences of Drosophila eag and cyclic nucleotide-gated channels. Science 254:730.
Hegle AP, Marble DD, Wilson GF (2006) A voltage-driven switch for ion-independent signaling by ether-a-go-go K+ channels. Proc Natl Acad Sci U S A 103:2886-2891.
Hille B (2001) Ion Channels of Excitable Membranes.814.
Hodgkin AL, Huxley AF (1945) Resting and action potentials in single nerve fibres. J Physiol 104:176-195.
Hoglund M, Siden T, Rohme D (1992) The isolation of evolutionarily conserved Eag I end-clones from mouse chromosome 17 using cloned DNA. DNA Cell Biol 11:613-619.
Holmes TC, Berman K, Swartz JE, Dagan D, Levitan IB (1997) Expression of voltage-gated potassium channels decreases cellular protein tyrosine phosphorylation. J Neurosci 17:8964-8974.
Hoshi T, Zagotta WN, Aldrich RW (1990) Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250:533-538.
Huang K, El-Husseini A (2005) Modulation of neuronal protein trafficking and function by palmitoylation. Curr Opin Neurobiol 15:527-535.
Jan LY, Jan YN (1997) Cloned potassium channels from eukaryotes and prokaryotes. Annu Rev Neurosci 20:91-123.
Jeng CJ, Chang CC, Tang CY (2005) Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons. Neuroreport 16:229-233.
Jenke M, Sanchez A, Monje F, Stuhmer W, Weseloh RM, Pardo LA (2003) C-terminal domains implicated in the functional surface expression of potassium channels. Embo J 22:395-403.
Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R (2002) The open pore conformation of potassium channels. Nature 417:523-526.
Johnston D, Christie BR, Frick A, Gray R, Hoffman DA, Schexnayder LK, Watanabe S, Yuan LL (2003) Active dendrites, potassium channels and synaptic plasticity. Philos Trans R Soc Lond B Biol Sci 358:667-674.
Jow GM, Jeng CJ (2008) Differential localization of rat Eag1 and Eag2 potassium channels in the retina. Neurosci Lett 431:12-16.
Kaczmarek LK (2006) Non-conducting functions of voltage-gated ion channels. Nat Rev Neurosci 7:761-771.
Kaplan WD, Trout WE, 3rd (1969) The behavior of four neurological mutants of Drosophila. Genetics 61:399-409.
Kistner U, Wenzel BM, Veh RW, Cases-Langhoff C, Garner AM, Appeltauer U, Voss B, Gundelfinger ED, Garner CC (1993) SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A. J Biol Chem 268:4580-4583.
Knaus HG, Garcia-Calvo M, Kaczorowski GJ, Garcia ML (1994) Subunit composition of the high conductance calcium-activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels. J Biol Chem 269:3921-3924.
Lai HC, Jan LY (2006) The distribution and targeting of neuronal voltage-gated ion channels. Nat Rev Neurosci 7:548-562.
Lastraioli E, Guasti L, Crociani O, Polvani S, Hofmann G, Witchel H, Bencini L, Calistri M, Messerini L, Scatizzi M, Moretti R, Wanke E, Olivotto M, Mugnai G, Arcangeli A (2004) herg1 gene and HERG1 protein are overexpressed in colorectal cancers and regulate cell invasion of tumor cells. Cancer Res 64:606-611.
Lesage F, Reyes R, Fink M, Duprat F, Guillemare E, Lazdunski M (1996) Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge. Embo J 15:6400-6407.
Lim ST, Antonucci DE, Scannevin RH, Trimmer JS (2000) A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons. Neuron 25:385-397.
Ludwig J, Owen D, Pongs O (1997) Carboxy-terminal domain mediates assembly of the voltage-gated rat ether-a-go-go potassium channel. Embo J 16:6337-6345.
Ludwig J, Terlau H, Wunder F, Bruggemann A, Pardo LA, Marquardt A, Stuhmer W, Pongs O (1994) Functional expression of a rat homologue of the voltage gated either a go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart. Embo J 13:4451-4458.
Ludwig J, Weseloh R, Karschin C, Liu Q, Netzer R, Engeland B, Stansfeld C, Pongs O (2000) Cloning and functional expression of rat eag2, a new member of the ether-a-go-go family of potassium channels and comparison of its distribution with that of eag1. Mol Cell Neurosci 16:59-70.
MacLean JN, Zhang Y, Johnson BR, Harris-Warrick RM (2003) Activity-independent homeostasis in rhythmically active neurons. Neuron 37:109-120.
Martin S, Lino de Oliveira C, Mello de Queiroz F, Pardo LA, Stuhmer W, Del Bel E (2008) Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain. Neuroscience 155:833-844.
Migliore M, Shepherd GM (2002) Emerging rules for the distributions of active dendritic conductances. Nat Rev Neurosci 3:362-370.
Morais Cabral JH, Lee A, Cohen SL, Chait BT, Li M, Mackinnon R (1998) Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain. Cell 95:649-655.
Napp J, Monje F, Stuhmer W, Pardo LA (2005) Glycosylation of Eag1 (Kv10.1) potassium channels: intracellular trafficking and functional consequences. J Biol Chem 280:29506-29512.
Occhiodoro T, Bernheim L, Liu JH, Bijlenga P, Sinnreich M, Bader CR, Fischer-Lougheed J (1998) Cloning of a human ether-a-go-go potassium channel expressed in myoblasts at the onset of fusion. FEBS Lett 434:177-182.
Pardo LA (2004) Voltage-gated potassium channels in cell proliferation. Physiology (Bethesda) 19:285-292.
Pardo LA, Contreras-Jurado C, Zientkowska M, Alves F, Stuhmer W (2005) Role of voltage-gated potassium channels in cancer. J Membr Biol 205:115-124.
Phartiyal P, Jones EM, Robertson GA (2007) Heteromeric assembly of human ether-a-go-go-related gene (hERG) 1a/1b channels occurs cotranslationally via N-terminal interactions. J Biol Chem 282:9874-9882.
Piros ET, Shen L, Huang XY (1999) Purification of an EH domain-binding protein from rat brain that modulates the gating of the rat ether-a-go-go channel. J Biol Chem 274:33677-33683.
Ponting CP, Aravind L (1997) PAS: a multifunctional domain family comes to light. Curr Biol 7:R674-677.
Preisig-Muller R, Schlichthorl G, Goerge T, Heinen S, Bruggemann A, Rajan S, Derst C, Veh RW, Daut J (2002) Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome. Proc Natl Acad Sci U S A 99:7774-7779.
Rivera JF, Chu PJ, Arnold DB (2005) The T1 domain of Kv1.3 mediates intracellular targeting to axons. Eur J Neurosci 22:1853-1862.
Rivera JF, Ahmad S, Quick MW, Liman ER, Arnold DB (2003) An evolutionarily conserved dileucine motif in Shal K+ channels mediates dendritic targeting. Nat Neurosci 6:243-250.
Roux B, MacKinnon R (1999) The cavity and pore helices in the KcsA K+ channel: electrostatic stabilization of monovalent cations. Science 285:100-102.
Saganich MJ, Machado E, Rudy B (2001) Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain. J Neurosci 21:4609-4624.
Saganich MJ, Vega-Saenz de Miera E, Nadal MS, Baker H, Coetzee WA, Rudy B (1999) Cloning of components of a novel subthreshold-activating K(+) channel with a unique pattern of expression in the cerebral cortex. J Neurosci 19:10789-10802.
Schonherr R, Gessner G, Lober K, Heinemann SH (2002) Functional distinction of human EAG1 and EAG2 potassium channels. FEBS Lett 514:204-208.
Schwalbe RA, Wang Z, Wible BA, Brown AM (1995) Potassium channel structure and function as reported by a single glycosylation sequon. J Biol Chem 270:15336-15340.
Shi W, Wymore RS, Wang HS, Pan Z, Cohen IS, McKinnon D, Dixon JE (1997) Identification of two nervous system-specific members of the erg potassium channel gene family. J Neurosci 17:9423-9432.
Shi W, Wang HS, Pan Z, Wymore RS, Cohen IS, McKinnon D, Dixon JE (1998) Cloning of a mammalian elk potassium channel gene and EAG mRNA distribution in rat sympathetic ganglia. J Physiol 511 ( Pt 3):675-682.
Stuhmer W, Alves F, Hartung F, Zientkowska M, Pardo LA (2006) Potassium channels as tumour markers. FEBS Lett 580:2850-2852.
Sun XX, Hodge JJ, Zhou Y, Nguyen M, Griffith LC (2004) The eag potassium channel binds and locally activates calcium/calmodulin-dependent protein kinase II. J Biol Chem 279:10206-10214.
Torres YP, Morera FJ, Carvacho I, Latorre R (2007) A marriage of convenience: beta-subunits and voltage-dependent K+ channels. J Biol Chem 282:24485-24489.
Wang Z, Wilson GF, Griffith LC (2002) Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel. J Biol Chem 277:24022-24029.
Warmke J, Drysdale R, Ganetzky B (1991) A distinct potassium channel polypeptide encoded by the Drosophila eag locus. Science 252:1560-1562.
Watanabe I, Zhu J, Recio-Pinto E, Thornhill WB (2004) Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking. J Biol Chem 279:8879-8885.
Watanabe I, Wang HG, Sutachan JJ, Zhu J, Recio-Pinto E, Thornhill WB (2003) Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism. J Physiol 550:51-66.
Whitaker GM, Angoli D, Nazzari H, Shigemoto R, Accili EA (2007) HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels. J Biol Chem 282:22900-22909.
Xu J, Yu W, Jan YN, Jan LY, Li M (1995) Assembly of voltage-gated potassium channels. Conserved hydrophilic motifs determine subfamily-specific interactions between the alpha-subunits. J Biol Chem 270:24761-24768.
Xu M, Cao R, Xiao R, Zhu MX, Gu C (2007) The axon-dendrite targeting of Kv3 (Shaw) channels is determined by a targeting motif that associates with the T1 domain and ankyrin G. J Neurosci 27:14158-14170.
Yellen G (2002) The voltage-gated potassium channels and their relatives. Nature 419:35-42.
Zhang L, Foster K, Li Q, Martens JR (2007) S-acylation regulates Kv1.5 channel surface expression. Am J Physiol Cell Physiol 293:C152-161.
Zhou M, Morais-Cabral JH, Mann S, MacKinnon R (2001) Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature 411:657-661.
Zhu J, Recio-Pinto E, Hartwig T, Sellers W, Yan J, Thornhill WB (2008) The Kv1.2 potassium channel: The position of an N-glycan on the extracellular linkers affects its protein expression and function. Brain Res.