大鼠Eag鉀離子通道次細胞分佈差異之結構性基礎

Structural Basis of the Differential Subcellular Localization of Rat Eag K+ Channels

10.6342/NTU.2006.02036

林惠敏

Eag ； 鉀離子通道 ； Eag ； potassium channel

臺灣大學生理學研究所學位論文

2006年

碩士

湯志永

繁體中文

EAG鉀離子通道屬於電位控制鉀離子通道大家族之一，而根據胺基酸序列之相異性，又可細分為eag、erg、elk三種次家族。在哺乳動物中eag次家族成員包含Eag1以及Eag2鉀離子通道，此二離子通道主要分佈於中樞神經系統中。本實驗室先前針對大鼠Eag1（rEag1）與Eag2（rEag2）在海馬迴神經細胞中之次細胞分佈情形的研究結果顯示，rEag1主要分佈於細胞本體、近端樹突、及遠端樹突；而rEag2則主要分佈於細胞本體及近端樹突。此外rEag1在樹突上具有明顯的點狀（cluster）分佈，由於這些點狀分部與突觸標地（marker）具有colocalization之現象，顯示rEag1鉀離子通道還分佈於神經突觸之位置。 本篇論文之目的為探討rEag1與rEag2此二離子通道在神經細胞中的次細胞分佈狀況不同的分子機制為何。由胺基酸序列可知，rEag1與rEag2在core region之extracellular loops中，所包含之N-linked glycosylation sites有所不同，其中rEag1在胺基酸序列中388及406兩個位置具有glycosylation sites，而在rEag2中此相對位置不具glycosylation consensus sequence。另外，rEag1與rEag2之胺基酸序列，其最大差異處在於C端。因此綜合以上，我們首先要驗證之假設為rEag1在胺基酸序列388或406 glycosylation pattern可能與rEag1與rEag2在神經細胞之次細胞分佈之差異有關。第二，我們也將驗證決定rEag1與rEag2之targeting的差異亦可能在於C端。再者，內源性鉀離子通道中，同一次家族成員彼此間可能形成hetero-tetramer，過去研究證實rEag1與rEag2在大鼠中樞神經系統中之分佈確實具有部分重疊性，因此我們將驗證之假設三為，若rEag1與rEag2在in vivo當中能夠彼此聚合，當rEag1與rEag2 subunits共同形成四聚體時，能由某方主導次細胞之分佈位置。 在實驗方式上，我們利用GFP tagged之rEag1（GFP-rEag1）於388、406此二位置作點突變，使之廢除glycosylation sites，再將此二mutant transfect到培養之神經細胞當中，利用confocal image觀察其在神經細胞中之subcellular localization。接者為證實是否為C端決定targeting之所在，我們將GFP-rEag1與GFP-rEag2之C端序列作交換，使產生reag2-chimera 1、reag2-chimera 2、reag1-chimera 1之結構，亦將其transfect到培養之神經細胞當中，觀察其表現模式，結果證實rEag1與rEag2 C端之差異，確實可能為造成兩者在神經細胞內差異的主要因素。再者為驗證rEag1與rEag2在in vivo中彼此聚合之可能性，我們將rEag1與GFP-rEag2共同在HEK293T細胞中表達，以及將腦組織研磨後純化出細胞膜蛋白後，再以免疫沉澱的方式作為佐證依據，結果顯示，在HEK293T以及鼠腦組織膜蛋白萃取物中，皆顯示出rEag1與rEag2能夠相互結合，證實兩者在in vivo中形成heterotetramer之可能性。 綜合以上，我們證實rEag channels之C端為影響神經細胞內次細胞分佈之重要因子，且rEag1與rEag2在in vivo當中確實能彼此聚合，形成hetero-tetramer之離子通道結構。

The EAG potassium channel is a member of the superfamility of voltage- gated potassium channels, and can be subdivided into three different subfamilies based on sequence homology：eag, erg and elk. In mammalians, the eag subfamily includes Eag1 and Eag2 K channels, which have been shown to express exclusively in central nervous system. Previous studies from our lab have demonstrated that rat Eag1 and rat Eag2 displayed differential subcellular localization patterns in hippocampus neurons. In the present study, we aim to determine the structural basis of differential localization of rEag1 and rEag2 in neurons. We intend to test the following hypotheses：(1) the differential localization pattern may be related to the different glycosylation patterns between rEag1 and rEag2. (2) the intracellular carboxyl terminal（C-terminal） region of rEag1 and rEag2 may contain the targeting signal that dictates the subcellular localization patterns of the two channels, and (3) rEag1 and rEag2 may form hetero-tetramers in neurons, suggesting the existence of a dominancy in the subcellular targeting within hetero-tetramer. We first studied the effect of abolishing either of the two known glycosylation sites in GFP-rEag1. Our results did not suppory a role of glycosylation in the subcellular localization of rEag1. We next engineered several chimeras that involved the C terminal regions of rEag1 and rEag2. By studing the confocal image of neuron transfected chimeric GFP constructs, we found evidence showing the C –terminal regions of rEag channels may confer the differential localization patterns of the two channels. Finally, we performed biochemical experiments to confirm that rEag1 and rEag2 are capable of forming hetero-tetramers in vivo. In conclusion, the C terminals of rEag channels play an important role in differential localization in neurons and rEag1 and rEag2 can assemble with each other in vivo.

1. Bauer CK, Schwarz JR (2001) Physiology of EAG K+ channels. J Membr Biol 182:1-15.
   連結：
2. Baukrowitz T, Yellen G (1995) Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15:951-960.
   連結：
3. Burack MA, Silverman MA, Banker G (2000) The role of selective transport in neuronal protein sorting. Neuron 26:465-472.
   連結：
4. Craig AM, Banker G (1994) Neuronal polarity. Annu Rev Neurosci 17:267-310.
   連結：
5. del Camino D, Yellen G (2001) Tight steric closure at the intracellular activation gate of a voltage-gated K(+) channel. Neuron 32:649-656.
   連結：
6. Demo SD, Yellen G (1991) The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker. Neuron 7:743-753.
   連結：
7. Deng Q, Rashid AJ, Fernandez FR, Turner RW, Maler L, Dunn RJ (2005) A C-terminal domain directs Kv3.3 channels to dendrites. J Neurosci 25:11531-11541.
   連結：
8. 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.
   連結：
9. Francesconi A, Duvoisin RM (2002) Alternative splicing unmasks dendritic and axonal targeting signals in metabotropic glutamate receptor 1. J Neurosci 22:2196-2205.
   連結：
10. Garrido JJ, Fernandes F, Giraud P, Mouret I, Pasqualini E, Fache MP, Jullien F, Dargent B (2001) Identification of an axonal determinant in the C-terminus of the sodium channel Na(v)1.2. Embo J 20:5950-5961.
    連結：
11. Grabner M, Dirksen RT, Beam KG (1998) Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca2+ channels expressed in dysgenic myotubes. Proc Natl Acad Sci U S A 95:1903-1908.
    連結：
12. Gu C, Jan YN, Jan LY (2003) A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301:646-649.
    連結：
13. Guy HR, Seetharamulu P (1986) Molecular model of the action potential sodium channel. Proc Natl Acad Sci U S A 83:508-512.
    連結：
14. Helenius A, Aebi M (2001) Intracellular functions of N-linked glycans. Science 291:2364-2369.
    連結：
15. Hille B, Schwarz W (1978) Potassium channels as multi-ion single-file pores. J Gen Physiol 72:409-442.
    連結：
16. Hille B. (2001) Ionic Channels of Excitable Membranes.
    連結：
17. Horton AC, Ehlers MD (2003) Neuronal polarity and trafficking. Neuron 40:277-295.
    連結：
18. Hoshi T, Zagotta WN, Aldrich RW (1990) Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250:533-538.
    連結：
19. Hsueh YP, Kim E, Sheng M (1997) Disulfide-linked head-to-head multimerization in the mechanism of ion channel clustering by PSD-95. Neuron 18:803-814.
    連結：
20. Huang K, El-Husseini A (2005) Modulation of neuronal protein trafficking and function by palmitoylation. Curr Opin Neurobiol 15:527-535.
    連結：
21. Imamura F, Maeda S, Doi T, Fujiyoshi Y (2002) Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel. J Biol Chem 277:3640-3646.
    連結：
22. Jareb M, Banker G (1998) The polarized sorting of membrane proteins expressed in cultured hippocampal neurons using viral vectors. Neuron 20:855-867.
    連結：
23. Jeng CJ, Chang CC, Tang CY (2005) Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons. Neuroreport 16:229-233.
    連結：
24. 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.
    連結：
25. Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M (1995) Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378:85-88.
    連結：
26. Lee TE, Philipson LH, Kuznetsov A, Nelson DJ (1994) Structural determinant for assembly of mammalian K+ channels. Biophys J 66:667-673.
    連結：
27. 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.
    連結：
28. 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.
    連結：
29. 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.
    連結：
30. Meyer R, Heinemann SH (1998) Characterization of an eag-like potassium channel in human neuroblastoma cells. J Physiol 508 ( Pt 1):49-56.
    連結：
31. 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.
    連結：
32. Nehring RB, Wischmeyer E, Doring F, Veh RW, Sheng M, Karschin A (2000) Neuronal inwardly rectifying K(+) channels differentially couple to PDZ proteins of the PSD-95/SAP90 family. J Neurosci 20:156-162.
    連結：
33. 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.
    連結：
34. Pardo LA, del Camino D, Sanchez A, Alves F, Bruggemann A, Beckh S, Stuhmer W (1999) Oncogenic potential of EAG K(+) channels. Embo J 18:5540-5547.
    連結：
35. Rivera JF, Chu PJ, Arnold DB (2005) The T1 domain of Kv1.3 mediates intracellular targeting to axons. Eur J Neurosci 22:1853-1862.
    連結：
36. 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.
    連結：
37. 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.
    連結：
38. Schonherr R, Gessner G, Lober K, Heinemann SH (2002) Functional distinction of human EAG1 and EAG2 potassium channels. FEBS Lett 514:204-208.
    連結：
39. Stowell JN, Craig AM (1999) Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains. Neuron 22:525-536.
    連結：
40. Tanemoto M, Fujita A, Higashi K, Kurachi Y (2002) PSD-95 mediates formation of a functional homomeric Kir5.1 channel in the brain. Neuron 34:387-397.
    連結：
41. Tsunoda S, Sun Y, Suzuki E, Zuker C (2001) Independent anchoring and assembly mechanisms of INAD signaling complexes in Drosophila photoreceptors. J Neurosci 21:150-158.
    連結：
42. 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.
    連結：
43. 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.
    連結：
44. 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.
    連結：
45. Yellen G (2002) The voltage-gated potassium channels and their relatives. Nature 419:35-42.
    連結：
46. 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.
47. Farias LM, Ocana DB, Diaz L, Larrea F, Avila-Chavez E, Cadena A, Hinojosa LM, Lara G, Villanueva LA, Vargas C, Hernandez-Gallegos E, Camacho-Arroyo I, Duenas-Gonzalez A, Perez-Cardenas E, Pardo LA, Morales A, Taja-Chayeb L, Escamilla J, Sanchez-Pena C, Camacho J (2004) Ether a go-go potassium channels as human cervical cancer markers. Cancer Res 64:6996-7001.
48. 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.