臺灣博碩士論文加值系統

本論文主要目的係藉由定點突變的方式探討KChIP2.2 (Kv channel
interacting protein2.2)四個EF-hands 與鈣離子及鎂離子結合能力對其結構之影響，及對Kv channel 結合反應之參與機制。CD spectra 顯示相較於突變EF-hands 1、2、3 及4，之重組蛋白之二級結構與wild-type 有明顯差異，但wild-type 及所有mutated KChIP2.2 之二級結構均可因結合鈣離子及鎂離子而有明顯變化。螢光實驗結果證實EF3 及EF4 為具有高親和性鈣離子結合的位置，但四個EF-hands 突變後均不影響KChIP2.2 與鎂離子之結合能力。膠體層析法結果顯示情形EF1、EF2 及EF3 突變後可使KChIP2.2 分子間引起oligomerization，而鈣離子及鎂離子可使其程度增加。突變蛋白及wild-type KChIP2.2 與猪腦組織萃取膜及liposome 之結合能力無明顯差異，且不受鈣離子及鎂離子的調控。Pull-down assay 結果顯示突變蛋白與KChIP2.2 在沒有離子存在下皆會與Kv4.2 結合，雖然鈣離子及鎂離子均會增加KChIP2.2 與Kv4.2 的結合，但 KChIP2.2 突變EF-hands 之後與Kv4.2結合能力明顯下降。細胞分佈實驗結果顯示突變EF1 後 KChIP2.2 集中於細胞核內，而其他EF-hand 突變蛋白及wild-type KChIP2.2 則分佈於細胞質與細胞核，提高細胞內鈣離子濃度會使突變後EF1 分佈至細胞質但對其他蛋白之分佈則無明顯改變。綜合上述結果顯示，四個EF-hands 扮演穩定KChIP2.2 構形之角色以利與Kv4.2 之結合反應，但此功能角色與細胞膜之結合能力無直接相關性。

Mutagenesis studies on the four EF-hands of KChIP2.2 (Potassium channel-interacting protein 2.2) were carried out to explore the conformational transition upon the binding of Ca2+ and Mg2+ and the subsequent effect on the interaction between KChIP2.2 and Kv4.2. CD spectra indicated that Ca2+- and Mg2+-loaded wild-type and mutated KChIP2.2 altered the secondary structure contents. In contrast to other mutants, mutation on EF1 caused a notably change in the secondary structure of KChIP2.2. Fluorescence measurement revealed that EF-hands 3 and 4 were high affinity Ca2+-binding sites within KChIP2.2 molecule, but the binding of Mg2+ with KChIP2.2 was marginally affected by EF-hand mutations. The results of size-exclusion chromatography showed that mutations on EF-1, EF-2 and EF-3 induced the oligomerization of KChIP2.2 and the extent of oligomerization was enhanced by Ca2+ and Mg2+. No significant differences were noted when wild-type and mutated KChIP2.2 bound with porcine brain membrane and liposome either in the absence or presence of Ca2+- and Mg2+. Pull down assay showed that KChIP2.2 and EF-hand mutants could bind with Kv4.2 in the absence of Ca2+ and Mg2+, but the interaction was enhanced by Ca2+ and Mg2+. However, the binding capability of mutants for Kv4.2 was notably lower than that observed for wild-type KChIP2.2. It was found that, in sharp contrast to that EF1 mutant exclusively localized in the nucleus, the other EF-hand mutants and wild-type protein distributed within nucleus as well as cytoplasm. Elevating intracellular Ca2+ concentration caused the translocation of EF1 mutant to cytoplasm but no appreciable effect on other mutants and wild-type KChIP2.2. . Taken together, these results suggest that the integrity of the four EF-hands are involved in function to stabilize conformation for binding with Kv channel, but this conformational transition is not essential for the binding to cell membrane.

目錄
中文摘要 ----------------------------------- 1
英文摘要 ----------------------------------- 2
英文縮寫表 --------------------------------- 3
序言 --------------------------------------- 4
實驗材料 ----------------------------------- 8
實驗方法 ----------------------------------- 11
實驗結果 ----------------------------------- 28
討論 --------------------------------------- 36
表 ----------------------------------------- 40
圖 ----------------------------------------- 45
參考文獻 ----------------------------------- 62

An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000). Modulation of A-type potassium channels by a family of calcium sensors. Nature, 403, 553-556.

Bahring, R., Dannenberg, J., Peters, H. C., Leicher, T., Pongs, O., and Isbrandt, D. (2001). Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating. J. Biol. Chem., 276, 23888-23894.

Boland, M. L., Jiang, M., Lee, Y. S., Fahrenkrug, C. S., Harnett, T. M., and O''Grady, M. S. (2003) Functional properties of a brain-specific NH2-terminally spliced modulator of Kv4 channels. J. Physiol. Cell., 285, 161-170.

Burgoyne, R. D. and Weiss, J. L. (2001) The neuronal calcium sensor family of Ca2+-binding proteins. Biochem. J., 353, 1-12.

Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E. and Wasco, W. (1998) Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment. Nat. Med., 4, 1177-81.

Callsen B., Isbrandt, D., Sauter, K., Hartmann, L.S., Pongs, O., and Bahring, R. (2005). Contribution of N- and C-terminal channel domains to Kv4.2 domains to KChIP interaction. J. Physiol., 568, 397-412.

Carrion, A. M., Link, W. A., Ledo, F., Mellstrom, B. and Naranjo, J. R. (1999) DREAM is a Ca2+-regulated transcriptional repressor. Nature, 398, 80-84.

Choe, S. (2002). Potassium channel structures. Nat. Rev. Neurosci., 3, 115-121.

Decher, N., Uyguner, O., Scherer, C. R., Karaman, B., Yuksel-Apak, M., Busch, A. E., Steinmeyer, K. and Wollnik, B. (2001) hKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant. Cardiovasc. Res., 52, 255-264.

Decher, N., Barth, A. S., Gonzalez, T., Steinmeyer, K., and Sanguinetti, M. C. (2004). Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents. J. Physiol., 557, 761-772.

Deschenes, I., DiSilvestre, D., Juang, G. J., Wu, R. C., An, W. F. and Tomaselli, G. F. (2002a) Regulation of Kv4.3 current by KChIP2 splice variants: a component of native cardiac I(to) Circulation, 106, 423-429.

Deschenes, I., and Tomaselli, G. F. (2002b). Modulation of Kv4.3 current by accessory subunits. FEBS Lett., 528, 183-188.

Dixon, J. E., Shi, W., Wang, H. S., McDonald, C., Yu, H., Wymore, R. S., Cohen, I. S., and McKinnon, D. (1996). Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ. Res., 79, 659-668.

Doronin, V. S., Potapova, A. I., Lu, Z, and Cohen, S. I. (2004). Angiotensin receptor type 1 forms a complex with the transient outward potassium channel Kv4.3 and regulates its gating properties and intracellular localization. Biochem. J., 279, 48231-48237.

Gorczyaca, W. A., Kobialka, M., and Takamatsu, K. ( 2001) Ca2+ diffetently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin. Aata. Biochim., 50,367-376.

Han, W., Nattel, S., Noguchi, T., and Shrier, A. (2006). C-terminal domain of Kv4.2 and associated KChIP2 interactions regulate functional expression and gating of Kv4.2. . J. Biol. Chem., Published on July 4


Hwang. J.Y., Schlesinger, R., and Koch, K.W. (2004). Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation. Eur. J. Biochem., 271,3785-3793

Jerng, H.H., Qian, Y., and Pfaffinger, P.J. (2004). Modulation of Kv4.2 channel expression and gating by dipeptidyl peptidase 10 (DPP10). Biophys. J., 87, 2380-2396

Jiang, B., Sun, X., Cao, K., and Wang, R. (2002). Endogenous Kv channels in human embryonic kidney (HEK-293) cells. Mol. Cell. Biochem., 238, 69-79.

Kim, L.A., Furst, J., Gutierrez, D., Butler, M.H., Xu, S., Goldstein, S.A., and Grigorieff, N. (2004). Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution. Neuron, 41, 513-519.

Kobayashi, M., Takamatsu, K., Saitoh, S., and Noguchi, T. (1993) Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties.

Kuo, H.C., Cheng, C.F., Clark, R.B., Lin, J.J., Lin, J.L., Hoshijima, M. Nguyen-Tran, V.T., Gu, Y., Ikeda, Y., Chu, P.H., Ross, J., Giles, W.R., and Chien, K.R. (2001). A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia. Cell , 107, 801-813

Lin, Y. L., Chen, C. Y., Cheng, C. P., and Chang, L. S. (2004). Protein-protein interactions of KChIP proteins and Kv4.2. Biochem. Biophys. Res. Commun., 321, 606-610.

Lodish, H., Berk, A., Zipursky, S. L., Matudaria, P., Baltimore, D., and Darnell, J. E. (1999a) in Molecular Cell Biology (Sara, T., ed) Vol. 21, pp. 936, W. H. Freeman and company, New York.

Lodish, H., Berk, A., Zipursky, S. L., Matudaria, P., Baltimore, D., and Darnell, J. E. (1999b) in Molecular Cell Biology (Sara, T., ed) Vol. 15, pp. 585, W. H. Freeman and company, New York.

Morohashi, Y., Hatano, N., Ohya, S., Takikawa, R., Watabiki, T., Takasugi, N., Imaizumi, Y., Tomita, T., and Iwatsubo, T. (2002). Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4. J. Biol. Chem., 277, 14965-14975.

O''Callaghan, D. W., Hasdemir, B., Leighton, M., and Burgoyne, R. D. (2003). Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels. J. Cell Sci., 116, 4833-4845.

Ohya, S., Morohashi, Y., Muraki, K., Tomita, T., Watanabe, M., Iwatsubo, T., and Imaizumi, Y. (2001). Molecular cloning and expression of the novel splice variants of K(+) channel-interacting protein 2. Biochem. Biophys. Res. Commun., 282, 96-102.

Osawa, M., Dace, A., Tong, K. I., Valiveti, A., Ikura, M., and Ames, J. B. (2005). Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM. J. Biol. Chem., 280, 18008-18014

Patel, S.P., Campbell, D.L., Morales, M.J., and Strauss, H.C. (2002a). Heterogeneous expression of KChIP2 isoforms in the ferret heart. J. Physiol., 539, 649-656.

Patel, S.P., Campbell, D.L., and Strauss, H.C. (2002b). Elucidating KChIP effects on Kv4.3 inactivation and recovery kinetics with a minimal KChIP2 isoform. J. Physio., 545, 5-11

Pourrier, M., Herrera, D., Caballero, R., Schram, G., Wang, Z., and Nattel, S. (2004). The Kv4.2 N-terminal restores fast inactivation and confers KChlP2 modulatory effects on N-terminal-deleted Kv1.4 channels. Pflugers Arch., 449, 235-247.

Ren, X., Stuart, H., and Takimoto, K. (2003). Effective association of Kv channel-interactiong proteins with Kv4 channel is Mediated with their unique core peptide. Biochem. J., 278,43564-43570.

Scannevin, R.H., Wang, K., Jow, F., Megules, J., Kopsco, D.C., Edris, W., Carroll, K.C., Lu, Q., Xu, W., Xu, Z., Katz, A.H., Olland, S., Lin, L., Taylor, M., Stahl, M., Malakian, K., Somers, W., Mosyak, L., Bowlby, M.R., Chanda, P., and Rhodes, K.J. ( 2004). Two N-terminal domains of Kv4 K(+) channels regulate binding to and modulation by KChIP1. Neuron, 41, 587-598.

Schultz, J.H., Janzen, C., Volk, T., and Ehmke, H. (2005). Kv4.2 and KChIP2 transcription in individual cardiomyocytes from the rat left ventricular free wall. J. Mol. Cell Cardiol., 39, 269-275.

Serodio, P., Kentros, C., and Rudy, B. (1994). Identification of molecular components of A-type channels activating at subthreshold potentials. J. Neurophysiol., 72, 1516-1529.

Shibata, R., Misonou, H., Campomanes, C. R., Anderson, A. E., Schrader, L. A., Doliveira, L. C., Carroll, K. I., Sweatt, J. D., Rhodes, K. J., and Trimmer, J. S. (2003). A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels. J. Biol. Chem., 278, 36445-36454.

Suzuki, T., and Takimoto, K. (2005). Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy. Am. J. Physio. Endocrinol. Metab., 288, E335-341.

Takimoto, K., Yang, E. K., and Conforti, L. (2002). Palmitoylation of KChIP splicing variants is required for efficient cell surface expression of Kv4.3 channels. J. Biol. Chem., 277, 26904-26911.

Tatulian, S.A. (2003) Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites. Biophys. J., 84, 1173-1183

Xiong, H., Kovacs, I., and Zhang, Z. (2004). Differential distribution of KChIPs mRNAs in adult mouse brain. Brain Res. Mol. Brain Res., 128, 103-111.

Zhou, W., Qian, Y., Kunjilwar, K., Pfaffinger, P.J., and Choe, S. (2004). Structural insights into the functional interaction of KChIP1 with Shal-type K(+) channels. Neuron, 41, 573-586.