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研究生(外文):Po-Wen Cheng
論文名稱(外文):Effects of Calneuron in Modulating L-type and P/Q-type Calcium Currents
指導教授(外文):Chien-Yuan Pan
外文關鍵詞:calcium channelcalcium binding proteinCaBPcalneuronP/Q-type calcium channelL-type Calcium channel
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Calneuron為CaBP (calcium binding protein) 家族中的一員,主要表現在神經組織中,於N端有兩個能與鈣離子結合的EF-hand motif,其C端並具有一個疏水性結構。之前研究指出CaBP在細胞內鈣離子訊息傳遞路徑中扮演了重要角色,甚至可以直接調控鈣離子電流,而目前對於calneuron在生理上的相關研究卻非常少。電壓依賴性鈣離子通道( Voltage-dependent calcium channel )能調控細胞外鈣離子的流入進而影響鈣離子在細胞內所引發的各種反應。L型鈣離子通道廣泛地表現在各種類型的組織,而P/Q型鈣離子通道則主要表現於神經組織中。本實驗中,我們將大鼠calneuron選殖出來並與L型或P/Q型鈣離子通道同時表現於HEK293t細胞中,發現calneuron可抑制高鉀溶液引發的胞內鈣離子濃度上升。若以電生理技術實驗,結果顯示calneuron可以直接抑制P/Q型鈣離子電流峰值。相較於抑制L型鈣離子電流的大小,calneuron傾向改變開啟L型鈣離子通道的機制。而喪失疏水型C端的calneuron突變型不能發揮calneuron原有功能。由此結果可推測Calneuron能調控胞內鈣離子的訊息傳遞,進而影響胞吐作用與神經細胞間訊息傳導。
Calneuron belongs to calcium binding protein family and is predominantly expressed in neurons. Calneuron has two N-terminal functional EF-hand motifs and a hydrophobic C-terminus. When co-expressed with P/Q-type or L-type Ca(2+) channel in HEK293t cells, calneuron inhibited the intracellular Ca(2+) responses stimulated by high K(+) solution. The P/Q-type Ca(2+) currents measured by whole-cell patch-clamp were inhibited by calneuron; on the other hand, the opening kinetics of L-type Ca(2+) channels was modulated by calneuron. Calneuron mutants with the deletion of their hydrophobic C-terminus did not have the same effect as calneuron in modulating these Ca(2+) currents. These results suggested that calneuron may modulate intracellular Ca(2+) signaling pathway and participate in neuronal function by interacting with the Ca(2+) channels
誌謝 i
縮寫表 ii
中文摘要 iv
英文摘要 v
1 前言
1.1 鈣離子的重要性 1
1.2 L型及P/Q型鈣離子通道 1
1.3 鈣離子結合蛋白家族 3
1.4 實驗目標 5
2 材料與方法
2.1 化學藥品 7
2.2 溶液 7
2.3 分子生物技術 8
2.4 細胞培養繼代 9
2.5 細胞轉染 9
2.6 螢光鈣離子顯影 9
2.7 電生理 10
2.8 統計 11
3 結果
3.1 Calneuron 和CaBPs序列有高度相對應性 12
3.2 Calneuron 具有2個EF-hand motif及一個疏水性C端 12
3.3 Calneuron 能抑制高鉀溶液引發的胞內鈣離子濃度上升 13
3.4 Calneuron 抑制了P/Q型鈣離子電流 14
3.5 Calneuron 影響了L型鈣離子通道的啟動機制 14
3.6 去除C端的calneuron突變型影響了其抑制功能 16
4 討論 18
5 參考文獻 22
6 表 29
7 圖 30
Augustine GJ, Charlton MP, Smith SJ (1987) Calcium action in synaptic transmitter release. Annu Rev Neurosci 10:633-693.

Bahi N, Friocourt G, Carrie A, Graham ME, Weiss JL, Chafey P, Fauchereau F, Burgoyne RD, Chelly J (2003) IL1 receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis. Hum Mol Genet 12:1415-1425.

Barclay JW, Morgan A, Burgoyne RD (2005) Calcium-dependent regulation of exocytosis. Cell Calcium 38:343-353.

Burgoyne RD (2007) Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat Rev Neurosci 8:182-193.

Calin-Jageman I, Lee A (2008) Ca(v)1 L-type Ca2+ channel signaling complexes in neurons. J Neurochem 105:573-583.

Chen ML, Chen YC, Peng IW, Kang RL, Wu MP, Cheng PW, Shih PY, Lu LL, Yang CC, Pan CY (2008) Ca2+ binding protein-1 inhibits Ca2+ currents and exocytosis in bovine chromaffin cells. J Biomed Sci 15:169-181.

Cifuentes F, Licona, II, De Leon L, Medina P, De-Miguel FF, Morales MA (2004) Contribution of different calcium channels to long-term potentiation in superior cervical ganglion of the rat. Neuroscience 129:647-653.

Clark NC, Nagano N, Kuenzi FM, Jarolimek W, Huber I, Walter D, Wietzorrek G, Boyce S, Kullmann DM, Striessnig J, Seabrook GR (2003) Neurological phenotype and synaptic function in mice lacking the CaV1.3 alpha subunit of neuronal L-type voltage-dependent Ca2+ channels. Neuroscience 120:435-442.

Dolmetsch RE, Xu K, Lewis RS (1998) Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392:933-936.

Ertel EA, Campbell KP, Harpold MM, Hofmann F, Mori Y, Perez-Reyes E, Schwartz A, Snutch TP, Tanabe T, Birnbaumer L, Tsien RW, Catterall WA (2000) Nomenclature of voltage-gated calcium channels. Neuron 25:533-535.

Few AP, Lautermilch NJ, Westenbroek RE, Scheuer T, Catterall WA (2005) Differential regulation of CaV2.1 channels by calcium-binding protein 1 and visinin-like protein-2 requires N-terminal myristoylation. J Neurosci 25:7071-7080.

Freir DB, Herron CE (2003) Inhibition of L-type voltage dependent calcium channels causes impairment of long-term potentiation in the hippocampal CA1 region in vivo. Brain Res 967:27-36.

Gomez-Ospina N, Tsuruta F, Barreto-Chang O, Hu L, Dolmetsch R (2006) The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor. Cell 127:591-606.

Grabarek Z (2006) Structural basis for diversity of the EF-hand calcium-binding proteins. J Mol Biol 359:509-525.

Haeseleer F, Sokal I, Verlinde CL, Erdjument-Bromage H, Tempst P, Pronin AN, Benovic JL, Fariss RN, Palczewski K (2000) Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin. J Biol Chem 275:1247-1260.

Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP, Catterall WA (1993) Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J Cell Biol 123:949-962.

Hirst RA, Harrison C, Hirota K, Lambert DG (1999) Measurement of [Ca2+]i in whole cell suspensions using fura-2. Methods Mol Biol 114:31-39.

Huxley HE (1969) The mechanism of muscular contraction. Science 164:1356-1365.

Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105-132.

Lacerda AE, Kim HS, Ruth P, Perez-Reyes E, Flockerzi V, Hofmann F, Birnbaumer L, Brown AM (1991) Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature 352:527-530.

Lee A, Westenbroek RE, Haeseleer F, Palczewski K, Scheuer T, Catterall WA (2002) Differential modulation of Ca(v)2.1 channels by calmodulin and Ca2+-binding protein 1. Nat Neurosci 5:210-217.

Mikhaylova M, Sharma Y, Reissner C, Nagel F, Aravind P, Rajini B, Smalla KH, Gundelfinger ED, Kreutz MR (2006) Neuronal Ca2+ signaling via caldendrin and calneurons. Biochim Biophys Acta 1763:1229-1237.

Moosmang S, Haider N, Klugbauer N, Adelsberger H, Langwieser N, Muller J, Stiess M, Marais E, Schulla V, Lacinova L, Goebbels S, Nave KA, Storm DR, Hofmann F, Kleppisch T (2005) Role of hippocampal Cav1.2 Ca2+ channels in NMDA receptor-independent synaptic plasticity and spatial memory. J Neurosci 25:9883-9892.

Mori Y, Friedrich T, Kim MS, Mikami A, Nakai J, Ruth P, Bosse E, Hofmann F, Flockerzi V, Furuichi T, et al. (1991) Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350:398-402.

Nakayama S, Kretsinger RH (1994) Evolution of the EF-hand family of proteins. Annu Rev Biophys Biomol Struct 23:473-507.

Nelson MR, Chazin WJ (1998) Structures of EF-hand Ca(2+)-binding proteins: diversity in the organization, packing and response to Ca2+ binding. Biometals 11:297-318.
O''Callaghan DW, Burgoyne RD (2003) Role of myristoylation in the intracellular targeting of neuronal calcium sensor (NCS) proteins. Biochem Soc Trans 31:963-965.

Reid CA, Bekkers JM, Clements JD (2003) Presynaptic Ca2+ channels: a functional patchwork. Trends Neurosci 26:683-687.
Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31:3381-3385.

Starr TV, Prystay W, Snutch TP (1991) Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc Natl Acad Sci U S A 88:5621-5625.

Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 51:187-200.

Tippens AL, Lee A (2007) Caldendrin, a neuron-specific modulator of Cav/1.2 (L-type) Ca2+ channels. J Biol Chem 282:8464-8473.

Turner TJ, Adams ME, Dunlap K (1993) Multiple Ca2+ channel types coexist to regulate synaptosomal neurotransmitter release. Proc Natl Acad Sci U S A 90:9518-9522.

Wang CY, Yang F, He X, Chow A, Du J, Russell JT, Lu B (2001) Ca(2+) binding protein frequenin mediates GDNF-induced potentiation of Ca(2+) channels and transmitter release. Neuron 32:99-112.

Weiss JL, Archer DA, Burgoyne RD (2000) Neuronal Ca2+ sensor-1/frequenin functions in an autocrine pathway regulating Ca2+ channels in bovine adrenal chromaffin cells. J Biol Chem 275:40082-40087.

West AE, Chen WG, Dalva MB, Dolmetsch RE, Kornhauser JM, Shaywitz AJ, Takasu MA, Tao X, Greenberg ME (2001) Calcium regulation of neuronal gene expression. Proc Natl Acad Sci U S A 98:11024-11031.

Wingard JN, Chan J, Bosanac I, Haeseleer F, Palczewski K, Ikura M, Ames JB (2005) Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator of calcium channels. J Biol Chem 280:37461-37470.

Wu YQ, Lin X, Liu CM, Jamrich M, Shaffer LG (2001) Identification of a human brain-specific gene, calneuron 1, a new member of the calmodulin superfamily. Mol Genet Metab 72:343-350.

Zhou H, Yu K, McCoy KL, Lee A (2005) Molecular mechanism for divergent regulation of Cav1.2 Ca2+ channels by calmodulin and Ca2+-binding protein-1. J Biol Chem 280:29612-29619.
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