臺灣博碩士論文加值系統

TRP 通道為一種非專一性陽離子通道，在視覺、味覺、嗅覺、聽覺或觸覺等感覺生理上扮演重要角色，協助生物體偵測周圍環境或壓力的變化。2-APB是大部份TRP通道的抑制劑，但在我們爪蟾神經肌-突觸培養中，利用whole-cell patch clamp記錄方式發現，單獨施予2-APB反而造成運動神經自發性神經傳遞物質釋放量的上升。進一步將細胞外溶液置換成不含鈣離子的生理緩衝液(Ca2+ free Ringer)、加入廣效性鈣離子通道抑制劑Cd2+或膜通透性高的鈣離子螯合劑BAPTA-AM，再觀察2-APB的作用情形，發現皆能有效地抑制2-APB促進神經傳遞物質釋放作用，顯示2-APB促進神經活性與細胞外鈣離子有關。於SKF96365、flufenamic acid或RuR (TRP 通道的抑制劑)存在時，2-APB的促進作用亦消失。利用cell-attached patch-clamp單一離子通道電流紀錄方法觀測2-APB開啟離子通道後所造成的電流訊號，但是當廣效性TRP通到抑制劑SKF96365存在的情況下，2-APB所活化的離子通道活性明顯減小。因此我們認為2-APB影響突觸前神經傳遞物質的釋放，與TRP通道活性有極大的關係。
而當我們進行實驗前，事先投予各種不同訊息傳遞路徑的抑制劑至培養基中，再觀察2-APB的作用，發現當PLC抑制劑（U73122)，tyrosine kinase 抑制劑(Genistein)或DAG類似物(PMA)存在的情況下，2-APB促進神經活性的作用受到抑制。將培養液置換成不含胎小牛血清（fetal calf serum）的生理緩衝溶液，2-APB的促進作用不再產生。總括以上結果我們認為在爪蟾神經肌細胞培養中，培養液中的血清含有成長因子，能活化膜上受體使tyrosine kinase活化，啟動PI3K和PLC訊息路徑產生DAG，進而打開TRP通道，加入2-APB可能增強TRP通道的活化作用，造成神經細胞末梢內的鈣離子濃度上升，而促進神經傳遞物質大量釋放。

The transient receptor potential (TRP) channel superfamily is a non-selective Ca2+-permeable cation channels involved in sensory physiology. Here we show that 2-aminoethoxydiphenyl borate (2-APB), a compound commonly used as TRP channel inhibitor, dose-dependently induce a significant facilitation on the frequency of spontaneous neurotransmitter release at developing Xenopus neuromuscular junction through, surprisingly, TRP channel activation. Bath application of universal TRP channel inhibitors either SKF96365, flufenamic acid or RuR cease the 2-APB-induced synaptic facilitation. Exclusion of Ca2+ from culture medium or bath application of the pharmacological Ca2+ channel inhibitor cadmium, membrane-permeable Ca2+ chelator BAPTA-AM, effectively hampered the facilitation of neurotransmitter release induced by 2-APB, suggesting Ca2+ influx is requisite for 2-APB-induced synaptic facilitation. Blockade of the voltage-dependent Ca2+ channel with either nifedipine, verapamil or ω-CTX failed to abolish the SSC facilitating effect of 2-APB. Electrophysiological recording of 2-APB induced single channel currents by using cell-attached patch-clamp technique reveals 2-APB evoked a robust single channel activity recorded at different pipette voltages. Furthermore, the 2-APB-evoked single-channel events are significantly abolished in the presence of SKF96365.
Either pretreatment of the cultures with inhibitor of phospholipase C (U73122) or tyrosine kinase (Genistein) abolishes 2-APB induced potentiation of synaptic transmission. The structure of PMA is analogous to diacylglycerol (DAG), which abolishes 2-APB induced synaptic facilitation. 2-APB no longer elicited any changes in SSC frequency when serum is eliminated from culture medium. Overall, results from our current study provide evidences that 2-APB induces the opening of TRP channels and Ca2+ influx which resulting in facilitation of spontaneous neurotransmitter release at developing Xenopus neuromuscular synapse. Serum may activate tyrosine kinase to turn on PI3K and phospholipase C. Then phospholipase C cleavage PIP2 to IP3 and diacylglycerol, and diacylglycerol induced TRP channel opening. 2-APB potentiates and sensitizes the TRP channel, increasing Ca2+ inffux. Elevated [Ca2+]i resulted in enhancement of neurotransmitter release from presynaptic nerve terminal.

縮寫表 1
中文摘要 2
英文摘要 4
緒論 6
實驗材料 13
實驗方法 15
§ 電生理紀錄方法 15
§ 單一離子通道電流紀錄方法 16
§ 量測細胞內鈣離子濃度 17
§ 胚胎顯微注射 18
結果 21
討論 35
參考文獻 44
圖表 52
圖 表 順 序
附圖1 52
附圖2 53
附圖3 54
附圖4 55
Fig 1. 57
Fig 2. 58
Fig 3. 60
Fig 4. 62
Fig 5. 64
Fig 6. 67
Fig 7. 69
Fig 8. 71
Fig 9. 73
Fig 10. 75
Fig 11. 78
Fig 12. 80
Fig 13. 82
Fig 14. 84
Fig 15. 85

Anderson, M.J., Cohen, M.W., and Zorychta, E. (1977). Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol 268, 731-756.
Banker, G., and Goslin, K. (1998). Culturing nerve cells, 2nd ed (Cambridge, Mass.: MIT Press).
Berridge, M.J., Bootman, M.D., and Roderick, H.L. (2003). Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4, 517-529.
Bezzerides, V.J., Ramsey, I.S., Kotecha, S., Greka, A., and Clapham, D.E. (2004). Rapid vesicular translocation and insertion of TRP channels. Nat Cell Biol 6, 709-720.
Bi, G., and Poo, M. (2001). Synaptic modification by correlated activity: Hebb''s postulate revisited. Annu Rev Neurosci 24, 139-166.
Bobanovic, L.K., Laine, M., Petersen, C.C., Bennett, D.L., Berridge, M.J., Lipp, P., Ripley, S.J., and Bootman, M.D. (1999). Molecular cloning and immunolocalization of a novel vertebrate trp homologue from Xenopus. Biochem J 340 ( Pt 3), 593-599.
Boels, K., Glassmeier, G., Herrmann, D., Riedel, I.B., Hampe, W., Kojima, I., Schwarz, J.R., and Schaller, H.C. (2001). The neuropeptide head activator induces activation and translocation of the growth-factor-regulated Ca(2+)-permeable channel GRC. J Cell Sci 114, 3599-3606.
Bootman, M.D., Collins, T.J., Mackenzie, L., Roderick, H.L., Berridge, M.J., and Peppiatt, C.M. (2002). 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release. FASEB J 16, 1145-1150.
Catterall, W.A. (2000). Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16, 521-555.
Chung, C., Deak, F., and Kavalali, E.T. (2008). Molecular substrates mediating lanthanide-evoked neurotransmitter release in central synapses. J Neurophysiol 100, 2089-2100.
Chung, M.K., Lee, H., Mizuno, A., Suzuki, M., and Caterina, M.J. (2004). 2-aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J Neurosci 24, 5177-5182.
Clapham, D.E., Runnels, L.W., and Strubing, C. (2001). The TRP ion channel family. Nat Rev Neurosci 2, 387-396.
Colton, C.K., and Zhu, M.X. (2007). 2-Aminoethoxydiphenyl borate as a common activator of TRPV1, TRPV2, and TRPV3 channels. Handb Exp Pharmacol, 173-187.
Evers, J., Laser, M., Sun, Y.A., Xie, Z.P., and Poo, M.M. (1989). Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents. J Neurosci 9, 1523-1539.
Greka, A., Navarro, B., Oancea, E., Duggan, A., and Clapham, D.E. (2003). TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nat Neurosci 6, 837-845.
Gu, Q., Lin, R.L., Hu, H.Z., Zhu, M.X., and Lee, L.Y. (2005). 2-aminoethoxydiphenyl borate stimulates pulmonary C neurons via the activation of TRPV channels. Am J Physiol Lung Cell Mol Physiol 288, L932-941.
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85-100.
Hofmann, T., Obukhov, A.G., Schaefer, M., Harteneck, C., Gudermann, T., and Schultz, G. (1999). Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259-263.
Hu, H.Z., Gu, Q., Wang, C., Colton, C.K., Tang, J., Kinoshita-Kawada, M., Lee, L.Y., Wood, J.D., and Zhu, M.X. (2004). 2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem 279, 35741-35748.
Kanzaki, M., Zhang, Y.Q., Mashima, H., Li, L., Shibata, H., and Kojima, I. (1999). Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nat Cell Biol 1, 165-170.
Kim, B.J., Kim, M.T., Jeon, J.H., Kim, S.J., and So, I. (2008). Involvement of phosphatidylinositol 4,5-bisphosphate in the desensitization of canonical transient receptor potential 5. Biol Pharm Bull 31, 1733-1738.
Kwan, H.Y., Huang, Y., and Yao, X. (2006). Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G. J Cell Physiol 207, 315-321.
Li, H.B., Mao, R.R., Zhang, J.C., Yang, Y., Cao, J., and Xu, L. (2008). Antistress effect of TRPV1 channel on synaptic plasticity and spatial memory. Biol Psychiatry 64, 286-292.
Li, Y., Jia, Y.C., Cui, K., Li, N., Zheng, Z.Y., Wang, Y.Z., and Yuan, X.B. (2005). Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434, 894-898.
Lievremont, J.P., Bird, G.S., and Putney, J.W., Jr. (2005). Mechanism of inhibition of TRPC cation channels by 2-aminoethoxydiphenylborane. Mol Pharmacol 68, 758-762.
Ma, H.T., Patterson, R.L., van Rossum, D.B., Birnbaumer, L., Mikoshiba, K., and Gill, D.L. (2000). Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science
287, 1647-1651.
Maruyama, T., Kanaji, T., Nakade, S., Kanno, T., and Mikoshiba, K. (1997). 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem 122, 498-505.
Meir, A., Ginsburg, S., Butkevich, A., Kachalsky, S.G., Kaiserman, I., Ahdut, R., Demirgoren, S., and Rahamimoff, R. (1999). Ion channels in presynaptic nerve terminals and control of transmitter release. Physiol Rev 79, 1019-1088.
Morenilla-Palao, C., Planells-Cases, R., Garcia-Sanz, N., and Ferrer-Montiel, A. (2004). Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity. J Biol Chem 279, 25665-25672.
Odell, A.F., Scott, J.L., and Van Helden, D.F. (2005). Epidermal growth factor induces tyrosine phosphorylation, membrane insertion, and activation of transient receptor potential channel 4. J Biol Chem 280, 37974-37987.
Penna, A., Juvin, V., Chemin, J., Compan, V., Monet, M., and Rassendren, F.A. (2006). PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane. Cell Calcium 39, 495-507.
Prescott, E.D., and Julius, D. (2003). A modular PIP2 binding site as a determinant of capsaicin receptor sensitivity. Science 300, 1284-1288.
Provan, S.D., and Miyamoto, M.D. (1992). Subcellular mechanism and site of action of ionic lanthanum at the motor nerve terminal. Neuroreport 3, 101-104.
Rahamimoff, R., Erulkar, S.D., Lev-Tov, A., and Meiri, H. (1978). Intracellular and extracellular calcium ions in transmitter release at the neuromuscular synapse. Annals of the New York Academy of Sciences 307, 583-598.
Ramsey, I.S., Delling, M., and Clapham, D.E. (2006). An introduction to TRP channels. Annu Rev Physiol 68, 619-647.
Reiser, J., Polu, K.R., Moller, C.C., Kenlan, P., Altintas, M.M., Wei, C., Faul, C., Herbert, S., Villegas, I., Avila-Casado, C., et al. (2005). TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet 37, 739-744.
Rohacs, T., Lopes, C.M., Michailidis, I., and Logothetis, D.E. (2005). PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci 8, 626-634.
Runnels, L.W., Yue, L., and Clapham, D.E. (2002). The TRPM7 channel is inactivated by PIP(2) hydrolysis. Nat Cell Biol 4, 329-336.
Saito, S., and Shingai, R. (2006). Evolution of thermoTRP ion channel homologs in vertebrates. Physiol Genomics 27, 219-230.
Schlingmann, K.P., Weber, S., Peters, M., Niemann Nejsum, L., Vitzthum, H., Klingel, K., Kratz, M., Haddad, E., Ristoff, E., Dinour, D., et al. (2002). Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 31, 166-170.
Shatz, C.J. (1990). Impulse activity and the patterning of connections during CNS development. Neuron 5, 745-756.
Singh, B.B., Lockwich, T.P., Bandyopadhyay, B.C., Liu, X., Bollimuntha, S., Brazer, S.C., Combs, C., Das, S., Leenders, A.G., Sheng, Z.H., et al. (2004). VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx. Mol Cell 15, 635-646.
Spitzer, N.C., and Lamborghini, J.E. (1976). The development of the action potential mechanism of amphibian neurons isolated in culture. Proc Natl Acad Sci U S A 73, 1641-1645.
St Pierre, M., Reeh, P.W., and Zimmermann, K. (2009). Differential effects of TRPV channel block on polymodal activation of rat cutaneous nociceptors in vitro. Exp Brain Res 196, 31-44.
Strubing, C., Krapivinsky, G., Krapivinsky, L., and Clapham, D.E. (2001). TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron 29, 645-655.
Tanaka, R., Muraki, K., Ohya, S., Yamamura, H., Hatano, N., Itoh, Y., and Imaizumi, Y. (2008). TRPV4-like non-selective cation currents in cultured aortic myocytes. J Pharmacol Sci 108, 179-189.
Tang, J., Lin, Y., Zhang, Z., Tikunova, S., Birnbaumer, L., and Zhu, M.X. (2001). Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels. J Biol Chem 276, 21303-21310.
Trebak, M., Lemonnier, L., DeHaven, W.I., Wedel, B.J., Bird, G.S., and Putney, J.W., Jr. (2009). Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels. Pflugers Arch 457, 757-769.
Trebak, M., St, J.B.G., McKay, R.R., Birnbaumer, L., and Putney, J.W., Jr. (2003). Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels. J Biol Chem 278, 16244-16252.
Tsiokas, L. (2009). Function and regulation of TRPP2 at the plasma membrane. Am J Physiol Renal Physiol.
Ueda, T., Yamada, T., Ugawa, S., Ishida, Y., and Shimada, S. (2009). TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium. Biochem Biophys Res Commun 383, 130-134.
Vazquez, G., Bird, G.S., Mori, Y., and Putney, J.W., Jr. (2006). Native TRPC7 channel activation by an inositol trisphosphate receptor-dependent mechanism. J Biol Chem 281, 25250-25258.
Vazquez, G., Lievremont, J.P., St, J.B.G., and Putney, J.W., Jr. (2001). Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci U S A 98, 11777-11782.
Venkatachalam, K., and Montell, C. (2007). TRP channels. Annu Rev Biochem 76, 387-417.
Venkatachalam, K., Zheng, F., and Gill, D.L. (2003). Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 278, 29031-29040.
Verkhrats''kyi, O.N., and Fedulova, S.A. (2004). Endoplasmic reticulum and regulation of neuromediator release in presynaptic terminals. Fiziol Zh 50, 142-149.
Wang, G.X., and Poo, M.M. (2005). Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434, 898-904.
Wegierski, T., Lewandrowski, U., Muller, B., Sickmann, A., and Walz, G. (2009). Tyrosine phosphorylation modulates the activity of TRPV4 in response to defined stimuli. J Biol Chem 284, 2923-2933.
Winn, M.P., Conlon, P.J., Lynn, K.L., Farrington, M.K., Creazzo, T., Hawkins, A.F., Daskalakis, N., Kwan, S.Y., Ebersviller, S., Burchette, J.L., et al. (2005). A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 308, 1801-1804.
Xie, Z.P., and Poo, M.M. (1986). Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neuron. Proc Natl Acad Sci U S A 83, 7069-7073.
Xu, X.Z., and Sternberg, P.W. (2003). A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization. Cell 114, 285-297.
Young, S.H., and Poo, M.M. (1983). Spontaneous release of transmitter from growth cones of embryonic neurones. Nature 305, 634-637.
Zhang, L.I., Tao, H.W., Holt, C.E., Harris, W.A., and Poo, M. (1998). A critical window for cooperation and competition among developing retinotectal synapses. Nature 395, 37-44.
Zhang, W., Tong, Q., Conrad, K., Wozney, J., Cheung, J.Y., and Miller, B.A. (2007). Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1. Am J Physiol Cell Physiol 292, C1746-1758.