調鈣素與Calneuron 1對電壓依賴性離子通道之調控

Regulation of Voltage-gated Ion Channels by Calmodulin and Calneuron 1

紀志弘(Chih-Hung Chi)

指導教授 ： 潘建源

國立臺灣大學/生命科學院/生命科學系/博士(2017年)

https://doi.org/10.6342/NTU201704249

摘要

細胞內鈣離子的提升能活化細胞內各種生理作用，其主要來源是透過細胞外鈣離子通過細胞膜上的電壓依賴型鈣離子通道進入細胞內。此鈣離子依賴型不活化已知與調鈣素偵測鈣離子濃度有關。調鈣素如何調節電壓依賴型鈣離子通道CaV2.2仍保有未知，因此我們在胚胎腎臟皮質細胞中表現CaV2.2與調鈣素或其突變型並記錄其電流來探討此鈣離子依賴不活化現象。結果顯示CaV2.2在鈣離子溶液中能快速的不活化，但是並不存在於鋇離子為溶液時。當與缺乏鈣離子結合能力的調鈣素突變型共同表現時，此鈣離子依賴型不活化效果隨之減小。利用谷胱甘肽轉移酶標記之調鈣素或突變型為餌，我們發現在不論鈣離子有無的情況下，調鈣素能和胞內的電壓依賴型鈣離子通道之C端交互作用。然而，當此突變CaV2.2上，已知與調鈣素接合的高保留性氨基酸的片段時，調鈣素或型無法失去與之交互作用。調鈣素接合位置的突變導致此CaV2.2的的電流減小但能被調鈣素Ｎ端突變型拯救。然而，我們發現N-端突變型的調鈣素可以增加CaV2.2的的總體表現量，卻不會增加細胞膜上的表現比例。總的來看，我們的結果認為調鈣素不只能調節CaV2.2的不活化；同時，藉由調控其表現量，已達控制細胞內鈣離子濃度對生理活動的影響。
實驗室之前的研究發現與調鈣素結構類似的鈣離子結合蛋白，Calneuron 1 (Caln1)，能夠抑制腎上腺髓質嗜鉻細胞上的電壓依賴型鈣離子電流。此外，酵母菌雙雜交實驗中發現，Caln1在能與Scyl1交互作用。本篇研究發現，Caln1能夠抑制表現於胚胎腎臟細胞中的電壓依賴型鈣離子通道CaV1.3、CaV2.2、CaV3.1以及電壓依賴型鈉離子通道NaV1.4之電流。此外，若將Caln1表現於初級培養之胚胎大腦皮質神經細胞中，由鈣離子影像實驗與動作電位紀錄發現，Caln1同樣能導致神經細胞活性受到抑制。此外，Caln1的表現並不會影響到CaV2.2的總體蛋白質表現量，卻能增加其在細胞膜上的表現量，而且在體外培養14日之初級培養的大腦皮質神經細胞中，Caln1的表現可以透過化學誘導之長期增強作用下被增強表現。然而，透過免疫沈澱試驗發現，Caln1並沒有辦法與CaV2.2或CaV3.1直接作用。前人研究發現，Caln1與Scyl1都會參與細胞內小泡的運輸，且分別影響由高基氏體到細胞膜上的運輸以及內質網到高基氏體的運輸過程。綜合之前的研究，此篇研究發現，Scyl1與Caln1皆能抑制CaV2.2之鈣離子電流，且共同表現於胚胎腎臟細胞中時，具有協同作用，使鈣離子電流完全被抑制。此外，也透過免疫染色實驗發現，Caln1與Scyl1能同時座落在細胞內部形成小泡狀構造，猜測此一構造是在高基氏體上。當同時將Caln1上激素相似構造與自體聚集構造突變時，Caln1與Scyl1共同表現時造成的小泡堆積結構便不存在。因此，Caln1對於調節細胞內電壓依賴型通道的運輸與電流控制進而影響神經細胞活性皆扮演重要的角色。然而，Caln1在電壓依賴型通道的運輸與電流調控上之關係仍需要更多的研究與發現。

關鍵字

電壓依賴型鈣離子通道 ； 電壓依賴型鈉離子通道 ； 調鈣素 ； 鈣離子依賴型不活化 ； Calneuron 1 ； Scyl-like 1

並列摘要

The Ca2+ influx through the voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major extracellular factor responsible for the elevation in intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. The inactivation phase of CaVs determines the amount of Ca2+ that enters the cell, and calmodulin (CaM) is known to be involved in the Ca2+-dependent inactivation process. However, how CaM modulates CaV2.2 is still unclear. Here, we expressed CaV2.2 with CaM or CaM mutants in HEK293T cells and measured the currents to characterize the inactivation. The results showed that CaV2.2 had a fast inactivation with Ca2+, but not Ba2+, as the charge carrier; when it was co-expressed with CaM mutants with a Ca2+-binding deficiency, the levels of inactivation decreased. Using GST-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of CaV2.2 in the presence or absence of Ca2+. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. The mutations in the CaM-binding site greatly reduced the current of CaV2.2 but could be rescued by CaM12 (Ca2+-binding deficiency at the N-lobe) overexpression; in addition, CaM12 enhanced the total expression level of CaV2.2, but the ratios between the membrane and total fractions remained unchanged. Together, our data suggest that CaM not only modulates the inactivation of CaV2.2 but also regulates its expression to control [Ca2+]i elevation for physiological activities.
Previous studies in our lab showed that calneuron 1 (Caln1), a Ca2+ binding protein with structure similar to CaM, inhibits the inward current of CaVs in chromaffin cells and interacts with Scyl1 verified by yeast-2-hybrid. Here we showed Caln1 inhibited the inward currents of CaV1.3, 2.2, 3.1, and NaV1.4 expressed in HEK293T cells, also, its overexpression suppressed neuronal activities in primary cultured cortical neurons. Caln1 did not directly interact with CaV2.2 and CaV3.1, however, the chemical LTP decreased the presence of channel proteins on the plasma membrane. Caln1 also can be upregulated in primary cortical neuron. Both Caln1 and Scyl1 has been reported to regulate the vesicle tracking from Golgi to plasma membrane and ER to Golgi, respectively. Here, the patch clamping results showed that Scyl1 inhibited the currents of CaV2.2 with Caln1. In the immunostaining experiment, Scyl1 and Caln1 colocalized and accumulated at the intracellular vesicles, considered to be the Golgi apparatus, however, Scyl1ΔKLHT, lacking kinase function and self-associated manner, eliminated this appearance. Therefore, Caln1 is important in the trafficking of CaV2.2 and inward current regulation of VGICs (voltage-gated ion channels), however, the correlation between trafficking and modulation of VGICs in controlling neuronal activities still remains the open questions.

並列關鍵字

CaV ； NaV ； Calmodulin ； Calcium-dependent inactivation ； Calneuron 1 ； Scyl-like 1

參考文獻

Alseikhan, B.A., DeMaria, C.D., Colecraft, H.M., and Yue, D.T. (2002). Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation. Proceedings of the National Academy of Sciences of the United States of America 99, 17185-17190.

Ariel, P., Hoppa, M.B., and Ryan, T.A. (2012). Intrinsic variability in Pv, RRP size, Ca2+ channel repertoire, and presynaptic potentiation in individual synaptic boutons. Front Synaptic Neurosci 4, 9.

Babu, Y.S., Bugg, C.E., and Cook, W.J. (1988). Structure of calmodulin refined at 2.2 A resolution. J Mol Biol 204, 191-204.

Bangaru, M.L., Meng, J., Kaiser, D.J., Yu, H., Fischer, G., Hogan, Q.H., and Hudmon, A. (2015). Differential expression of CaMKII isoforms and overall kinase activity in rat dorsal root ganglia after injury. Neuroscience 300, 116-127.

Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W., and Bax, A. (1992). Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31, 5269-5278.