Regulation of cell surface expression of voltage-dependent sodium channels by multiple calcium signalings : their mRNA levels and intracellular trafficking

通过多种钙信号传导调节电压依赖性钠通道的细胞表面表达：mRNA水平和细胞内运输

• 批准号: 12670092
• 负责人: WADA Akihiko
• 金额: $ 2.24万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670092/
• 关键词: Voltage-dependent Na^+ channels; Down-regulation; Ca^<2+> oscillation; Ca^<2+> signaling; Protein kinase; Protein phosphatase; Calpain; Neuroprotective drug; Ca^<2+>oscillation; Ca^<2+>signaling; Voltage-dependent sodium channels; Up-and down-r

In cultured bovine adrenal chromaffin cells, we studied this project, by using ^3H-saxitoxin binding, ^<22>Na influx, Western and Northern blot analyses, nuclear run-on assay, and cytoplasmic [Ca^<2+>]i measurement.(1) Protein kinas C (PKC) isoform-specific mechanisms for Na channel down-regulation : PKC-α accelerated internalization of cell surface Na channels. PKC-ε increased degradation of Na channel α- subunit mRNA (without changing its transcriptional rate) via de novo synthesis of short-lived protein(s), thus lowering α- subunit mRNA level.(2) Na channel down-regulation by [Ca^<2+>]i rise : its amplitude and duration : (a) Moderate and relatively prolonged rise of [Ca^<2+>]i activated cPKC-α, calcineurin and calpain, thus promoting internalization of Na channels via clathrin-coated vesicles. Large and sustained rise of [Ca^<2+>]i decreased Na channel α- and β_1- subunit mRNA levels. Small and transient rise of [Ca^<2+>]i had no effect on cell surface density of Na channels, (b) Calcineurin, and FKBP- and rapamycin-associated protein (FKBP), a serine/threonine protein kinase, were involved in the suppression of vesicular externalization of newly-synthesized Na channels from the trnas-Golgi network. (C) Allosteric gating of Na channels by veratridine, α- and β-scorpion venom and brevetoxin was not impaired in the down-regulated Na channels.(3) Neuroprotective NS-7 : gating inhibition and up-regulation of Na channels : NS-7 bound to domain I segment 6 of Na channel a-subunit, and inhibited Na channel gating, thus reducing gating of voltage-dependent Ca channels and exocytic secretion of catecholamines. Long-term treatment with NS-7 accelerated Na channel externalization, while inhibiting Na channel internalization (without changing Na channel subunit mRNA levels), thus up-regulating Na channels.

在培养的牛肾上腺嗜铬细胞中，我们通过^3 H-石房蛤毒素结合、^<22>Na内流、Western和北方印迹分析、核运行分析和胞浆[Ca^&lt;2+&gt;]i测量研究了这一项目。(1)蛋白激酶C（PKC）亚型特异性Na通道下调机制：PKC-α加速细胞表面Na通道的内化。PKC-ε通过重新合成短寿命蛋白，增加钠通道α亚基mRNA的降解（不改变其转录速率），从而降低α亚基mRNA水平。(2)[Ca^2+]i升高对Na通道的下调：幅度和持续时间：（a）[Ca^2+]i的中度和相对延长的升高激活cPKC-α、钙调神经磷酸酶和钙蛋白酶，从而促进Na通道通过网格蛋白包被的囊泡的内化。[Ca^&lt;2+&gt;]i的持续升高使Na通道α-和β_1-亚基mRNA水平降低。[Ca^&lt;2+&gt;]i的短暂小幅升高对Na通道的细胞表面密度没有影响。（B）钙调神经磷酸酶、FKBP和雷帕霉素相关蛋白（FKBP）（一种丝氨酸/苏氨酸蛋白激酶）参与了对trnas-Golgi网络中新合成的Na通道囊泡外化的抑制。(C)在下调的Na通道中，藜芦碱、α-和β-蝎毒和短尾蝎毒素对Na通道的别构门控没有受损。(3)神经保护NS-7：Na通道的门控抑制和上调：NS-7与Na通道α-亚基的结构域I片段6结合，抑制Na通道门控，从而减少电压依赖性Ca通道的门控和儿茶酚胺的胞外分泌。NS-7长期处理加速Na通道外化，同时抑制Na通道内化（不改变Na通道亚基mRNA水平），从而上调Na通道。

项目成果

Shiraishi S et al.: "Up-regulation of cell surface sodium channels by cyclosporin A, FK506, and rapamycin in adrenal chromaffin cells."Journal of Pharmacology and Experimental Therapeutics. 297・2. 657-665 (2001)

Shiraishi S 等人：“肾上腺嗜铬细胞中环孢菌素 A、FK506 和雷帕霉素对细胞表面钠离子通道的上调”。《药理学和实验治疗学杂志》297・2（2001 年）。

Yanagita T et al.: "Protein kinase C-α and -ε down-regulate cell surface sodium channels via differential mechanisms in adrenal chromaffin cells"Journal of Neurochemistry. 74. 1674-1684 (2000)

Yanagita T 等人：“蛋白激酶 C-α 和 -ε 通过肾上腺嗜铬细胞中的差异机制下调细胞表面钠通道”《神经化学杂志》74. 1674-1684 (2000)。

Hideyuki Kobayashi et al.: "Regulation of voltage-dependent sodium channel expression in adrenal chromaffin cells : involvement, of multiple calcium signaling pathways"Annals of The New York Academy of Science. (in press). (2002)

Hideyuki Kobayashi 等人：“肾上腺嗜铬细胞中电压依赖性钠通道表达的调节：多种钙信号传导途径的参与”纽约科学院年鉴。

Kobayashi H et al.: "Regulation of voltage-dependent sodium channel expression in adrenal chromaffin cells : involvement, of multiple calcium signaling pathways"Annals of The New York Academy of Sciences. (in press).

Kobayashi H 等人：“肾上腺嗜铬细胞中电压依赖性钠通道表达的调节：多种钙信号传导途径的参与”纽约科学院年鉴。

Seiji Shiraisdi et al.: "Up-regulation of cell surface sodium channels by cyclosporin A, FK506 and rapamycin in adrenal chromaffin cells."Journal of Pharmacology and Experimental Therapeutics. (accepted for publication on January 31,2001).

Seiji Shiraisdi 等人：“肾上腺嗜铬细胞中环孢菌素 A、FK506 和雷帕霉素上调细胞表面钠通道。”药理学和实验治疗学杂志。