Intracellular mechanisms regulating cell surface expression of Na channels : Na channel subunit mRNA levels and intracellular trafficking.

调节 Na 通道细胞表面表达的细胞内机制：Na 通道亚基 mRNA 水平和细胞内运输。

• 批准号: 09670097
• 负责人: WADA Akihiko
• 金额: $ 2.05万
• 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: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670097/
• 关键词: Sodium channels; Up-and down-regulation; Gene expression; Northern blot; Western blot; Protein kinases; Calcium; Neuroprotective drugs; Sodium channel; C kinase family; Antiepileptics; Binding; Exocytosis

In cultured bovine adrenal medullary chromaffin cells (embryologically derived from the neural crest), we examined the mechanisms that regulate the density/function of cell surface Na channels, and Na channel alpha- and (beta_i-subunit mRNA levels, using ^3H-saxitoxin binding, ^<22>Na influx, Western and Northern blot analyses, nuclear run-on assay, and cytosolic [Ca]_i measurement.1 . Regulation by protein kinase C (PKC) : Thymeleatoxin (an activator of conventional PKC), Go6976 (an inhibitor of conventional PKC), phorbol esters and brefeldin A (an inhibitor of ADP ribosylation factor-l) were employed.(1) Translocation of PKC-alpha from cytosol to membranes accelerated endocytosis of cell surface Na channels. (2) Membrane association of PKC-epsilon decreased Na channel alpha-subunit mRNA level, followed by a rise of beta1-subunit mRNA level. (3) Fall of alpha-subunit mRNA level was due to the elevated degradation rate of its mRNA, but not to the reduction of its gene transcriptional r … More ate. (4) These effects of PKC-epsilon were dependent on the de novo synthesis of protein(s).2. Regulation by A-kinase : An activation of A-kinase raised the number of cell surface Na channels, which was dependent on protein synthesis, but not accompanied by changes in Na channel alpha- and beta_1-subunit mRNA levels. Na channels newly-incorporated into plasma membrane exhibited the same allosteric gating mechanism, as did the native Na channels.3. Regulation by [Ca]_i : A sustained rise of [Ca]_i caused by thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca-ATPase, lowered levels of cell surface Na channels, a- and betai-subunit mRNAs, whereas a transient increase of [Ca]_i caused by 2,5-di-(t-butyl)-l, 4-benzohydroquinone (DBHQ) had no effect.4. Regulation by neuroprotective drugs : Riluzole, and NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride] bound to the transmembrane segment 6 of domain I in Na channel a-subunit, and inhibited Na channel opening, thereby resulting in the reduction of Ca channel gating and catecholamine secretion. We are now studying whether/how these drugs could alter the density of cell surface Na channels. Less

在培养的牛肾上腺髓质嗜铬细胞（胚胎来源于神经嵴）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、细胞核连续试验和胞浆[Ca] i测定，研究了调节细胞表面Na通道密度/功能以及Na通道α和β i亚基mRNA水平的机制。蛋白激酶C（PKC）的调节：使用胸腺毒素（常规PKC的激活剂）、Go 6976（常规PKC的抑制剂）、佛波酯和布雷菲德菌素A（ADP核糖基化因子-1的抑制剂）。(1)PKC-α从胞浆到细胞膜的转运加速了细胞表面Na通道的内吞作用。(2)PKC-ATP的膜结合使Na通道α亚基mRNA水平降低，随后使β 1亚基mRNA水平升高。(3)α-亚基mRNA水平的下降是由于其mRNA降解速率的增加，而不是由于其基因转录水平的降低。 ...更多信息 吃过了(4)PKC-β的这些作用依赖于蛋白质的从头合成. A激酶的调节：A-激酶的激活增加了细胞表面Na通道的数量，这依赖于蛋白质的合成，但不伴随着Na通道α-和β_1-亚基mRNA水平的变化。新进入质膜的钠通道与天然钠通道具有相同的变构门控机制. [Ca]_i的调节：肌质网Ca-ATP酶抑制剂毒胡萝卜素（thapsigargin）引起[Ca]_i的持续升高，降低细胞表面Na通道、α-和β-亚基mRNA的水平，而2，5-二-（叔丁基）-1，4-苯并氢醌（DBHQ）引起的[Ca]_i的短暂升高没有影响.神经保护药物的调节：阿曲唑和NS-7 [4-（4-氟苯基）-2-甲基-6-（5-哌啶基戊氧基）嘧啶盐酸盐]与Na通道α-亚基结构域I的跨膜片段6结合，抑制Na通道开放，从而导致Ca通道门控和儿茶酚胺分泌减少。我们现在正在研究这些药物是否/如何改变细胞表面Na通道的密度。少

项目成果

Hideyuki Kobayashi et al.: "Inhibition by riluzole and NS-7 of voltage-dependent Na channels and catecholamine secretion in adrenal chromaffin cells." Japanese Journal of Pharmacology. 79(Suppl.I). 138 (1999)

Hideyuki Kobayashi 等人：“利鲁唑和 NS-7 抑制电压依赖性 Na 通道和肾上腺嗜铬细胞中儿茶酚胺的分泌。”

Akihiko Wada et al.: "Modulation of voltage-dependent sodium channel subunit mRNAs and their cell surface expression by extra- and intra-cellular signals. The adrenal chromaffin cells : archetype and exempler of cellular signalling in secretory control."

Akihiko Wada 等人：“通过细胞外和细胞内信号调节电压依赖性钠通道亚基 mRNA 及其细胞表面表达。肾上腺嗜铬细胞：分泌控制中细胞信号传导的原型和范例。”

Akihiko Wada et al.: "Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells." Naunyn-Schmiedeberg's Archives of Pharmacology. 357 (5). 526-531 (1998)

Akihiko Wada 等人：“利鲁唑选择性抑制电压依赖性钠通道和肾上腺嗜铬细胞中儿茶酚胺的分泌。”

Ryuichi Yamamoto et al.: "Up-regulation of sodium channel subunit mRNAs and their cell surface expression by antiepileptic valproic acid:activation of calcium channel and catecholamine secretion in adrenal chromaffin cells." Journal of Neurochemistry. 68.

Ryuichi Yamamoto 等人：“抗癫痫丙戊酸上调钠通道亚基 mRNA 及其细胞表面表达：激活肾上腺嗜铬细胞中的钙通道和儿茶酚胺分泌。”

Toshihiko Yanagita al.: "Novel role of protein kinase C α and ε in cell surface expression of sodium channel subunit in neural crest-derived cells." Japanese Journal of Pharmacology. 73(Suppi.I). 96P- (1998)

Toshihiko Yanagita 等人：“蛋白激酶 C α 和 ε 在神经嵴衍生细胞的细胞表面表达中的新作用”，《日本药理学杂志》73（Suppi.I）。