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

在培养的牛肾上腺染色质细胞中，我们研究了这个项目，通过^ 3h -石蜡毒素结合，^<22>Na内流，Western和Northern印迹分析，核运行试验和细胞质[Ca^<2+>]i测量。(1)蛋白激酶C （PKC）亚型特异性下调Na通道的机制：PKC-α加速细胞表面Na通道内化。PKC-ε通过从头合成短寿命蛋白增加Na通道α亚基mRNA的降解（不改变其转录率），从而降低α亚基mRNA水平。(2) [Ca^<2+>]升高对Na通道的下调：其幅度和持续时间：(a) [Ca^<2+>]的适度和相对延长的升高激活了cPKC-α、钙调磷酸酶和钙蛋白酶，从而促进了Na通道通过网格蛋白包被囊泡的内化。[Ca^<2+>]i的持续大幅升高降低了Na通道α-和β_1-亚基mRNA水平。(b)钙调磷酸酶和丝氨酸/苏氨酸蛋白激酶FKBP-和雷帕霉素相关蛋白（FKBP）参与抑制反式高尔基网络新合成的Na通道的囊泡外化。(C)缬草碱、α-和β-蝎毒和短尾蛇毒对Na通道的变构门控在下调的Na通道中不受损害。(3)神经保护性NS-7：门控抑制和上调Na通道：NS-7结合Na通道a亚基I结构域第6段，抑制Na通道门控，从而降低电压依赖性Ca通道的门控和儿茶酚胺的胞外分泌。长期使用NS-7可加速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。

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.