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。

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.