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

在培养的牛肾上腺髓质嗜铬细胞(胚胎学上来源于神经脊)中，我们通过~3H-萨曲霉毒素结合、Na内流、Western和Northern印迹分析、核启动试验和胞浆[Ca]_i测定，研究了细胞表面Na通道密度/功能和Na通道α和(β_i)亚单位mRNA水平的调节机制。蛋白激酶C的调节：胸腺细胞毒素(常规蛋白激酶C的激活剂)、Go6976(常规蛋白激酶C的抑制剂)、佛波酯和布雷菲尔丁A(腺苷二磷酸核糖基化因子的抑制剂-L)：(1)蛋白激酶C-α从胞浆转位到膜上，促进细胞表面钠通道的内吞。(2)膜结合的PKC-epsilon使Na通道α-亚单位mRNA水平降低，β1-亚单位mRNA水平升高。(3)α-亚基基因表达水平的下降是由于其基因转录的r-…降低，而是由于其基因的降解速率增加所致。吃得更多了。(4)PKC-epsilon的这种作用依赖于蛋白质的从头合成(S)。A-激酶的调节：A-激酶的激活增加了细胞表面钠通道的数量，这依赖于蛋白质的合成，但不伴随着钠通道α和β_1亚单位mRNA水平的变化。新掺入质膜的钠通道与天然钠通道表现出相同的变构门控机制。[Ca]_i的调节：肌浆网Ca-ATPase抑制剂thapsigargin引起的[Ca]_i持续升高，细胞表面Na通道、α和β亚基mRNAs水平降低，而2，5-二(叔丁基)-L，4-苯氢二酚引起的细胞内[Ca]_i的一过性升高无明显作用。神经保护药物利鲁唑和NS-7[4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine盐酸盐与钠通道α亚基结构域I跨膜段6结合，抑制钠通道开放，从而减少钙通道门控和儿茶酚胺的分泌。我们正在研究这些药物是否/如何改变细胞表面钠通道的密度。较少

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