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。 关于我们 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：毒胡萝卜素（肌质网Ca-ATP酶抑制剂）引起的[Ca]_i持续升高，降低了细胞表面Na通道、α和β亚基mRNA的水平，而2，5-二-（叔丁基）-1，4-苯并对苯二酚（DBHQ）引起的[Ca]_i的短暂升高没有影响。4.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

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