ORGANELLE TRANSPORT OF ION CHANNELS IN EXCITABLE CELLS

可兴奋细胞中离子通道的细胞器运输

• 批准号: 6111843
• 负责人: JOHN CLAY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6111843
• 关键词: alternatives to animals in research; axoplasm; cell motility; confocal scanning microscopy; fluorescence microscopy; fluorescent dye /probe; interference microscopy; ion transport; membrane channels; microtubules; neurofilament; neuronal transport; organelles; squid

The classical voltage-gated potassium ion channel in squid giant axons has been localized using an affinity purified polyclonal antibody. Widely dispersed spots of intense immunofluorescence were observed throughout the axonal membrane: ~1 per 25 square microns of membrane surface area. We also observed punctate immunofluorescence in the axoplasm which was localized to a ~25 micron wide column down the length of the nerve (axon diameter ~500 microns). Immuno-electron microscopy revealed potassium ion channel containing transport vesicles ~20-30 nm in diameter in linear arrays within this column. Transport vesicles were isolated from axoplasm using novel techniques described in previous annual reports. Approximately 1% of all such vesicles contained a potassium ion channel. These preparations lacked synaptobrevin, the classical v-snare of synaptic vesicles. Synaptobrevin was observed in another axoplasm fraction. Incorporation of transport vesicles into artificial lipid bilayers revealed potassium ion channel activity similar to that recorded directly from the axonal membrane. Transport vesicles may be involved in recycling of axonal proteins (potassium ion channels and other proteins) via constituitive fusion. We also have isolated another axoplasm fraction containing larger vesicles (d ~ 150 nm) - possibly endocytotic in origin - which may take potassium ion channels back to the cell body.

我国经典电压门控钾离子通道的研究 利用纯化的亲和力定位了鱿鱼巨型轴突 多克隆抗体。广泛分散的强烈的斑点 免疫荧光在整个轴突中观察到。 膜：每25平方微米的膜表面积~1。 我们还观察到轴浆内的点状免疫荧光。 它被定位在一根约25微米宽的柱子上 神经(轴突直径~500微米)。免疫电子 显微镜显示钾离子通道含有转运 该柱内的囊泡呈线状排列，直径约20-30 nm。 从轴浆中分离运输囊泡 在以前的年度报告中描述的技术。大约1% 在所有这些囊泡中都含有一个钾离子通道。这些 准备工作缺乏突触的经典v-陷阱--突触短缩蛋白 水泡。突触素存在于轴浆的另一部分。 将转运囊泡掺入人工脂双层 显示的钾离子通道活动与记录的相似 直接从轴突细胞膜。运输囊泡可能是 参与轴突蛋白(钾离子通道)的循环 和其他蛋白质)。我们还孤立了 另一个轴浆部分含有较大的小泡(d~150 nm)- 可能来源于内吞--可能需要钾离子 通道回到细胞体。