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: 6501276
• 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/6501276
• 关键词: alternatives to animals in research; axoplasm; immunoelectron microscopy; immunofluorescence technique; ion transport; lipid bilayer membrane; membrane fusion; neuronal transport; organelles; potassium channel; squid; synaptic vesicles; voltage gated channel

This project is concerned with turnover of ion channel proteins in nerve cells. Electrical signaling in the body occurs primarily by propagation of nerve impulses along axons. Therefore, an understanding of the mechanisms by which the ion channels that underlie the impulse are maintained in axons is important. We have been using the squid giant axon as a model system for this work. The classical voltage-gated potassium ion channel in this preparation has been localized using a 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.

这个项目涉及神经细胞中离子通道蛋白的周转。体内的电信号主要是通过神经冲动沿着轴突传播而发生的。因此，了解轴突中维持冲动背后的离子通道的机制是很重要的。我们一直使用鱿鱼巨型轴突作为这项工作的模型系统。用多克隆抗体定位了该制剂中经典的电压门控钾离子通道。轴索膜上可见广泛分散的强免疫荧光斑点：每25平方微米的膜表面积约有1个。我们还在轴浆内观察到点状免疫荧光，定位于沿神经长度方向的约25微米宽的柱(轴突直径~500微米)。免疫电子显微镜显示该柱内钾离子通道含有直径约20-30 nm的线性排列的转运小泡。使用以前的年度报告中描述的新技术从轴浆中分离出运输囊泡。在所有这些囊泡中，大约1%含有钾离子通道。这些准备工作缺乏突触小泡的经典v-陷阱--突触短缩蛋白。突触素存在于轴浆的另一部分。将转运囊泡掺入人工脂质双层，发现钾离子通道的活动与直接从轴突膜记录到的相似。运输囊泡可能通过构成性融合参与轴突蛋白(钾离子通道和其他蛋白)的循环。我们还分离到另一个含有较大小泡(d~150 nm)的轴浆组分--可能是内吞的--它可能将钾离子通道带回细胞体。