ORGANELLE TRANSPORT OF ION CHANNELS IN EXCITABLE CELLS

本项目涉及神经细胞中离子通道蛋白的周转。体内的电信号主要通过神经冲动沿着轴突传播而发生。 因此，了解轴突中维持冲动的离子通道的机制是很重要的。 我们一直在使用鱿鱼的巨大轴突作为这项工作的模型系统。 经典的电压门控钾离子通道在此制备已被本地化使用的多克隆抗体。在整个轴突膜上观察到广泛分散的强免疫荧光斑点：约1/25平方微米的膜表面积。 我们还观察到轴浆中的点状免疫荧光，其定位于神经长度下的~25微米宽的柱（轴突直径~500微米）。 免疫电子显微镜显示在该柱内线性阵列中含有约20-30 nm直径的转运囊泡的钾离子通道。 运输囊泡分离轴质使用新的技术在以前的年度报告中描述。 大约1%的囊泡含有钾离子通道。 这些制剂缺乏突触小泡蛋白，突触囊泡的经典v形圈套。在另一个轴浆组分中观察到小突触素。将运输囊泡纳入人工脂质双层显示钾离子通道活性类似于直接从轴突膜记录。 运输囊泡可能通过组成性融合参与轴突蛋白（钾离子通道和其他蛋白）的再循环。 我们还分离出另一个轴质部分，其中含有较大的囊泡（d ~ 150 nm）-可能起源于内吞作用-这可能会将钾离子通道带回细胞体。

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.