ANALYSIS OF ION CHANNELS IN AXOPLASMIC ORGANELLES

轴浆细胞器离子通道分析

• 批准号: 3760255
• 负责人: J R 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/3760255
• 关键词: alternatives to animals in research; animal tissue; axoplasm; interference microscopy; ion transport; membrane channels; neurofilament; neuronal transport; organelles; potassium channel; sodium channel; transmission electron microscopy; voltage /patch clamp; western blottings

This project concerns the transport of ion channels in nerve axoplasm by intracellular organelles. Previous work in this project has demonstrated to distinct populations of organelles isolated from the axoplasm of squid giant axons; a putative anterograde organelle population having diameters in the 40-60 nm range, and a putative retrograde population having diameters in the 100-150 nm range. During the past year these two populations have been analyzed with electrophysiological and biochemical procedures. One key finding has been that immunoblots of the anterograde organelles display cross-reactivity to an antibody against kinesin, a 120 kD ATPase which translocates these organelles along microtubules in the nerve. No cross-reactivity to kinesin was observed in the putative retrograde organelles even though the antibody has been shown with differential interference contrast light microscopy to block transport of organelles in both the anterograde and retrograde directions. Preliminary work has also been carried out with antibodies raised against various portions of the C-terminal region of the rat brain potassium channel, Kv 2.1. One particular antibody shows specific crossreactivity in immunoblots to the retrograde organelles. However, the antibody does not block potassium current, l/K, in axons using the standard axial wire voltage-clamp technique, whereas other antibodies which do block l/K in the axon do not appear to exhibit cross-reactivity with either population of organelles in immunoblots. These results suggest that the l/K channel has biochemical differences depending upon its location, be it either in anterograde or retrograde organelles, or in the axolemma itself.

这项研究涉及神经轴浆中离子通道的运输。 胞内细胞器。这个项目之前的工作已经证明 从鱿鱼的轴浆中分离出不同种群的细胞器 巨大的轴突；一个假定的具有直径的顺行细胞器群 在40-60 nm范围内，并且假定的逆行种群具有 直径在100-150 nm范围内。在过去的一年里，这两位 对种群进行了电生理和生化分析 程序。一个关键的发现是顺行的免疫印迹 细胞器对激动素A_(120)抗体有交叉反应 KD-ATPase将这些细胞器沿微管运输到 很有胆量。未观察到与激动素的交叉反应 细胞器逆行，尽管抗体已被证实为 差示干涉对比光学显微镜阻断输运 细胞器的顺行和逆行方向。 初步的工作也已经开展，用来对抗的抗体 大鼠脑内钾离子C-末端的不同部分 Kv 2.1频道。一种特殊的抗体表现出特异性的交叉反应。 在免疫印迹中进入逆行细胞器。然而，抗体会 用标准轴丝不阻断轴突内钾电流L/K 电压钳技术，而其他阻断L/K的抗体 轴突似乎没有表现出与这两个群体的交叉反应 免疫印迹中细胞器的数量。这些结果提示，L/K通道 有不同的生化差异取决于它的位置，无论是在 顺行或逆行细胞器，或在轴膜本身。