ION TRANSPORT IN NERVE AND FIBROBLASTS

在这项建议中，我们将研究膜离子传输过程， 特别强调涉及氯离子的机理。我们会 不仅要研究与这种离子传输有关的基本的、机械的问题 过程，以及与这些离子传输过程相关的问题 人类的健康和疾病。 使用内部透析的鱿鱼巨型轴突，它可以直接进入 对于轴膜的内外表面，有几个重要的 与Na-K-Cl共转运体运行有关的机制问题 将会得到解决。将使用放射性同位素通量和电压钳制 来解决这些问题。我们将测试(A)高[Cl-]i是否作用于 更改外部和内部结合位点的表观亲和力 共转运的离子；(B)[Cl-]i升高的影响是否合理 对一种蛋白激酶的抑制。对可能产生的影响的审查 细胞内K+对Na-K-Cl共转运体功能的影响。这个离子 在假定的结合和释放顺序中占据关键位置。 因此，仔细检查它的影响将会产生相当大的影响。 关于离子结合和释放的实际机制 辅助传送器。该共转运蛋白的细胞收缩激活将是 学习。电压钳位和同位素磁通将结合起来检查 共传输通量的可能电压敏感性。 我们实验室的一个新方向是研究离子传输的作用 人成纤维细胞肿胀的发生机制 (巨细胞增大症)对人类巨细胞病毒感染的反应。在……里面 特别是，我们将讨论Na/H交换和Cl/HCO3的可能作用 在这种病理学的发展过程中。感染似乎会导致 MRC-5成纤维细胞内源性Na/H交换器对细胞的敏感性 缩水。我们将跟进一项初步调查结果，这表明 [CL-]我在这个效应中扮演了一个角色。病毒感染对人类健康的影响 将研究MRC-5细胞的音量调节能力。这些研究 涉及离子浓度和细胞的荧光探针的使用 以及放射性同位素的使用。

In this proposal we will study membrane ion transport processes, with particular emphasis on mechanisms involving the chloride anion. We will study not only basic, mechanistic issues related to such ion transport processes, but also issues that relate these ion transport processes to human health and disease. Using the internally dialyzed squid giant axon, which gives direct access to both the inner and outer faces of the axolemma, several important mechanistic issues related to the operation of the Na-K-Cl cotransporter will be addressed. Radioisotopic fluxes and voltage clamping will be used to address these issues. We will test (a) whether high [Cl-]i acts to change the apparent affinity of external and internal binding sites for the cotransported ions; (b) whether the effect of elevated [Cl-]i is due to inhibition of a protein kinase. An examination of possible effects of intracellular K+ on Na-K-Cl cotransporter function will be made. This ion occupies a key spot in the putative order of binding and release. Therefore, a careful examination of its effects will shed considerable light on the actual mechanism of ion binding and release by this cotransporter. Activation by cell shrinkage of this cotransporter will be studied. Voltage-clamping and isotopic fluxes will be combined to examine possible voltage sensitivity of cotransport fluxes. A new direction for our lab is to examine the role of ion transport mechanisms in the development of human fibroblast cell swelling (cytomegaly) in response to infection with the human cytomegalovirus. In particular, we will address the possible role of Na/H exchange and Cl/HCO3 in the development of this pathology. Infection appears to cause the endogenous Na/H exchanger in MRC-5 fibroblasts to become sensitive to cell shrinkage. We will follow up a preliminary finding that suggests a fall of [Cl-]i plays a role in this effect. The effects of the viral infection on the MRC-5 cell's ability to volume regulate will be studied. These studies involve the use of fluorescent probes for ion concentrations and cell volume as well as the use of radioisotopes.