ION TRANSPORT IN NERVE AND FIBROBLASTS

在这个建议中，我们将研究膜离子转运过程， 特别强调涉及氯阴离子的机制。我们将 不仅研究与这种离子传输有关的基本的、机械的问题， 过程，而且还涉及这些离子传输过程的问题， 人类健康和疾病。 使用内部透析的鱿鱼巨大轴突，它可以直接进入 在轴膜的内表面和外表面， 与Na-K-Cl协同转运蛋白的运作有关的机制问题 将得到解决。将使用放射性同位素通量和电压箝位 来解决这些问题。我们将测试（a）高[Cl-] i是否会 改变外部和内部结合位点的表观亲和力 （B）[Cl-] i升高的影响是否是由于 抑制蛋白激酶。 对可能产生的影响的审查 细胞内K+对Na-K-Cl协同转运蛋白功能的影响。该离子 在结合和释放的假定顺序中占据了关键位置。 因此，仔细研究其影响， 光的离子结合和释放的实际机制， 协同转运蛋白通过细胞收缩激活这种协同转运蛋白， 研究了将结合电压箝位和同位素通量来检查 共转运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 人成纤维细胞肿胀形成的机制 （巨细胞），以响应感染人巨细胞病毒。在 特别是，我们将讨论Na/H交换和Cl/HCO3的可能作用 在这种病理的发展过程中。感染似乎导致了 MRC-5成纤维细胞中的内源性Na/H交换器变得对细胞敏感 收缩我们将跟进一项初步调查结果， [Cl-] i在这种效应中起作用。病毒感染对 将研究MRC-5细胞体积调节能力。这些研究 涉及使用荧光探针检测离子浓度和细胞 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.