GASTROINTESTINAL AND RENAL CHLORIDE TRANSPORT PROCESSES

胃肠道和肾氯转运过程

• 批准号: 2144537
• 负责人: William Paul Dubinsky
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144537
• 关键词: biological transport; biomarker; chloride channels; electrolytes; epithelium; gastrointestinal absorption /transport; gel electrophoresis; membrane channels; membrane proteins; membrane transport proteins; molecular cloning; polymerase chain reaction; protein purification; protein reconstitution; protein sequence; renal tubular transport; sodium potassium exchanging ATPase; western blottings

Electrolyte transport by gastrointestinal and renal epithelial cells is mediated by an array of coupled transporters, ATPases and ion channels. Absorption generally occurs via coupled solute entry across the lumenal border driven by an inwardly directed Na+ gradient established by the (Na+ + K+)-ATPase on the serosal surface. Secretion of Cl- into the lumenal compartment is mediated by specific ion channels and provides the driving force for the osmotic movement of water. It is likely that similar elements mediate the volume regulatory responses to osmotic perturbations. Although the stimuli for these responses are well- defined, the modes of activation are only partly understood. Recently we have isolated a subcellular membrane fraction that is enriched in a single type of Cl- channel. The membrane has a characteristic protein composition thus antibodies that are specific for these membrane proteins allow their identification and localization to specific cell types. Although we do not know the function of these channels it is interesting to note that all of the cells and tissues thus far demonstrated to have these antigens are involved in Cl- secretion or absorption. In particular, T84, cells, a model secretory cell derived from a human colonic tumor, possess a comparable membrane fraction exhibiting Cl- channel activity. The hypothesis to be tested is that there are two levels of control of ion transport in these tissues: (1) direct activation of channels in the plasma membrane and (2) a change in the overall number of transporters in the plasma membrane by recruitment or cycling through a subcellular pool. The hypothesis will be tested by focusing on T84 cell Cl- transport. Antibodies and biochemical probes to specific membrane proteins will be used to examine membrane turnover in confluent, functionally intact T84 cell monolayers grown on permeable supports. The antibodies will be generated by purification of membranes and proteins from more readily accessible bovine tissue. The channel protein(s) will be purified using reconstitution to estimate purity. Relevant proteins, identified as having a functional role in reconstitution experiments, will be partially sequenced to provide information to synthesize oligonucleotide primers. Purified mRNA from bovine trachea will be used as template in the polymerase chain reaction to generate a cDNA specific for channel protein. The cDNA will provide true sequence information for synthesis of specific oligonucleotide probes to screen cDNA expression libraries.

胃肠和肾上皮细胞的电解质转运是 由一系列偶联转运蛋白、ATP酶和离子通道介导。 吸收通常通过偶联溶质穿过管腔进入而发生 边界由向内的Na+梯度驱动， (Na+ + K+）-ATP酶。 Cl-分泌到 内腔室由特定的离子通道介导， 水渗透运动的驱动力。 很可能 类似的元件介导对渗透压的体积调节反应， 扰动 虽然这些反应的刺激是很好的- 定义，激活的模式只是部分理解。 最近 我们已经分离出了一种亚细胞膜组分， Cl-通道单一。 膜上有一种特征性的蛋白质 因此，组合物中的抗体特异于这些膜 蛋白质允许它们被识别和定位到特定的细胞 类型 虽然我们不知道这些渠道的功能，但它是 有趣的是，到目前为止， 证明这些抗原参与Cl-分泌，或 吸收。 特别是，T84细胞，一种模型分泌细胞衍生的 来自人类结肠肿瘤，具有可比较的膜分数 表现出Cl-通道活性。 要检验的假设是，有两个层次的控制， 离子在这些组织中的转运：（1）直接激活通道， 质膜和（2）转运蛋白总数的变化 通过募集或循环通过亚细胞膜 池 将通过关注T84细胞Cl-来检验该假设。 运输 针对特定膜的抗体和生化探针 蛋白质将被用于检查融合中的膜更新， 功能完整的T84细胞单层生长在可渗透的支持。 抗体将通过膜纯化产生， 更容易获得的牛组织中的蛋白质。 信道 将使用重构来纯化蛋白质以估计纯度。 相关蛋白，被鉴定为在以下方面具有功能性作用： 重组实验，将部分测序，以提供 合成寡核苷酸引物的信息。 纯化的mRNA来自 牛气管将被用作聚合酶链反应的模板 以产生对通道蛋白特异的cDNA。 cDNA将提供 用于合成特定寡核苷酸的真实序列信息 探针筛选cDNA表达文库。