GASTROINTESTINAL AND RENAL CHLORIDE TRANSPORT PROCESSES

胃肠和肾上皮细胞的电解质转运 由一系列偶联转运蛋白、ATPase和离子通道介导。 吸收通常通过耦合的溶质进入管腔进行。 边界由向内定向的钠梯度驱动，由 浆膜表面(Na，K)-ATPase。将氯离子分泌到细胞内 腔室由特定的离子通道介导，并提供 水渗透运动的驱动力。很可能是 相似元件介导对渗透压的体积调节反应 微扰。尽管这些反应的刺激物很好- 定义，激活的模式只是部分了解。最近 我们已经分离出一种亚细胞膜组分，它富含一种 单一类型的氯离子通道。膜上有一种特有的蛋白质 因此，这些膜的特异性抗体的组成 蛋白质使它们能够识别和定位到特定的细胞 类型。虽然我们不知道这些通道的功能，但它是 有趣的是，到目前为止所有的细胞和组织 证明有这些抗原参与了氯离子的分泌或 吸收。特别是T84细胞，是一种模型分泌细胞 来自人类结肠肿瘤，具有类似的膜组分 表现出氯离子通道活性的。 需要检验的假设是，有两个水平的控制 这些组织中的离子传输：(1)直接激活这些组织中的通道 质膜和(2)转运体总数的变化 在质膜中通过重新募集或循环通过亚细胞 游泳池。这一假说将通过聚焦T84细胞Cl-来检验。 运输。针对特定膜的抗体和生化探针 蛋白质将被用来检查合流中的膜周转， 在可渗透的载体上生长的功能完整的T84细胞单层。 抗体将通过提纯细胞膜和 从更容易获得的牛组织中提取蛋白质。航道 蛋白质(S)将采用重组法进行纯化，以估计纯度。 相关蛋白质，被鉴定为在 重建实验，将进行部分测序以提供 合成寡核苷酸引物的信息。纯化的mRNAs 牛气管将作为聚合酶链式反应的模板 目的：获得通道蛋白的特异性cDNA.该cDNA将提供 合成特定寡核苷酸的真实序列信息 筛选c DNA表达文库的探针。

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.