CLONING & EXPRESSION OF EPITHELIAL NA+/H+ EXCHANGERS

大多数毛囊炎是通过抑制绒毛细胞刷毛而发生的 边界Na+/H+交换。 我们克隆了两个50%相同的兔回肠 绒毛细胞Na+/H+交换器：NHE-1和NHE-2。 NHE-1是基底外侧 Na+/H+交换体定位于回肠绒毛细胞基底外侧 在免疫印迹和免疫细胞化学研究中， 对阿米洛利抑制的敏感性，以及它被促分裂原激活。 NHE-2 最可能是兔回肠绒毛细胞Na+/H+的顶端亚型 NHE-2在兔肾脏和回肠中大量表达， 对阿米洛利不敏感。 本提案的总体目标是克隆人类根尖上皮细胞， Na+/H+交换器，检测其在人肠道中的表达及其 通过已知抑制刷状缘Na+/H+的第二信使进行调节 交易所 我们将首先证明兔NHE-2是根尖上皮细胞， 通过证明其在兔根尖膜上的定位， 回肠的免疫印迹和免疫细胞化学研究，通过显示， NHE-2仅在中性NaCl吸收上皮细胞中表达， 测定NHE-2对阿米洛利的K1和其内部pH设定点。 我们将 用NHE-2筛选人肾皮质和空肠cDNA文库， 试图克隆人类根尖交换器。 一个完整的克隆将是 在Na+/H+交换缺陷的中国人肺成纤维细胞中表达 线PS120以证明其编码为功能性Na+/H+交换剂， 允许成纤维细胞进行阿米洛利敏感的Na+/H+交换。 使用从人NHE-2构建的探针，在定量方法中， 逆转录-PCR分析，我们将表征 胃肠道组织活检中的人NHE-2信使RNA 正常成人和儿童空肠顶端Na+/H+ 交换缺乏型先天性慢性分泌性腹泻 我们的最终目标 将通过确定人NHE-2的第二信使调节， 如果在PS120成纤维细胞中稳定表达的人NHE-2可以被调节， 已知的抑制剂刷状缘Na+？H+交换，如cAMP、蛋白质 激酶C和Ca++/CaM激酶II。 我们将确定这种抑制是否 伴随着交换剂的磷酸化。 然后我们将 确定哪些是功能性推定的蛋白激酶共识 序列位于NHE-2的C-末端胞质尾，通过检查 这些激酶共有序列的单向连续缺失 序列对第二信使调节人NHE-2的能力的影响。

Most diarrheal diseases occur through inhibition of villus cell brush border Na+/H+ exchange. We have cloned two 50% identical rabbit ileal villus cell Na+/H+ exchangers: NHE-1 and NHE-2. NHE-1 is the basolateral Na+/H+ exchanger by its localization to the ileal villus cell basolateral membrane in immunoblotting and immunocytochemical studies, its marked sensitivity to amiloride inhibition, and its activation by mitogens. NHE-2 is most likely the apical isoform of the rabbit ileal villus cell Na+/H+ exchangers; NHE-2 is greatly expressed in rabbit kidney and ileum and it is less sensitive to amiloride. The overall GOALS of this proposal is to clone the human apical epithelial Na+/H+ exchanger, examine its expression in the human gut and its regulation by second messengers known to inhibit brush border Na+/H+ exchange. We will first prove that rabbit NHE-2 is the apical epithelial isoform by demonstrating its localization to the apical membrane of rabbit ileum in immunoblotting and immunocytochemical studies, by showing that NHE-2 is solely expressed in neutral NaC1 absorbing epithelia and by determining NHE-2's K1 of amiloride and its internal pH set point. We will use NHE-2 to screen human kidney cortex and jejunum cDNA libraries in an attempt to clone the human apical exchanger. A full length clone will be expressed in Na+/H+ exchange deficient Chinese Hamster Lung fibroblast cell line PS120 to demonstrate that it codes for a functional Na+/H+ exchanger, allowing the fibroblasts to perform amiloride sensitive Na+/H+ exchange. Using a probe constructed from human NHE-2 in a quantitative method of Reverse Transcription-PCR analysis we will characterize the expression of human NHE-2 messenger RNA in tissue biopsies from the gastrointestinal tract of normal adults and from the jejunum of children with apical Na+/H+ exchange deficient congenital chronic secretory diarrhea. Our final goal will be to define second messenger regulation of human NHE-2 by determining if human NHE-2, as stably expressed in PS120 fibroblasts, can be regulated by known inhibitors of brush border Na+?H+ exchange, such as cAMP, protein kinase C, and Ca++/CaM kinase II. We will determine if such inhibition is accompanied by the phosphorylation of the exchanger. We will then determine which are the functional putative protein kinase consensus sequences located on NHE-2's C-terminus cytoplasmic tail, by examining the effects that unidirectional serial deletions of these kinase consensus sequences have on the ability of second messengers to regulate human NHE-2.