CLONING AND REGULATION OF INTESTINAL NA+/H+ EXCHANGERS

这项建议的长期目标是确定和研究 回肠Na/H交换的调节。兔回肠绒毛细胞 两种功能截然不同的Na/H交换器 (跨细胞钠吸收与ph值/容量调节)，药理学 (阿米洛利敏感性)和定位(根尖与基底外侧)。这个 根尖膜钠氢交换器是中性氯化钠吸收的一部分 它在腹泻疾病中被抑制。我们已经隔离了 在Na/H交换缺陷的成纤维细胞中功能性表达 编码兔回肠绒毛基侧膜Na~*/H~*的基因 并克隆了编码Na/H交换器的部分基因 可能是顶膜Na/H的相关蛋白(NHERP) 交易所。因此，我们建议获得一个全长的编码基因 NHERP；分析NHERP的组织分布；并利用基因组 Southern印迹分析进一步证实NHE和NHERP编码 由两个不同的基因决定。我们将确认NHE和NHERP的功能 通过瞬时和稳定地将cDNA导入Na/H交换器 Na/H交换缺陷的成纤维细胞；Na/H交换活性将 通过证明依赖于钠的酸回收来确定 通过细胞内pH敏感染料的荧光监测负荷， BCECF.与回肠绒毛细胞相比，隐窝细胞没有 顶膜Na/H交换器。为了将它们关联起来 NHE和NHERP在回肠上皮中的生理作用，我们将 将NHE和NHERP信息定位于绒毛隐窝轴线上的细胞 用NHE和NHERP cDNA进行Northern杂交和原位杂交 通过Western blotting和免疫印迹技术定位NHE和NHERP 使用针对NHE和NHERP的抗体进行免疫细胞化学。这个 顶膜Na/H交换器受蛋白激酶的调节。我们 将确定NHERP的磷酸化在 丹参对回肠钠吸收细胞Na/H交换的调节作用 证明NHERP是一种磷酸化蛋白，并对其进行了研究 第二信使激动剂对其体外磷酸化的影响 体内磷酸化结合Western blotting。要提供 确定肌动蛋白磷酸化位点(S)的依据 Na/H交换器，我们将鉴定NHERP中 对C和A激酶升高的反应形式。那么，我们会 通过确认NHE和NHERP的构效关系来研究NHE和NHERP的构效关系 NHE和NHERP是糖蛋白和识别离子转运 NHE和NHE参与蛋白激酶调节的结构域和结构域 NHERP通过缺失/表达分析。

The long term GOAL of this proposal is to identify and study the regulation of ileal Na+/H+ exchangers. Rabbit ileal villus cells have two forms of Na+/H+ exchangers which are distinct in terms of function (transcellular Na absorption vs ph/volume regulation), pharmacology (amiloride sensitivity) and localization (apical vs basolateral). The apical membrane Na+/H+ exchanger is part of neutral NaCl absorption which is inhibited in diarrheal diseases. We have isolated and functionally expressed in fibroblasts deficient in Na+/H+ exchange, a cDNA encoding a rabbit ileal villus basolateral membrane Na*/H* exchanger (NHE) and cloned a partial cDNA encoding a Na+/H+ exchanger related protein (NHERP) which may be the apical membrane Na+/H+ exchanger. Therefore, we propose to obtain a full-length cDNA encoding the NHERP; analyze the tissue distribution of NHERP; and use genomic Southern blot analysis to further confirm that NHE and NHERP are encoded by two separate genes. We will confirm that NHE and NHERP function as Na+/H+ exchangers by transient and stable transfection of the cDNAs into the Na+/H+ exchange deficient fibroblasts; Na+/H+ exchange activity will be determined by demonstration of Na+-dependent recovery from an acid load monitored with fluoresence by the intracellular ph-sensitive dye, BCECF. In contrast to ileal villus cells, crypt cells do not have apical membrane Na+/H+ exchangers. In order to correlate the physiological roles of NHE and NHERP in the ileal epithelium, we will localize the NHE and NHERP messages in cells along the villuscrypt axis by Northern blotting and in situ hybridization using NHE and NHERP cDNAs as probes and localize NHE and NHERP by Western blotting and immunocytochemistry using antibodies raised against NHE and NHERP. The apical membrane Na+/H+ exchanger is regulated by protein kinases. We will establish the functional role of the phosphorylation of NHERP in regulation of Na+/H+ exchange in ileal Na+ absorbing cells by demonstrating that the NHERP is a phosphoprotein and studying the effects of second messenger agonists on its phosphorylation by in vitro and in vivo phosphorylation combined with Western blotting. To provide the basis for identification of the phosphorylation site(s) of the Na+/H+ exchanger, we will identify the phosphoamino acids in NHERP which form in response to elevation in C kinase and A kinase. Then, we will study the structure-funtion relationship of NHE and NHERP by confirming that NHE and NHERP are glycoproteins and identifying ion transporting domains and domains involved in protein kinase regulation of NHE and NHERP by deletion/expression analysis.