CLONING & REGULATION OF INTESTINAL NA+/H+ EXCHANGERS

本提案的长期目标是确定和研究 回肠Na+/H+交换器的调节。兔回肠绒毛细胞具有 功能不同的两种Na+/H+交换剂 （跨细胞Na吸收vs ph/容量调节），药理学 （阿米洛利敏感性）和定位（顶侧vs基底侧）。 的 顶膜Na+/H+交换器是中性NaCl吸收的一部分 其在牙周病中被抑制。 我们已经分离出 在Na+/H+交换缺陷的成纤维细胞中功能性表达， 编码兔回肠绒毛基底外侧膜Na ~*/H ~* 的cDNA 通过构建Na+/H+交换器（NHE）的cDNA文库，克隆了编码Na+/H+交换器的部分cDNA 相关蛋白（NHERP），可能是顶膜Na+/H+ 交换器 因此，我们建议获得全长cDNA编码 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是编码的 两个不同的基因。 我们将确认NHE和NHERP的功能， Na+/H+交换器通过瞬时和稳定转染cDNA进入 Na+/H+交换缺陷成纤维细胞; Na+/H+交换活性将 通过证明从酸中回收Na+依赖性来确定 通过细胞内pH敏感染料用荧光监测负荷， BCECF。 与回肠绒毛细胞相反，隐窝细胞不具有 顶膜Na+/H+交换器。 为了将 NHE和NHERP在回肠上皮中的生理作用，我们将 将NHE和NHERP信息定位于沿着绒毛隐窝轴的细胞中 通过使用NHE和NHERP cDNA的北方印迹和原位杂交 以NHE和NHERP为探针，通过Western blotting进行定位， 使用针对NHE和NHERP的抗体进行免疫细胞化学。 的 顶膜Na+/H+交换受蛋白激酶调节。 我们 将确定NHERP磷酸化在以下方面的功能作用： 药物对回肠钠吸收细胞钠/氢交换的调节 证明NHERP是一种磷蛋白， 第二信使激动剂对其磷酸化的影响 和体内磷酸化结合蛋白质印迹。 提供 鉴定磷酸化位点的基础 Na+/H+交换器，我们将确定NHERP中的磷酸氨基酸， C激酶和A激酶的升高。 那就 通过验证NHE和NHERP的结构与功能关系， NHE和NHERP是糖蛋白， 结构域和参与NHE的蛋白激酶调节的结构域， 通过缺失/表达分析的NHERP。

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.