MOLECULAR REGULATION AND FUNCTION OF NA/H EXCHANGER-2

电刷边缘(BB)Na+/H+交换器(NHE)是中性的一部分 食盐吸收过程对肠道基础钠的影响 吸收，并在腹泻疾病中变得异常。NHE3已经被 根据其阿米洛利显示为功能性肠道BB亚型 蛋白激酶C的抗性和抑制作用一项新的观察显示 我们认为NHE2对肠道钠吸收的贡献是基于 肠道BB Na~+/H~+交换分离NHE2和NHE3活性 使用阿米洛利类似物H0E694。因此，我们建议进行研究，以 确定NHE2和NHE3对小肠(兔)的贡献 回肠)和结肠(大鼠降结肠)BB Na+/H+交换 基本条件和使用模型条件来模拟 肠道Na+/H+交换和Na~+/H~+交换 消化和腹泻疾病(刺激(表皮生长 因素)或通过抑制(通过蛋白激酶C的卡巴胆碱)。为了进一步 了解生物化学和分子机制 生理调节NHE2活性，我们建议使用三个细胞 模型兔回肠BB NHE_2、HT29/NH_2和PS120/NH_2(NH_2稳定 肠上皮HT29/19A细胞和NHE缺陷型PS的表达 120细胞)，探讨NHE2的结构/功能关系：(1) 研究NHE2磷酸化在调节NHE2活性中的作用 分离细胞表面的二维磷酸肽图 和胞内形式的NHE2使用生物素化；(2)确定 生长因子/蛋白激酶是否增加NHE2的Vmax 是由于细胞表面NHE2的周转数增加 和/或增加细胞表面交换器的数量； 辅助蛋白对NHE2调控机制的研究：(A) 钙调素(CaM)：磷酸化、周转数与膜 NHE2的改组将被作为抑制NHE2的机制进行探讨 用CaM研究CaM与NHE2的体内结合 通过免疫共沉淀法。NHE2的CaM结合部位为 通过截断和定点突变研究进行鉴定。(B)第32页及 P34：在体内，这两种磷酸蛋白与NHE2的结合已经被证实。 通过免疫共沉淀法建立。我们建议确定 鉴定这两个潜在的调控蛋白；(4)定位 生长因子/蛋白激酶调节亚域的组织 通过截断突变研究推测NHE2的C-末端。这些 亚区是潜在的磷酸化和/或结合位点 介导生长因子/蛋白激酶的相关蛋白 对NHE2的调控。

The brush border (bb) Na+/H+ exchanger (NHE) is part of the neutral NaCl absorptive process which accounts for intestinal basal Na absorption and becomes abnormal in diarrheal diseases. NHE3 has been shown to be the functional intestinal bb isoform based on its amiloride resistance and inhibition by protein kinase C. A new observation shown by us is the contribution of NHE2 to intestinal Na absorption based on separation of NHE2 and NHE3 activity on intestinal bb Na+/H+ exchange using an amiloride analogue, H0E694. Therefore, we propose studies to define the contribution of NHE2 and NHE3 to small intestinal (rabbit ileum) and colonic (rat descending colon) bb Na+/H+ exchange under basal conditions and using model conditions to mimic changes in intestinal NaCl absorption and bb Na+/H+ exchange which occur in digestion and in diarrheal diseases (stimulation (epidermal growth factor) or by inhibition (carbachol via protein kinase C)). To further understand the biochemical and molecular mechanisms that physiologically modulate NHE2 activity, we propose to use three cell models, rabbit ileal bb NHE2, HT29/NHE2 and PS12O/NHE2 (NHE2 stably expressed in intestinal epithelial HT29/19A cells and NHE deficient PS 120 cells), to probe structure/function relationship of NHE2: (1) to study the role of NHE2 phosphorylation in regulating NHE2 activity by two dimensional phosphopeptide mapping with separating the cell surface and intracellular forms of NHE2 using biotinylation; (2) to determine whether the increase in V max of NHE2 by growth factors/protein kinases is due to an increase in the turnover number of cell surface NHE2 and/or an increase in the amount of cell surface exchangers; (3) to study the mechanism of NHE2 regulation by accessory proteins: (a) calmodulin (CaM): phosphorylation, turnover number and membrane shuffling of NHE2 will be probed as mechanisms of inhibition of NHE2 by CaM as a consequence of in vivo binding of CaM to NHE2 demonstrated by co-immunoprecipitation. The CaM binding site of NHE2 will be identified by truncation and site directed mutant studies. (b) p32 and p34: In vivo association of these two phosphoproteins to NHE2 has been established by co-immunoprecipitation. We propose to determine the identity of these two potential regulatory proteins; (4) to map the organization of growth factor/protein kinase regulator subdomains on the putative C-terminus of NHE2 by truncation mutant studies. These subdomains are potential sites for phosphorylation and/or binding of associated proteins which mediate growth factor/protein kinase regulation of NHE2.