MOLECULAR REGULATION AND FUNCTION OF NA/H EXCHANGER-2

刷状缘（bb）Na+/H+交换器（NHE）是中性粒细胞的一部分。 说明肠基础钠的NaCl吸收过程 吸收，并成为不正常的牙周病。NHE 3已经 显示为基于其阿米洛利的功能性肠BB同种型 抵抗和蛋白激酶C的抑制。一项新的观察显示， NHE 2对肠钠吸收的贡献是基于 分离NHE 2和NHE 3对肠bb Na+/H+交换的活性 使用阿米洛利类似物H 0 E694。因此，我们建议进行研究， 确定NHE 2和NHE 3对小肠（兔）的贡献 回肠）和结肠（大鼠降结肠）bb Na+/H+交换 基础条件和使用模型条件来模拟 小肠对NaCl的吸收和bb型Na+/H+交换 消化和消化道疾病（刺激（表皮生长 因子）或通过抑制（卡巴胆碱通过蛋白激酶C））。进一步 了解生物化学和分子机制， 生理调节NHE 2活性，我们建议使用三种细胞 模型，兔回肠bb NHE 2，HT 29/NHE 2和PS 12 O/NHE 2（NHE 2稳定 在肠上皮HT 29/19 A细胞和NHE缺陷型PS中表达 120个细胞），以探索NHE 2的结构/功能关系：（1）与 研究NHE 2磷酸化在调节NHE 2活性中的作用， 分离细胞表面的二维磷酸肽图 和细胞内形式的NHE 2使用生物素化;（2）以确定 是否通过生长因子/蛋白激酶增加NHE 2的Vmax 是由于细胞表面NHE 2的周转数增加 和/或细胞表面交换剂的量的增加;（3） 研究辅助蛋白对NHE 2的调节机制：（a） 钙调素（CaM）：磷酸化，周转数和膜 NHE 2的改组将作为NHE 2抑制的机制进行探讨 通过CaM作为CaM与NHE 2体内结合的结果，证实了 通过免疫共沉淀。NHE 2的CaM结合位点将是 通过截短和定点突变研究鉴定。(b)p32和 p34：这两种磷蛋白与NHE 2的体内结合已经被证实。 通过免疫共沉淀建立。我们建议确定 这两个潜在的调节蛋白的身份;（4）映射 生长因子/蛋白激酶调节子亚结构域的组织 通过截短突变体研究确定NHE 2的推定C-末端。这些 亚结构域是磷酸化和/或结合 介导生长因子/蛋白激酶的相关蛋白 NHE 2的调节。

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.