Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3.
Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3.
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DOI：
10.1371/journal.pone.0055623
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Alexander RT
中科院分区：
文献类型：
--
作者：
Hayashi H;Tamura A;Krishnan D;Tsukita S;Suzuki Y;Kocinsky HS;Aronson PS;Orlowski J;Grinstein S;Alexander RT
钠氢交换器亚型3（NHE 3）介导钠、碳酸氢盐和水从肾和肠腔的吸收。这种活性是维持生理血浆pH和血压的基础。为了实现这一功能，NHE 3必须存在于肾小管和肠上皮细胞的顶膜中。这种定位的分子决定因素尚未最终确定，虽然已提出通过ezrin连接到顶端肌动蛋白细胞骨架。我们开始评估这个假设。在ezrin敲除小鼠（Vil 2kd/kd）上进行NHE 3活性的功能研究，并且检测到与野生型动物相似的NHE 3活性。由于存在其他ERM（ezrin/radixin/moesin）蛋白，因此难以解释该结果。因此，我们产生了上皮细胞培养模型，其中埃兹蛋白是唯一可检测的ERM。用siRNA敲低ezrin表达后，radixin和膜突蛋白表达仍然检测不到。与动物超微结构数据一致，缺乏ezrin的细胞保留上皮表型，但具有缩短和较厚的微绒毛。NHE 3定位与用非靶向siRNA转染的细胞相同。NHE 3的顶端细胞骨架的附件是不变的，通过光漂白后的荧光恢复（FRAP）和NHE 3在Triton X-100中的溶解度进行评估。基线NHE 3活性不变，然而，尽管NHE 3在丝氨酸552和605处被磷酸化，但NHE 3的cAMP依赖性抑制在很大程度上丧失。因此，埃兹蛋白对于NHE 3的顶端定位、与细胞骨架的附着、基线活性或cAMP诱导的磷酸化不是必需的，而是cAMP介导的抑制所需要的。
The sodium hydrogen exchanger isoform 3 (NHE3) mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2kd/kd) and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin) proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP) and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.
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影响因子：
11.4
作者：
Dransfield, DT;Bradford, AJ;Goldenring, JR
通讯作者：
Goldenring, JR
DOI：
10.1111/j.1440-1681.2004.04001.x
发表时间：
2004-05-01
影响因子：
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