NHE8 mediates amiloride-sensitive Na+/H+ exchange across mosquito Malpighian tubules and catalyzes Na+ and K+ transport in reconstituted proteoliposomes
NHE8 mediates amiloride-sensitive Na+/H+ exchange across mosquito Malpighian tubules and catalyzes Na+ and K+ transport in reconstituted proteoliposomes
复制标题
DOI：
10.1152/ajprenal.00487.2005
复制
发表时间：
2007-05-01
影响因子：
4.2
通讯作者：
Gill, Sarjeet S.
中科院分区：
文献类型：
--
作者：
Kang'ethe, Wanyoike;Aimanova, Karlygash G.;Gill, Sarjeet S.
在一顿血餐之后，蚊子埃及伊蚊将获得巨大的钠负荷，必须迅速排泄才能恢复离子动态平衡。这是一个需要强大的钠和流体输送能力的过程。尽管参与蚊子马氏管上皮细胞离子转运的成分尚未完全确定，但电生理学研究表明，Na+/H+交换器将阳离子挤出到管腔内，继而由小管顶膜中的V型H+-ATPase产生的质子梯度驱动。我们已经确定了可能的交易所，并将其命名为AeNHE8。免疫定位研究表明，AeNHE8表达于马氏管、胃盲囊和直肠的顶膜。当AeNHE8在缺乏Na+排泄和Na+/H+交换蛋白的盐敏感酵母细胞中异源表达时，AeNHE8拯救了盐敏感的表型，并恢复了细胞在高盐介质中的生长能力。此外，在NHE缺乏的成纤维细胞中异源表达AeNHE8导致对阿米洛利敏感的Na-22(+)摄取。为了确定交换器的动力学性质，我们从酵母细胞中重组了表达该蛋白到脂蛋白脂质体中的膜，并用荧光法测定了阳离子依赖的H+交换。我们的结果表明，AeNHE8介导了Na+和K+对H+的饱和交换。我们认为，AeNHE8可能与跨马氏管的向内H+梯度偶联，并在主细胞内维持稳定的细胞内pH的同时，在排泄过量的钠和钾方面发挥作用。
Following a blood meal, the mosquito Aedes aegypti will have acquired an enormous sodium load that must be rapidly excreted to restore ion homeostasis. It is a process that demands robust sodium and fluid transport capabilities. Even though the identities of the components involved in this ion transport across the mosquito Malpighian tubule epithelia have not been completely determined, electrophysiological studies suggest the contribution of a Na+/H+ exchanger extruding cations into the lumen driven secondarily by the proton gradient created by the V-type H+-ATPase in the tubules' apical membrane. We have identified the putative exchanger and designated it AeNHE8. Immunolocalization studies demonstrated that AeNHE8 is expressed in the apical membranes of Malpighian tubules, gastric caecae, and rectum. When heterologously expressed in salt-sensitive yeast cells lacking Na+ extrusion and Na+/H+ exchange proteins, AeNHE8 rescues the salt-sensitive phenotype and restores the cells' ability to grow in high NaCl media. Furthermore, heterologous expression of AeNHE8 in NHE-deficient fibroblast cells results in an amiloride-sensitive Na-22(+) uptake. To determine the exchanger's kinetic properties, we reconstituted membranes from yeast cells expressing the protein into lipid proteoliposomes and assayed for cation-dependent H+ exchange by fluorimetric methods. Our results indicate that AeNHE8 mediates saturable exchange of Na+ and K+ for H+. We propose that AeNHE8 may be coupled to the inward H+ gradient across the Malpighian tubules and plays a role in the extrusion of excess sodium and potassium while maintaining steady intracellular pH in the principal cells.