Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 2011 Jul 1;286(26):22833-45. doi: 10.1074/jbc.M111.224998. Epub 2011 May 11.
ClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knock-out mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca(2+) (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO(3)(-), measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3.
ClC-5 是一种氯离子/质子交换体，主要表达和定位于肾近端小管的亚顶区内体。CLCN5 基因突变导致 Dent 病。Clcn5 敲除小鼠复制了 Dent 病的症状。小鼠中 ClC-5 的缺失与近端小管中 NHE3 的表面表达减少有关。这种变化的分子基础尚不完全清楚。在这项研究中，我们研究了 ClC-5 调节 NHE3 运输的机制。通过双光子显微镜确定了野生型 (WT) 和 Clcn5 KO 小鼠近端小管中 NHE3 的活性。与 WT 小鼠相比，Clcn5 KO 小鼠的基础和地塞米松刺激的 NHE3 活性降低，而增加细胞内 cAMP（forskolin）或 Ca（2+）（A23187）浓度对 NHE3 活性的抑制程度在 WT 和 Clcn5 KO 小鼠中没有差异。其次，通过单细胞管灌注测量 NHE3 依赖的 HCO₃⁻吸收，发现 Clcn5 KO 小鼠近端小管中的 NHE3 依赖性吸收减少。第三，通过细胞表面生物素化，在短发夹 RNA (shRNA) 质粒转染的袋貂肾细胞中研究了 NHE3 的运输。在 ClC-5 表达降低的袋貂肾细胞中，NHE3 的表面表达减少，而 NHE3 的总蛋白水平没有变化。甲状旁腺激素降低了 NHE3 的表面表达，但在 scramble 和 ClC-5 敲低细胞中观察到的减少程度和内吞作用的速率没有显著差异。然而，ClC-5 敲低细胞中 NHE3 的基础和地塞米松刺激的胞吐作用速率减弱。这些结果表明 ClC-5 在 NHE3 的胞吐作用中起关键作用。
