Abstract：
NaCCC2 transport proteins, including those from Drosophila melanogaster (Ncc83) and Aedes aegypti (aeCCC2), are an insect-specific clade with sequence similarity to Na+-K+-2Cl- cotransporters. Whereas the Na+-K+-2Cl- cotransporters and other cation-chloride cotransporters are electroneutral, recent work indicates that Ncc83 and aeCCC2 carry charge across membranes. Here, we further characterize the regulation and transport properties of Ncc83 and aeCCC2 expressed in Xenopus oocytes. In cation uptake experiments, Li+ was used as a tracer for Na+ and Rb+ was used as a tracer for K+. Li+ uptake of oocytes expressing either aeCCC2 or Ncc83 was greater than uptake in water-injected controls, activated by hypotonic swelling, and not inhibited by ouabain or ethyl cinnamate. Rb+ uptake of oocytes expressing either aeCCC2 or Ncc83 was not different than water injected controls. In oocytes expressing either aeCCC2 or Ncc83, Li+ uptake plateaued with increasing Li+ concentrations, with apparent Km values in the range of 10 to 20 mM. Following exposure to ouabain, intracellular [Na+] was greater in oocytes expressing aeCCC2 than in controls. Elevating intracellular cAMP (via 8-bromo-cAMP) in Ncc83 oocytes significantly stimulated both Li+ uptake and membrane conductances. Elevating intracellular cAMP in aeCCC2 oocytes did not affect Li+ uptake, but stimulated membrane conductances. Overall, these results confirm that the NaCCC2s resemble other cation-chloride cotransporters in their regulation and some transport properties. However, unlike other cation-chloride cotransporters, they carry charge across membranes.
摘要：
NaCCC2转运蛋白,包括果蝇（Ncc83）和埃及伊蚊（aeCCC2），是与Na-K-2Cl-协同转运蛋白具有序列相似性的昆虫特异性进化枝。而Na+-K+-2Cl-共转运蛋白和其他阳离子-氯化物共转运蛋白是电中性的，最近的工作表明，Ncc83和aeCCC2跨膜携带电荷。这里,我们进一步表征了在非洲爪鱼卵母细胞中表达的Ncc83和aeCCC2的调节和转运特性。在阳离子吸收实验中，Li+用作Na+的示踪剂,Rb+用作K+的示踪剂。表达aeCCC2或Ncc83的卵母细胞的Li摄取大于水注射对照的摄取，被低渗肿胀激活，不受哇巴因或肉桂酸乙酯抑制。表达aeCCC2或Ncc83的卵母细胞的Rb+摄取与水注射对照没有不同。在表达aeCCC2或Ncc83的卵母细胞中，Li+的摄取随着Li+浓度的增加而趋于稳定，表观Km值在10至20mM的范围内。在暴露于ouabain之后,表达aeCCC2的卵母细胞的细胞内[Na]比对照组高。Ncc83卵母细胞中细胞内cAMP的升高（通过8-溴-cAMP）显着刺激了Li摄取和膜电导。aeCCC2卵母细胞中细胞内cAMP的升高不影响Li的摄取，而是刺激膜电导。总的来说,这些结果证实，NaCCC2s在其调节和某些转运特性上类似于其他阳离子-氯化物协同转运蛋白。然而,与其他阳离子-氯化物共同转运者不同，它们携带电荷穿过膜。
