D'Andrea L, Lytle C, Matthews J B, Hofman P, Forbush B, Madara J L
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Nov 15;271(46):28969-76. doi: 10.1074/jbc.271.46.28969.
During intestinal chloride secretion, epithelial uptake of salts is accomplished largely by a bumetanide-sensitive Na:K:2Cl cotransporter designated here as NKCC. Using monoclonal antibodies directed against NKCC from the human crypt epithelial cell line, T84, we define its surface localization as a function of cotransporter activation. Immunoelectron microscopy, confocal localization, and selective surface biotinylation studies revealed that the 195-kDa NKCC protein is polarized to the basolateral domain. Following immunoprecipitation, several polypeptides coprecipitated with the 195-kDa cotransporter including two prominent proteins of molecular mass 160 and 130 kDa. Immunoblotting with three distinct anti-NKCC monoclonal antibodies in conjunction with deglycosylation experiments suggested that the 160- and 130-kDa bands represented novel proteins unrelated to the cotransporter. Stimulation of T84 monolayers with cAMP agonists, a condition which elicits chloride secretion and leads to microfilament-dependent NKCC activation, did not significantly increase the number of bumetanide-binding sites and only marginally increased surface expression of the 195-kDa cotransporter available for surface biotinylation. In contrast, cAMP agonist stimulation increased the surface expression of the coprecipitating 160- and 130-kDa proteins approximately 6-fold. The increase in surface 160- and 130-kDa proteins was attenuated by phalloidin preloading the cells, a condition which also prevents activation of NKCC without influencing the activity of other membrane transporters participating in chloride secretion. These studies define the polarized distribution of the NKCC protein on intestinal epithelia, indicate that NKCC may be associated with two other previously unidentified membrane proteins and such association is influenced by the F-actin cytoskeleton.
在肠道氯化物分泌过程中，上皮细胞对盐的摄取主要通过一种布美他尼敏感的钠钾氯共转运体（此处称为NKCC）来完成。利用针对人隐窝上皮细胞系T84中NKCC的单克隆抗体，我们确定了其表面定位是共转运体激活的一个函数。免疫电子显微镜、共聚焦定位和选择性表面生物素化研究表明，195 kDa的NKCC蛋白定位于基底外侧结构域。免疫沉淀后，有几种多肽与195 kDa的共转运体共沉淀，包括两种分子量分别为160 kDa和130 kDa的主要蛋白质。用三种不同的抗NKCC单克隆抗体进行免疫印迹并结合去糖基化实验表明，160 kDa和130 kDa的条带代表与共转运体无关的新蛋白质。用cAMP激动剂刺激T84单层细胞，这种情况会引发氯化物分泌并导致微丝依赖性NKCC激活，但并未显著增加布美他尼结合位点的数量，仅略微增加了可用于表面生物素化的195 kDa共转运体的表面表达。相比之下，cAMP激动剂刺激使共沉淀的160 kDa和130 kDa蛋白质的表面表达增加了约6倍。细胞预先加载鬼笔环肽可减弱表面160 kDa和130 kDa蛋白质的增加，这种情况也可防止NKCC激活，而不影响参与氯化物分泌的其他膜转运体的活性。这些研究确定了NKCC蛋白在肠道上皮细胞上的极化分布，表明NKCC可能与另外两种先前未鉴定的膜蛋白相关，并且这种关联受F-肌动蛋白细胞骨架的影响。
