Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells
Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells
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DOI：
10.1113/jphysiol.2004.064311
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发表时间：
2004-08-01
影响因子：
5.5
通讯作者：
Lapointe, JY
中科院分区：
文献类型：
--
作者：
Bissonnette, P;Coady, MJ;Lapointe, JY
肌醇是一种相容的渗透压调节剂，可被细胞（如肾髓质）使用，这些细胞受到细胞外渗透压变化的挑战。为了积累大量的这种多元醇，细胞依赖于Na+依赖性转运蛋白，如SMIT 1。我们最近已经确定了第二个Na+-肌醇协同转运蛋白，SMIT 2，它提出了相应的最近描述的肾近端小管顶膜的运输特性。为了进一步表征该转运系统，我们用兔SMIT 2 cDNA转染Madin-Darby犬肾（MDCK）细胞，并选择具有高表达水平的稳定克隆。该细胞系的放射性标记的肌醇的积累比在天然MDCK细胞中观察到的大20倍。用SMIT 2转染的MDCK细胞对肌醇的亲和力（K-m = 334 μ m）略低于在表达SMIT 2的电压钳位非洲爪蟾卵母细胞中发现的亲和力（K-m = 120 μ m）。使用半渗透性过滤器进行的转运研究显示SMIT 2转运蛋白完全以顶端为靶点。这种顶端定位的SMIT 2的纯化兔肾刷状缘膜囊泡（BBMV）的运输研究证实。使用纯化的抗SMIT 2抗体，我们还能够在从SMIT 2转染的MDCK细胞纯化的BBMV的蛋白质印迹中检测SMIT 2蛋白（分子量= 66 kDa）。当进行24小时高渗处理（+200 mosmol l（-1））时，SMIT 2活性也被刺激5倍。因此，SMIT 2-MDCK细胞系似乎是研究SMIT 2生物化学和调控的有前途的模型。
Myo-inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na+-dependent transporters such as SMIT1. We have recently identified a second Na+-myo-inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin-Darby canine kidney(MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo-inositol by this cell line is 20-fold larger than that seen in native MDCK cells. The affinity for myo-inositol of MDCK cells transfected with SMIT2 is slightly lower (K-m = 334 mum) than that found in voltage-clamped Xenopus laevis oocytes expressing SMIT2 (K-m = 120 mum). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2-transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5-fold when submitted to 24 h hypertonic treatment (+200 mosmol l(-1)). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.