Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16
Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16
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DOI：
10.1016/j.bbamem.2007.10.002
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发表时间：
2008-01-01
影响因子：
3.4
通讯作者：
Miwa, Masao
中科院分区：
文献类型：
--
作者：
Ikari, Akira;Okude, Chiaki;Miwa, Masao
肾镁主要通过Henle氏粗升支的细胞旁途径重吸收。claudin-16的表达增加Madin-Darby犬肾（MDCK）细胞中的镁转运。关于镁通过claudin-16转运的调控机制知之甚少。在这里，我们研究了多价阳离子敏感受体（CaSR）的细胞内分布和运输的镁的claudin-16的效果。使用Tet-OFF系统在MDCK细胞中稳定表达FLAG标记的claudin-16。镁、钙、新霉素和钆对CaSR的激活不影响FLAG标记的claudin-16、CaSR或紧密连接支架蛋白ZO-1的表达。这些激活剂降低了FLAG标记的密蛋白-16的磷酸丝氨酸水平以及FLAG标记的密蛋白-16与Z 0 -1的缔合。CaSR的激活导致PKA活性降低。免疫荧光显微镜显示，FLAG标记的claudin-16在未刺激的条件下分布在细胞-细胞边界，而它易位到细胞内区室，主要是溶酶体，与激活的CaSR。相反，ZO-1的分布不受活化的影响。FLAG标记的claudin-16的表达增加了跨上皮电阻（TER）和跨上皮镁转运，而不影响FITC-葡聚糖（MW 4000）通量。CaSR的激活降低TER和镁的转运，这是通过与二丁酰cAMP，一种膜渗透性cAMP类似物的共同治疗恢复。总之，CaSR激活可能降低PKA活性，导致磷酸化的claudin-16减少，claudin-16易位到溶酶体，镁重吸收减少。(c)2007 Elsevier B. V.保留所有权利。
Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption. (c) 2007 Elsevier B.V. All rights reserved.