Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells
Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells
复制标题
抗疟药伯氨喹在 Madin-Darby 犬肾细胞中拯救claudin-16突变体D97S的紧密连接定位
DOI：
10.1038/s41598-019-46250-4
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Ikari A
中科院分区：
文献类型：
--
作者：
Marunaka K;Fujii N;Kimura T;Furuta T;Hasegawa H;Matsunaga T;Endo S;Ikari A
镁离子（Mg 2+）通过肾脏中亨利氏袢的粗升支（TAL）中的密蛋白-16（CLDN 16）被细胞旁重吸收。CLDN 16的遗传病症引起CLDN 16的错误定位，导致低镁血症。低镁血症除了给予镁外没有有效的治疗方法。在这里，我们寻找一种新的药物来恢复CLDN 16突变体的紧密连接定位。在CLDN 16的第一胞外环（ECL）中具有突变的D97 S突变体主要与内体标记共定位，而野生型（WT）CLDN 16与紧密连接的衔接蛋白ZO-1共定位。D97 S突变体的蛋白质稳定性低于WT。蛋白酶体抑制剂lactacystin可提高D97 S突变体的表达水平。内吞抑制剂增加了D97 S突变体的紧密连接定位。我们发现，伯氨喹，抗疟药，增加了蛋白质的稳定性和细胞表面定位的D97 S突变体，但定位的其他突变体，其中有突变的胞质结构域或第二ECL，不受影响。伯氨喹可增加D97 S muR表达细胞的跨上皮Mg 2+通量。伯氨喹降低了伴侣蛋白的表达、蛋白酶体活性和乳酸脱氢酶的释放，增加了活细胞的比例。相反，在表达WT CLDN 16的细胞中未观察到这些作用。这些结果表明伯氨喹增加了D97 S突变体的紧密连接定位，导致ER应激和细胞损伤的减少。伯氨喹可能成为选定的突变型CLDN 16患者的有效治疗药物。
Magnesium ion (Mg2+) is paracellularly reabsorbed through claudin-16 (CLDN16) in the thick ascending limb (TAL) of Henle’s loop in the kidney. Genetic disorders of CLDN16 cause mislocalization of CLDN16, resulting in hypomagnesemia. There is no effective treatment for hypomagnesemia except for magnesium administration. Here, we searched for a novel drug to restore tight junctional localization of a CLDN16 mutant. A D97S mutant, which has a mutation in the first extracellular loop (ECL) of CLDN16, was mainly colocalized with endosome marker, whereas wild-type (WT) CLDN16 was colocalized with ZO-1, an adaptor protein of tight junctions. The protein stability of the D97S mutant was lower than that of WT. The expression level of the D97S mutant was increased by lactacystin, a proteasomal inhibitor. Endocytosis inhibitors increased the tight junctional localization of the D97S mutant. We found that primaquine, an antimalarial agent, increased the protein stability and cell surface localization of the D97S mutant, but the localization of other mutants, which have mutations in the cytosolic domain or second ECL, was not affected. Transepithelial Mg2+flux was increased by primaquine in D97S mutant-expressing cells. The expression of chaperon proteins, proteasome activity, and lactate dehydrogenase release were decreased by primaquine, and the proportion of viable cells increased. In contrast, these effects were not observed in WT CLDN16-expressing cells. These results suggested that primaquine increases the tight junctional localization of the D97S mutant, resulting in a reduction in ER stress and cellular injury. Primaquine may become an effective treatment drug for selected patients with mutant CLDN16.