The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion
The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion
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DOI：
10.1074/jbc.m603773200
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发表时间：
2006-09-29
影响因子：
4.8
通讯作者：
Gamba, Gerardo
中科院分区：
文献类型：
--
作者：
Pacheco-Alvarez, Diana;Cristobal, Pedro San;Gamba, Gerardo
肾脏 N-a+:Cl- 协同转运蛋白 rNCC 在人类疾病中发生突变，是噻嗪类利尿剂的治疗靶点，并且明显参与动脉血压调节。 rNCC 属于电中性阳离子偶联氯离子协同转运蛋白家族 (SLC12A)，该家族有两个具有逆生理功能和调节的主要分支：介导细胞 Cl- 流入的钠驱动协同转运蛋白（NCC 和 NKCC1/2）通过磷酸化激活，而介导细胞 Cl- 流出的钾驱动协同转运蛋白 (KCC) 通过去磷酸化激活。氨基末端结构域的三个苏氨酸残基簇与细胞内氯、细胞体积、加压素和 WNK/STE-20 激酶对 NKCC1/2 的调节有关。然而，rNCC 的监管机制尚不清楚。通过在非洲爪蟾卵母细胞中使用 rNCC 异源表达，我们发现两种独立的细胞内氯消耗策略使 rNCC 活性增加了 3 倍。两种策略的效果是协同且剂量依赖性的。增强型绿色荧光蛋白标记的 rNCC 的共聚焦显微镜显示 rNCC 细胞表面表达没有变化，而使用 R5-抗 NKCC1-磷酸抗体的免疫印迹分析显示 rNCC 氨基末端结构域苏氨酸残基 Thr(53) 和 Thr(58) 的磷酸化增加。消除这些苏氨酸与丝氨酸残基 Ser(71) 完全阻止了 rNCC 对细胞内氯消耗的反应。我们得出结论，rNCC 是通过涉及氨基末端结构域磷酸化的机制激活的。
The renal N-a+:Cl- cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl- influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl- efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.