Activation of the Bumetanide-sensitive Na+, K+,2Cl- Cotransporter (NKCC2) Is Facilitated by Tamm-Horsfall Protein in a Chloride-sensitive Manner
Activation of the Bumetanide-sensitive Na+, K+,2Cl- Cotransporter (NKCC2) Is Facilitated by Tamm-Horsfall Protein in a Chloride-sensitive Manner
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DOI：
10.1074/jbc.m111.222968
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发表时间：
2011-08-26
影响因子：
4.8
通讯作者：
Bachmann, Sebastian
中科院分区：
文献类型：
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作者：
Mutig, Kerim;Kahl, Thomas;Bachmann, Sebastian
Na+，K+，2Cl（-）协同转运蛋白（NKCC 2）是NaCl跨升支上皮的主动转运蛋白。NKCC 2的活性由加压素（AVP）或细胞内氯离子浓度决定，包括其氨基末端磷酸化。已提出共表达的Tamm-Horsfall蛋白（THP）与NKCC 2相互作用。我们假设THP调节TAL中的NKCC 2活性。THP缺陷型小鼠（THP-/-）显示位于亚顶端囊泡中的细胞内NKCC 2丰度增加（与野生型（WT）小鼠相比为+47%），而NKCC 2的基线磷酸化显著降低（与WT小鼠相比为-49%），表明在不存在THP的情况下转运蛋白活性降低。具有低内源性THP水平和低基线NKCC 2磷酸化的培养TAL细胞显示，在用THP转染后，NKCC 2磷酸化急剧增加（+38%）沿着细胞内氯离子浓度的显著变化。在表达NKCC 2的青蛙卵母细胞中，与THP cRNA共注射显著增强了NKCC 2在低氯低渗应激下的活化（+112%对+235%）。V2受体激动剂（脱氨基-顺式-D-Arg加压素）对加压素V2受体通路的短期（30分钟）刺激导致WT小鼠和用THP转染的培养TAL细胞中NKCC 2磷酸化增强，而在THP不存在的情况下，在两个系统中，脱氨基-顺式-D-Arg加压素后NKCC 2磷酸化减弱。速尿沿着THP-/-小鼠远曲小管功能和结构适应的减弱作用支持了缺乏THP的TAL中NaCl重吸收受损的观点。总之，这些结果与THP在调节NKCC 2依赖性TAL盐重吸收功能中的容许作用是一致的。
Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na+, K+,2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP-/-) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112% versus +235%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP-/- mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.