Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1).
Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1).
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DOI：
10.1074/jbc.m111.309211
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发表时间：
2012-01-13
期刊：
影响因子：
--
通讯作者：
Forbush, Biff
中科院分区：
文献类型：
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作者：
Monette, Michelle Y;Forbush, Biff
Na-K-Cl协同转运蛋白（NKCC 1）在大多数脊椎动物细胞中表达，并且在细胞体积和细胞内氯离子浓度的调节中至关重要。为了研究NKCC 1的结构和功能，我们在C末端内的两个位点用青色（CFP）和黄色（YFP）荧光蛋白标记该转运蛋白，并在稳定表达的人胚肾细胞系中测量荧光共振能量转移（FRET）。单标记和双标记NKCC 1均适当产生，运输至质膜，并表现出（86）Rb转运活性。当两种荧光探针被放置在同一个NKCC 1转运蛋白的C末端，我们记录了11%的FRET减少后，激活的转运蛋白。该结果清楚地证明了在对N末端磷酸化的调节反应期间C末端的移动。当我们在不同的NKCC 1构建体中分别引入CFP和YFP并在HEK细胞中共转染这些构建体时，我们观察到二聚体对之间的FRET，并且在转运蛋白激活后FRET分数降低为46%。定量地，这表明最大的FRET信号传导的移动是在二聚体对之间，这一观察结果得到了进一步实验的支持，其中双标记的构建体用未标记的NKCC 1共转染稀释。我们的研究结果表明，NKCC 1的调节伴随着二聚体协同转运蛋白C末端两个位置之间的大运动。我们认为NKCC 1 C末端参与了转运调控，二聚化可能在调控过程中发挥关键的结构作用。预计当与结构信息相结合时，我们的研究结果将为理解NKCC 1调控带来的构象变化提供一个模型。
The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.