Zifarelli Giovanni, Pusch Michael
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy.
EMBO Rep. 2009 Oct;10(10):1111-6. doi: 10.1038/embor.2009.159. Epub 2009 Aug 28.
ClC-5, an endosomal Cl(-)/H(+) antiporter that is mutated in Dent disease, is essential for endosomal acidification and re-uptake of small molecular weight proteins in the renal proximal tubule. Eukaryotic chloride channels (CLCs) contain two cytoplasmic CBS domains, motifs present in different proteins, the function of which is still poorly understood. Structural studies have shown that ClC-5 can bind to ATP at the interface between the CBS domains, but so far the potential functional consequences of nucleotide binding to ClC-5 have not been investigated. Here, we show that the direct application of ATP, ADP and AMP in inside-out patch experiments potentiates the current mediated by ClC-5 with similar affinities. The nucleotides increase the probability of ClC-5 to be in an active, transporting state. The residues Tyr 617 and Asp 727, but not Ser 618, are crucial for the potentiation. These results provide a mechanistic and structural framework for the interpretation of nucleotide regulation of a CLC transporter.
ClC-5是一种在内体性氯离子/氢离子反向转运体，在丹特病中发生突变，对于肾近端小管内体酸化和小分子蛋白质的再摄取至关重要。真核生物氯离子通道（CLCs）包含两个胞质CBS结构域，这些基序存在于不同蛋白质中，其功能仍知之甚少。结构研究表明，ClC-5可在CBS结构域之间的界面处与ATP结合，但迄今为止，尚未研究核苷酸与ClC-5结合的潜在功能后果。在此，我们表明，在inside-out膜片钳实验中直接应用ATP、ADP和AMP可增强由ClC-5介导的电流，且亲和力相似。核苷酸增加了ClC-5处于活性转运状态的概率。残基Tyr 617和Asp 727而非Ser 618对于增强作用至关重要。这些结果为解释CLC转运体的核苷酸调节提供了一个机制和结构框架。
