Waldegger Siegfried, Jeck Nikola, Barth Petra, Peters Melanie, Vitzthum Helga, Wolf Konrad, Kurtz Armin, Konrad Martin, Seyberth Hannsjörg W
Department of Pediatrics, Philipps University of Marburg, Deutschhausstr. 12, 35033 Marburg, Germany.
Pflugers Arch. 2002 Jun;444(3):411-8. doi: 10.1007/s00424-002-0819-8. Epub 2002 Apr 9.
The term Bartter syndrome encompasses a heterogeneous group of autosomal recessive salt-losing nephropathies that are caused by disturbed transepithelial sodium chloride reabsorption in the distal nephron. Mutations have been identified in the NKCC2 (Na(+)-K(+)-2Cl(-)) cotransporter and ROMK potassium channel, which cooperate in the process of apical sodium chloride uptake, and ClC-Kb chloride channels, which mediate basolateral chloride release. Recently, mutations in barttin, a protein not related to any known ion transporter or channel, were described in BSND, a variant of Bartter syndrome associated with sensorineural deafness. Here we show that barttin functions as an activator of ClC-K chloride channels. Expression of barttin together with ClC-K in Xenopus oocytes increased ClC-K current amplitude, changed ClC-K biophysical properties, and enhanced ClC-K abundance in the cell membrane. Co-immunoprecipitation revealed a direct interaction of barttin with ClC-K. We performed in situ hybridization on rat kidney slices and RT-PCR analysis on microdissected nephron segments to prove co-expression of barttin, ClC-K1 and ClC-K2 along the distal nephron. Functional analysis of BSND-associated point mutations revealed impaired ClC-K activation by barttin. The results demonstrate regulation of a CLC chloride channel by an accessory protein and indicate that ClC-K activation by barttin is required for adequate tubular salt reabsorption.
巴特综合征这一术语涵盖了一组由远曲小管上皮细胞跨膜氯化钠重吸收紊乱引起的常染色体隐性失盐性肾病。已在NKCC2(钠-钾-2氯)协同转运蛋白和ROMK钾通道中发现突变,它们在顶端氯化钠摄取过程中协同作用,还在介导基底外侧氯释放的ClC-Kb氯通道中发现突变。最近,在巴特综合征的一种与感音神经性耳聋相关的变异型BSND中,发现了与任何已知离子转运体或通道无关的蛋白质barttin中的突变。在此我们表明,barttin作为ClC-K氯通道的激活剂发挥作用。在非洲爪蟾卵母细胞中,barttin与ClC-K共同表达增加了ClC-K电流幅度,改变了ClC-K生物物理特性,并增强了ClC-K在细胞膜中的丰度。免疫共沉淀揭示了barttin与ClC-K之间的直接相互作用。我们对大鼠肾切片进行原位杂交,并对显微解剖的肾单位节段进行逆转录聚合酶链反应分析,以证明barttin、ClC-K1和ClC-K2在远曲小管中的共同表达。对与BSND相关的点突变进行功能分析,结果显示barttin对ClC-K的激活受损。这些结果证明了一种辅助蛋白对CLC氯通道的调节作用,并表明barttin对ClC-K的激活是肾小管充分重吸收盐分所必需的。