Kloch Monika, Milewski Michał, Nurowska Ewa, Dworakowska Beata, Cutting Garry R, Dołowy Krzysztof
Department of Biophysics, Warsaw University of Life Sciences SGGW, Warsaw, Poland.
Cell Physiol Biochem. 2010;25(2-3):169-80. doi: 10.1159/000276549. Epub 2010 Jan 12.
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.
囊性纤维化跨膜传导调节因子（CFTR）是一种ATP结合盒（ABC）转运蛋白，作为一种cAMP激活的氯离子通道发挥作用。最近的CFTR门控模型预测，CFTR通道的开放需要ATP与CFTR的两个核苷酸结合结构域（NBD1和NBD2）结合，而NBD2处的ATP水解会导致随后的通道关闭。在大多数ABC蛋白中，ATP的有效水解需要位于NBD C端部分的H环内存在不变的组氨酸残基。然而，迄今为止，NBD2相应区域（H环）对CFTR通道门控的贡献尚未得到研究。在这里，我们报告NBD2的H环中保守二肽HR基序的丙氨酸取代（HR→AA）导致CFTR通道的开放状态延长，表明H环是有效通道关闭所必需的。另一方面，在全细胞膜片钳分析中记录到，HR→AA取代导致转染的HEK 293细胞中CFTR介导的电流密度（pA/pF）大幅降低。这些结果表明，NBD2的H环除了是CFTR通道关闭所必需的外，可能还参与调节CFTR向细胞表面的转运。
