Zhang Zhihui, Baksh Michael M, Finn M G, Heidary David K, Richards Christopher I
Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506, United States.
Department of Chemistry, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
Biochemistry. 2017 Jan 10;56(1):240-249. doi: 10.1021/acs.biochem.6b00853. Epub 2016 Dec 21.
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the disease cystic fibrosis. Deletion of Phe508, the most prevalent mutation associated with this disease, disrupts trafficking of the protein. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase the level of trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. Real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low-temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however, the amount of channel in each vesicle remained the same. Direct binding measurements of VX-809 in native membranes using backscattering interferometry indicate tight binding to CFTR, which occurred in a manner independent of mutation. The similar values obtained for all forms of the channel indicate that the binding site is not compromised or enhanced by these mutations.
囊性纤维化跨膜传导调节因子（CFTR）的突变会导致囊性纤维化疾病。苯丙氨酸508缺失是与该疾病相关的最常见突变，它会破坏蛋白质的运输。小分子校正剂能使ΔF508 - CFTR向质膜的运输有适度改善。目前尚不清楚校正剂是通过改善运输囊泡的货物装载量还是通过直接与CFTR结合来提高运输水平。利用校正剂分子VX - 809、二次突变（I539T）和低温条件，对运输进行实时测量，以确定影响CFTR校正的化学、生化和热因素的作用机制细节。每种情况都能使ΔF508 - CFTR的运输改善至野生型水平的约10%。VX - 809与低温或I539T突变的组合可使质膜上CFTR的量增加至近40%，表明具有协同活性。到达表面的囊泡数量有显著变化；然而，每个囊泡中的通道量保持不变。使用背向散射干涉测量法对天然膜中VX - 809的直接结合测量表明，它与CFTR紧密结合，且这种结合方式与突变无关。所有形式通道获得的相似值表明，这些突变不会损害或增强结合位点。
