Direct Measurement of Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator to the Cell Surface and Binding to a Chemical Chaperone

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 AF508-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 Delta F508-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.