Citation

  • Authors: Chin, S., Ramjeesingh, M., Hung, M., Ereno-Oreba, J., Cui, H., Laselva, O., Julien, J. P., Bear, C. E.
  • Year: 2019
  • Journal: Cells 8
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK-293-F
    Description: Human embryonic kidney Fibroblast
    Known as: FreeStyle 293-F, HEK293-F, 293-F

Method

The DNA at 50 µg was mixed in a 1:1 ratio with transfection reagent FectoPRO® and added to each 200 mL suspension culture at 0.8 x 10^6 cells per mL, as previously described (Ereño-Orbea et al. Nat Comm 2017; Ereño-Orbea et al. J Vis Exp, 2018). The cells were grown at 37°C shaking at 180 rpm with 8% CO2 for 24 h. The next day, the cells were treated with 10 mM sodium butyrate at 37°C shaking for 24 h. The HEK293F cells transfected with FLAG-CFTR-His were collected and spun down at 6000 rpm at 4°C for 30 min.

Abstract

The recent cryo-electron microscopy structures of zebrafish and the human cystic fibrosis transmembrane conductance regulator (CFTR) provided unprecedented insights into putative mechanisms underlying gating of its anion channel activity. Interestingly, despite predictions based on channel activity measurements in biological membranes, the structure of the detergent purified, phosphorylated, and ATP-bound human CFTR protein did not reveal a stably open conduction pathway. This study tested the hypothesis that the functional properties of the detergent solubilized CFTR protein used for structural determinations are different from those exhibited by CFTR purified under conditions that retain associated lipids native to the membrane. It was found that CFTR purified together with phospholipids and cholesterol using amphipol: A8-35, exhibited higher rates of catalytic activity, phosphorylation dependent channel activation and potentiation by the therapeutic compound, ivacaftor, than did CFTR purified in detergent. The catalytic activity of phosphorylated CFTR detergent micelles was rescued by the addition of phospholipids plus cholesterol, but not by phospholipids alone, arguing for a specific role for cholesterol in modulating this function. In summary, these studies highlight the importance of lipid interactions in the intrinsic activities and pharmacological potentiation of CFTR.

Pubmed