Determining the Lipid Environment and Interactions of CFTR

Abstract

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the ABC-Transporter family's only chloride channel, whose deficiency results in Cystic Fibrosis (CF). Currently, the major cause of death for CF patients is lung failure, and thus focusing on the structure and function of CFTR in its pulmonary epithelial cell environment is of utmost relevance. While several high-resolution structures of CFTR have been solved, none of the structures in the activated state included an open pore enabling chloride conduction. Importantly, these structures were solved in a detergent environment, although some copurified lipids are resolved in some structures. Inspired by data indicating that CFTR's function is highly dependent on the lipid environment, our lab has set out to determine more specifically the lipid environment CFTR encounters and the effects of these lipids on CFTR activity. Thus, our hypothesis is that determining these lipid interactions will provide the information necessary to solve an open-pore CFTR structure and perform more accurate functional analyses. We are approaching this question using detergent-purified CFTR as well as styrene maleic acid lipid particle (SMALP)-purified CFTR. Detergent purification of CFTR would leave only lipids that are tightly bound to the protein, indicating their importance in structure and function. Expanding from these tightly bound lipids, SMALPs require no detergent, but rather “hole-punch” the cell membrane, granting insight into the native lipid neighborhood surrounding CFTR. After determining these lipids, we will conduct functional ATPase and Planar Lipid Bilayer reconstitution studies to determine the specific effects of the lipids on CFTR activity. Alongside these experiments, we are conducting experiments to determine the change in ceramide composition of cultured pulmonary epithelial cells from non-CF and CF subjects, to understand the role of lipid-mediated signaling on CFTR function. [CFF MCCART17G0, MCCART18G0; NIH F31-HL143863-01].