Structural/functional insights derived from studies of human and zebrafish CFTR orthologs
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels play a critical role in regulating the trans-epithelial movement of water and electrolyte in exocrine tissues and the malfunctions of CFTR result in cystic fibrosis, the most prevalent lethal autosomal recessive hereditary disease among the Caucasian populations. Despite decades of biochemical and biophysical studies of CFTR, our understanding of CFTR’s structure and gating mechanism remains limited. My dissertation research started with a focus on the structural and functional contribution of the fifth transmembrane segment (TM5) of human CFTR in forming the chloride permeation pathway and continued into tackling the role of the electrostatic profile in the pore for anion flux. These studies have led to the following conclusions: First, TM5 indeed contributes to forming the chloride permeation pathway but TM7 does not line the pore. Unlike the well-studied TM1, TM6, and TM12, the six identified pore-lining residues (A299, R303, N306, S307, F310 and F311) in TM5 only line the internal vestibule of the pore, not the narrow region nor the outer vestibule. Second, changing the side-chain properties of several pore-lining residues along TM5 resulted in channels with two distinct subconductance levels, small conductance O1 state and large conductance O2 state, respectively. Intriguingly, the preferred gating transition C→O1→O2→C over C→O2→O1→C (namely the O1O2 phenotype) as reported previously for R352 mutations suggests the existence of an irreversible gating process attributed to the input of the free energy from ATP hydrolysis. Then the timely solution of the cryo-EM structures of human and zebrafish CFTR offers a great opportunity to interweave the structural and functional data for a comprehensive understanding of CFTR. Taking a full advantage of this opportunity, I launched a thorough investigation of zebrafish CFTR to fill the blank of functional data for this ortholog. Although current cryo-EM data show minimal structural differences between human and zebrafish CFTR, my electrophysiological characterization of zebrafish CFTR revealed unexpected functional differences, which subsequently offer novel mechanistic insights regarding the mechanism of CFTR gating by ATP binding and hydrolysis.