Structural Basis For The State-Dependent Reactivity Of Engineered Cysteines In The Pore Of The Cftr Channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is the only known member of the ATP binding cassette transporter superfamily that functions as an ion channel. Its architecture is analogous to that of ABC exporters such as p-glycoprotein, MsbA and Sav1866, which export their substrates using an alternating access mechanism driven by catalytic cycles coupled to ATP hydrolysis. The opening and closing of the CFTR channel is also driven by ATP catalytic cycles. We studied the state-dependence of the reactivity of three engineered cysteines located at positions 334, 337 and 338 in transmembrane helix 6 (TM6) of CFTR toward externally-applied, thiol-directed reagents, in order to investigate the conformational changes associated with channel gating. The engineered cysteine at position 334 was more reactive in the closed state, consistent with the findings of Zhang et al. (2005; J. Biol. Chem. 280: 41997-42003), while the engineered cysteines at positions 337 and 338 were more reactive in the open state. These findings are consistent with the predictions of homology models of the CFTR channel based on the crystal structures of bacterial homologues captured in inward- and outward-facing states of the proteins. Funded by the National Institute of Diabetes and Digestive and Kidney Diseases, the Cystic Fibrosis Foundation, and the American Lung Association.