Probing Structure and Conformational Changes in the Extracellular Loops of CFTR

Abstract

CFTR is a member of the ABC transporter family, and as such bears closer homology to transporters than to other channels. However, it is unknown whether, like transporters, the transmembrane domains of CFTR undergo an inward-to-outward facing transition upon NBD dimerization. To test this, we made cysteine substitutions in the extracellular loops (ECL) 1 and 4, which appose each other in inward-facing but not outward-facing structures of ABC transporters. We found that the macroscopic current of D110C/K892C-CFTR channels (but not corresponding single mutants) was increased in the presence of DTT. Multichannel patch recordings revealed that DTT increased NPO of double cysteine mutant channels, indicating that they were locked into a closed state by a spontaneous disulfide bond prior to DTT. To further understand the kinetics of the interaction, we applied Cd2+ trapping and found that D110C alone is transiently bound by Cd2+, causing a rapidly reversible 33 +/- 4.6% inhibition of current. By contrast, Cd2+ inhibited D110C/K892C-CFTR irreversibly and to a greater extent (72.5 +/-6.8%) under identical cumulative exposure. Results were consistent in 2 mM, 20 uM and 200 nM Cd2+. K892C was reversibly modified to a small extent (1.5 +/- 0.9%) and E115C (near the end of ECL1) was not functionally modified by Cd2+, but was labeled by MTS-TAMRA, indicating accessibility. The data suggest: 1) ECL1 and ECL4 come into close proximity in a closed state of CFTR; 2) K892 is located away from the conductance pathway of open CFTR; and 3) ECL1 is oriented such that D110 is closer to the pore than E115. Experiments are currently investigating the single channel effect of Cd2+ on ECL mutants and state-dependent kinetics of Cd2+ coordination by D110C/K892C and other ECL pairs. (NIH R01-DK 056481).