Positioning of the First Extracellular Loop of CFTR Has Significant Effects on CFTR Gating

Abstract

The first extracellular loop (ECL1) of CFTR contains several residues involved in stabilizing the open state of CFTR. D110 is positioned on the side of ECL1 nearest to the CFTR pore, extracellular to several amino acids in the first transmembrane helix important for chloride permeation. In the present study, we utilized cysteine mutagenesis and electrophysiology to observe real time effects of chemical manipulation on D110C-CFTR and on a double mutant of D110C with K892C in ECL4, across the CFTR pore. Via whole Xenopus oocyte TEVC recording, we found that the reducing agent DTT increased the conductance of D110C-CFTR 3.21 +/− 1.16 fold and D110C/K892C-CFTR 13.0 +/−3.5 fold. Treatment of both variants after DTT with Copper (II) Phenanthroline quickly inhibited them to a similar extent (∼75%). Single channel recordings without DTT showed that both mutants contain full conductance comparable to WT-CFTR, but significantly decreased mean burst duration. We previously reported modification of D110C/K892C channels with DTT led to increased openings in multichannel patches, without apparent effects on single channel conductance or open burst duration, indicating that DTT likely breaks a closed-state linkage between D110C and K892C. Modification of D110C-CFTR with DTT resulted in an increase in mean burst duration from 154ms to 369ms. Finally, via TEVC, we found that 20uM Cadmium inhibited DTT-treated D110C-CFTR that reversed within 30s of washout, whereas D110C/K892C-CFTR was inhibited irreversibly in the same context. WT, K892C-, D112C/K892C-, and E115C/K892C-CFTR were unaffected by DTT or Cd2+. We interpret our results to indicate the positioning of the pore-facing end of ECL1 is important for CFTR gating, and the more profound effects of DTT and Cd2+ on D110C/K892C-CFTR versus D110C-CFTR may also indicate ECL1 and ECL4 must separate during CFTR channel opening. Support: NIH 5R01DK56481