Declaration of Independence by CFTR's Transmembrane Domains

Abstract

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) transporter superfamily in that it functions as an ATP-gated chloride channel. Recent cysteine scanning studies in our lab demonstrate that both the sixth transmembrane segment (TM6) and its C-terminal counterpart, TM12, play pivotal roles in chloride permeation and are also involved in gating conformational changes. Here, to study the functional role of TM1 in gating and permeation, we introduced cysteine residues into this TM and assessed their reactivity towards internally-applied thiol-directed methanethiosulfonate (MTS) reagents. Our initial cysteine scanning experiments (1-2 patches for each cysteine-substituted cysless-CFTR channels) identified four positive hits, including E92, K95, Q98 and L102, where the negatively-charged MTSES reacting with engineered cysteines diminishes ATP-induced current. Surprisingly, according to the CFTR topology based on hydropathy plots, all these positions reside in the external half of TM1- contrary to results with TM6 and TM12. We also found that modification by the positively-charged MTSET enhances macroscopic current in K95C-, Q98C-, and L102C-cysless-CFTR channels. Intriguingly, at the single-channel level, we observed that whereas the mutation L102C appears to destabilize the gate, deposition of the MTSET adduct decreases the single-channel current amplitude but increases the Po. Moreover, channel gating persisted even after a complete removal of ATP, raising the possibility that gating motion in the TMDs of modified L102C channels is independent of that in their NBDs. Finally, MTSET modification of L102C is state-dependent, meaning that L102C-cysless-CFTR channels do not respond to the treatment of MTSET in the absence of ATP. These findings suggest that either position 102 moves during gating or gating motion in other regions of CFTR alters its reactivity towards MTSET. Overall, our preliminary data reveal several interesting yet enigmatic aspects of TM1 that call for further studies.