**1. Introduction**

Cystic Fibrosis (CF), a life-threatening recessive disorder affecting ~80,000 individuals worldwide, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein present at the apical membrane of epithelial cells. This is the only member of the ATP-binding cassette (ABC) transporter family functioning as a channel, more precisely a cyclic Adenosine Monophosphate (cAMP)-dependent chloride (Cl <sup>−</sup>)/bicarbonate (HCO<sup>3</sup> −) channel. It consists of two membrane-spanning domains (MSD1/2), two nucleotide binding domains (NBD1/2) and a cytoplasmic regulatory domain (RD), which is unique to CFTR [1]. The MSDs are linked via intra- and extra-cellular loops (ICLs, ECLs, respectively). ATP binding promoting NBD1:NBD2 dimerization preceded by phosphorylation of RD at multiple sites lead to channel gating [2]. The NBD1:NBD2 and ICL4:NBD1

interfaces were shown to represent critical folding conformational sites [3–5] important for the gating and maturation of the CFTR protein [5,6].

Although, >2000 CFTR gene mutations were reported (http://www.genet.sickkids.on.ca), one mutation–F508del–occurs in 85% of CF patients. This NBD1 mutant fails to traffic to the plasma membrane (PM) due to protein misfolding and retention by the endoplasmic reticulum quality control that targets it for premature proteasomal degradation. F508del-CFTR folding is a complex and inefficient process but it can be rescued, at least partially, by several treatments. These include low temperature incubation [7], genetic revertants [4,5,8–13] or pharmacological agents, like "corrector" VX-809 (lumacaftor) [14], one of the first CFTR modulator drugs to receive approval from the United States Food and Drug Administration in combination with potentiator VX-770/ivacaftor. Determining the additive/ synergistic rescue of F508del-CFTR by small molecule correctors together with other rescuing agents/revertants is very valuable to determine the mechanism of action of these CFTR modulators [5].

Comparative studies of CFTR with other ABC transporters are very powerful to understand the uniqueness of some of its regions, their influence on CFTR maturation and function as well as how they affect distinctive binding of CFTR to modulator drugs. Two such unique regions are present in NBD1 of CFTR, which are absent in NBDs of other ABC transporters (Figure S1)—the regulatory extension (RE) and regulatory insertion (RI). Both regions were described as highly conformationally dynamic [3,15] following PKA phosphorylation at certain residues (660Ser, <sup>670</sup>Ser, and <sup>422</sup>Ser) [16,17]. Importantly, removal of the 32-amino acid RI (∆RI) was reported to rescue traffic of F508del-CFTR [13].

Our first goal was to assess the impact of removing the 30 amino acid RE (∆RE) alone or jointly with ∆RI on F508del-CFTR trafficking. Because there is some controversy regarding the RI and RE boundaries [3,13,15,18–20] which have structural implications, we tested the short and long versions of both regions (Figure S1). Secondly, we aimed to evaluate how RE and or RI removal from F508del-CFTR influences the rescue of this mutant by genetic revertants. Our third and final goal was to determine how the traffic and function of these combined variants of F508del-CFTR (∆RE, ∆RI plus genetic revertants) are rescued by CFTR modulator drugs VX-809 (corrector) and VX-770 (potentiator) to gain further insight into their mechanism of action.

Our data show that although F508del-CFTR without RE did not traffic to the PM, it showed a dramatic stabilization of its immature form (evidencing a turnover rate ~2× lower than that of wt-CFTR. Results also show that, while ∆RI further increased processing of F508del-CFTR with revertants to almost wt-CFTR levels, RE removal completely abolished their processing, thus highlighting the different impact of the two dynamic regions on revertant-rescued F508del-CFTR. Most strikingly, although VX-809 rescued ∆RI-F508del-CFTR and ∆RE-∆RI-F508del-CFTR processing to wt-CFTR levels, to achieve maximal function of F508del-CFTR, removal of just RI was insufficient, as both RI and RE had to be absent from F508del-CFTR. These data indicate that removal of these two regions has a positive effect on the rescuing efficacy of F508del-CFTR by CFTR modulators.
