**Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions**

**Inna Uliyakina , Hugo M. Botelho , Ana C. da Paula, Sara Afonso, Miguel J. Lobo, Verónica Felício , Carlos M. Farinha and Margarida D. Amaral \***

BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal; iiuliyakina@fc.ul.pt (I.U.); hmbotelho@fc.ul.pt (H.M.B.); acdapaula@fc.ul.pt (A.C.d.P.); scafonso@fc.ul.pt (S.A.); mglobo@fc.ul.pt (M.J.L.); vmfelicio@fc.ul.pt (V.F.); cmfarinha@fc.ul.pt (C.M.F.)

**\*** Correspondence: mdamaral@fc.ul.pt; Tel.: +351-217500861

Received: 22 May 2020; Accepted: 22 June 2020; Published: 25 June 2020

**Abstract:** Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region—the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region—regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing ∆RE and ∆RI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that ∆RI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.

**Keywords:** ABC transporters; drug action; regulatory extension; regulatory insertion; mechanism of action
