Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. In search of transformational drug therapies for CF patients, small-molecules have been identified that target defects in the expression and function of the commonest CF mutation F508del. However, relatively little is known about the mechanism of dysfunction of other CF mutations and their responses to small-molecules. Here, we investigate A561E, the second most common CF mutation in Portugal. CF patients homozygous for A561E have a clinical phenotype similar to that of F508del homozygotes (1, 2). Like F508del-CFTR, A561E-CFTR is a temperature-sensitive folding defect located in the first nucleotide-binding domain (1). To learn whether A561E perturbs CFTR thermostability and function, we investigated the single-channel activity of A561E-CFTR in excised membrane patches from BHK cells at 37 °C following rescue of its cell surface expression by low temperature incubation. Wild-type CFTR had robust channel activity that was stable over time. By contrast, A561E-CFTR exhibited paltry channel activity characterised by brief openings separated by prolonged closures that deactivated rapidly at 37 °C; like F508del-CFTR, A561E-CFTR rundown was complete within 8 minutes and irreversible. To learn whether small-molecules restore expression and function to A561E-CFTR, we studied the CFTR corrector VX-809 and the CFTR potentiators genistein, P2, P3, P4, P5 and P9 (all from Cystic Fibrosis Foundation Therapeutics (CFFT) except VX-809 (Selleck) and genistein (LC Laboratories)) using the iodide efflux technique. Incubation of BHK cells with VX-809 (3 μM) for 24-48 h at 37 °C restored some cell surface expression to F508del-CFTR, but little or none to A561E-CFTR. However, following low temperature incubation, all CFTR potentiators (genistein (50 μM), test potentiators (10 μM)) restored partial function to A561E- and F508del-CFTR. For wild-type CFTR, the rank order of potentiation was genistein ≥ P2 ≥ P9 ≥ P5 ≥ P4 > P3; for F508del-CFTR, it was genistein >> P2 ≥ P5 ≥ P4 ≥ P3 ≥ P9 and for A561E-CFTR, it was genistein > P4 > P9 ≥ P2 > P5 ≥ P3. We conclude that A561E perturbs CFTR thermostability and gating. Thus, the consequences of the A561E mutation are similar to those of F508del, despite the different impacts of the mutations on CFTR structure (3, 4). We further conclude that CFTR potentiators restore, albeit incompletely, function to A561E-CFTR, whereas the corrector tested had little or no effect. Therefore, transformational drug therapy for F508del- and A561E-CFTR will likely require different combinations of CFTR correctors and potentiators.