Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), such as F508del and G551D. Previous studies have demonstrated that the revertant mutations G550E and 4RK (the simultaneous mutation of four arginine-framed tripeptides: R29K, R516K, R555K and R766K) both rescue the processing and gating defects of F508del-CFTR [1]. Here we investigated the effects of G550E and 4RK on the CF mutation G551D, which disrupts profoundly channel gating [2]. To address our aim, we studied CFTR Cl- channels in inside-out membrane patches excised from BHK cells expressing wild-type (wt), G551D-, G550E-G551D- and 4RK-G551D-CFTR. The pipette (external) solution contained 10 mM Cl- and the bath (internal) solution contained 147 mM Cl-, 1 mM ATP and 75 nM PKA at 37 °C; voltage was -50 mV. When compared with wt-CFTR, the gating behaviour of G551D-CFTR is characterised by infrequent channel openings separated by very prolonged channel closures (wt, open probability (Po) = 0.467 ± 0.040, mean burst duration (MBD) = 149 ± 14 ms, interburst interval (IBI) = 166 ± 26 ms, mean ± SEM, n = 7; G551D, Po = 0.007 ± 0.001, MBD = 39 ± 7 ms, IBI = 5,452 ± 599 ms, n = 8). Although, neither G550E nor 4RK rescued the gating defects of G551D-CFTR, 4RK caused a significant increase in Po (G550E-G551D-, Po = 0.010 ± 0.001, n = 13; 4RK-G551D-, Po = 0.027 ± 0.005, n = 14; p < 0.05, Student’s t-test). However, both revertants altered dramatically the pattern of G551D channel gating. G550E- and 4RK-G551D-CFTR channels were characterised by very frequent, but very brief channel openings (G550E-G551D-, MBD = 27 ± 4 ms, IBI = 2,772 ± 301 ms, n = 13; 4RK-G551D-, MBD = 11 ± 1 ms, IBI = 535 ± 113 ms, n = 14; p < 0.05). In contrast to wt-CFTR, ATP fails to gate G551D-CFTR [2]. We therefore investigate whether revertant mutations restore ATP-dependence to G551D-CFTR. Like G551D, G550E-G551D was ATP-independent at low ATP concentrations. However, its activity was enhanced at 5 mM ATP unlike that of G551D-CFTR. Of note, the ATP-dependence of 4RK-G551D-CFTR was similar to that of wt-CFTR (wt, Kd = 210 ± 47 µM, n ≥ 5; 4RK-G551D, Kd = 209 ± 108 µM, n ≥ 6). In summary, the revertants G550E and 4RK altered the pattern of CFTR channel gating without rescuing channel activity. However, 4RK, but not G550E, restored the ATP-dependence of G551D-CFTR, suggesting that G550E and 4RK act by different mechanisms.