*2.7. VX-770-Stimulated Currents of CFTR Variants are Dramatically Decreased by* ∆*RI<sup>L</sup> but not by* ∆*RIL-*∆*RE<sup>S</sup>*

Given the interesting and diverse results observed for the ∆RE<sup>S</sup> and ∆RI<sup>L</sup> under VX-809, next we tested the effects of potentiator VX-770 (Ivacaftor), an approved drug for CF patients with gating mutations (Figure 5B) on these variants and in combination with VX-809, for F508del/F508del patients. Most strikingly, our data show that VX-770/Forskolin (Fsk) significantly decreased the function of <sup>∆</sup>RIL-F508del-CFTRin comparison with potentiation by Gen/Fsk (Figure 5B,C) from 92 <sup>±</sup> 7% to <sup>58</sup> <sup>±</sup> 7% of wt-CFTR (Figure 5B,C; black line in Figure S3J). As ∆RES-F508del-CFTR was not processed, we did

not test the effect of VX-770 on this variant, but when the ∆RES-∆RIL-F508del-CFTR variant was assessed for its function after acute application of VX-770 with Fsk, a tendency for increase in its function (to 87 ± 3% of wt-CFTR) was observed *versus* its function under Gen/Fsk (78 ± 7% of wt-CFTR) (Figure 5B,C; black dotted line in Figure S3J).

The most dramatic result, however, was observed for the ∆RIL-G550E-F508del-CFTR variant which under VX-770/Fsk only had 4 ± 1% function of wt-CFTR, while under Gen/Fsk it had 76 ± 8% (Figure 5B,C; black line in Figure S3K). For ∆RIL-R1070W-F508del-CFTR this decrease was not observed (Figure 5B,C; black line in Figure S3L), and in fact this variant had a slightly higher function under VX-770 stimulation (72 <sup>±</sup> 4%) than <sup>∆</sup>RIL-F508del-CFTR (58 <sup>±</sup> 7%) (Figure 5B,C, black line in Figure S3H). These results suggest that somehow removal of RI<sup>L</sup> can increase the chances of VX-770 blocking the channel pore.

The delay in peak response of F508del- comparing with wt-CFTR was not corrected for ∆RIL-F508del-CFTR (min = 3), but it was corrected for ∆RIL-∆RES-F508del-CFTR (min = 2) (black and dotted black lines in Figure S3J, respectively). Interestingly, on the other hand ∆RIL-R1070W-F508del-CFTR showed the quickest peak response (at min = 1), in contrast to either R1070W-F508del-CFTR or ∆RIL-F508del-CFTR, both at min = 3.

Similarly, VX-770 also significantly decreased the function of <sup>∆</sup>RIL-wt-CFTR from 127 <sup>±</sup> 15% under Gen to 57 ± 6% (Figure S4).

These results imply that, VX-770-stimulated currents of CFTR variants are dramatically decreased by ∆RI<sup>L</sup> but not by ∆RIL-∆RES.
