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. 2018 Dec 13;9:1464. doi: 10.3389/fphar.2018.01464

FIGURE 4.

FIGURE 4

Electrophysiological evaluation of F508del-CFTR inhibition by spautin-1. (A) TEEC measurement in FRT cells expressing F508del-CFTR. Cells were treated with or without VX-809 (1 μM) and, in the last 3 h, with spautin-1 (10 or 20 μM) or vehicle. The graph reports the ΔTEEC value, i.e., the difference between TEEC after maximal stimulation with forskolin plus genistein and TEEC after block with PPQ-102 (see text and Supplementary Figure 3 for further details). Data are from four independent experiments (∗∗∗p < 0.001). (B) Results from patch-clamp experiments. The top part shows data obtained in the whole-cell configuration on FRT cells expressing F508del or wild type CFTR (representative traces and graphs reporting the normalized current measured at +100 mV). Cells expressing mutant CFTR were previously treated for 24 h with VX-809 (1 μM). During experiments, cells were acutely stimulated with forskolin (20 μM) plus genistein (50 μM). After full stimulation of mutant or wild type CFTR, the currents were measured for 15 min to check that the activity was stable. Then spautin-1 (20 μM) was added to the extracellular solution and the currents were measured for further 30–40 min. Spautin-1 elicited a significant decrease in membrane currents in F508del-CFTR but not in wild type CFTR cells. The graphs show the currents measured at the three relevant time points. Arrow shows the time of spautin-1 addition. Spautin-1 caused a significant decrease in F508del-CFTR currents (∗∗∗p < 0.001). The bottom part of panel B shows representative currents obtained in the inside-out configuration on FRT cells expressing F508del-CFTR. Currents were significantly inhibited by CFTRinh-172 (10 μM) but not by spautin-1 (data are representative of three similar experiments). (C) Effect of spautin-1 (20 μM) on human bronchial epithelial cells from a F508del-CFTR patient. Representative traces show short-circuit recordings from epithelia treated with/without VX-809 (1 μM) for 24 h. Where indicated, spautin-1 was added in the last 3 h. During recordings, F508del-CFTR was activated with 20 μM forskolin plus 1 μM VX-770 (F+V) and then blocked with 10 μM CFTRinh-172 (I). The graph reports the amplitude of the current drop elicited by the CFTR inhibitor (n = 4 separate experiments per condition; ∗∗p < 0.01).