On the Mechanism of CFTR Inhibition by a Thiazolidinone Derivative

We employed inside-out patch-clamp technique to investigate the mechanism of CFTR inhibition by a thiazolidinone derivative, CFTRinh-172. We found that application of CFTRinh-172 results in an increase of the mean closed time, and a decrease of the mean open time of WT-CFTR channels. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. We have shown that though CFTRinh-172 can bind to both open and closed state of the channel, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance following binding of the inhibitor to the channel. Our data also indicate that stabilization of the open state leads to more potent inhibition: K1/2 is reduced to nanomolar range for locked-open CFTR channels. Interestingly, we found that although there is >95% homology between pig and human proteins, pig CFTR is significantly less sensitive to CFTRinh-172 (K1/2 = 8.05 ± 3.38 μM for pig CFTR vs K1/2 = 1.24 ± 0.14 μM for human CFTR). This result is especially puzzling since the open time for pig CFTR is ∼5 times longer than that of human CFTR. Like its human counterpart, pig CFTR, locked open with PPi, becomes more sensitive to CFTRinh-172 (K1/2 = 2.21 ± 0.41 μM). However, compared to human CFTR locked open with PPi, pig channels are >100 fold less sensitive to the inhibitor. We also found that dog CFTR behaves very similarly to pig CFTR, including a longer open time and a lower sensitivity to CFTRinh-172. Sequence analysis and chimera approach allowed us to reveal the region between the R- and NBD2- domains as largely responsible for this difference in sensitivity. Experiments further identifying responsible amino acid residue(s) are under way.