ABCMdb

PMID: 21078867

Kopeikin Z, Sohma Y, Li M, Hwang TC
On the mechanism of CFTR inhibition by a thiazolidinone derivative.
J Gen Physiol. 2010 Dec;136(6):659-71. Epub 2010 Nov 15., [PubMed]

Sentences

No. Mutations Sentence Comment

23 ABCC7 p.Glu1371Ser Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. Login to comment 66 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp However, it is strange that the degree of inhibition for G551D and G1349D was of the same order of magnitude as that of wild-type (WT)-CFTR, despite a very different closed time among these three channels (Bompadre et al., 2007). Login to comment 113 ABCC7 p.Asp1370Asn To further explore this relationship between the mean open time and the potency of CFTRinh-172, we tested CFTRinh-172 on a CFTR mutant, D1370N-CFTR, which exhibits an open time of 1 s (Bompadre et al., 2005). Login to comment 116 ABCC7 p.Asp1370Asn Fig. 4 B demonstrates the effects of 1 µM CFTRinh-172 on the macroscopic current of D1370N-CFTR. Login to comment 139 ABCC7 p.Glu1371Ser A similar observation was made with E1371S-CFTR channels locked open with ATP (Fig. 6 B). Login to comment 142 ABCC7 p.Asp1370Asn For D1370N-CFTR with an open time of 1 s, the IC50 is already in the nanomolar range. Login to comment 148 ABCC7 p.Glu1371Ser A similar observation was made with E1371S-CFTR, a hydrolysis-deficient mutant with an open time of 100 s (Fig. 5 B). Login to comment 149 ABCC7 p.Glu1371Ser Fig. 5 C shows the dose-response relationships of CFTRinh-172 for WT-CFTR locked open with ATP and PPi (green circles), or E1371S-CFTR locked open with ATP (blue squares). Login to comment 160 ABCC7 p.Asp1370Asn (A) Representative single-channel trace for D1370N-CFTR, a hydrolysis-deficient mutant with a mean open time of 1 s. Login to comment 161 ABCC7 p.Asp1370Asn (B) A continuous current recording showing a reversible inhibition of macroscopic D1370N-CFTR currents by 1 µM CFTRinh-172. Login to comment 162 ABCC7 p.Asp1370Asn (C) Dose-response relationships of CFTRinh-172 for WT-CFTR gated by ATP or P-ATP and D1370N-CFTR gated by ATP. Login to comment 165 ABCC7 p.Asp1370Asn Fitting parameters: for P-ATP-gated WT channels: IC50 = 0.37 ± 0.22 µM and Hill coefficient, n = 0.69 ± 0.38; for D1370N-CFTR: IC50 = 0.064 ± 0.007 µM and n = 1.26 ± 0.17. Login to comment 171 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser To further verify this slow recovery, we quantified this recovery phase using a phosphorylation-independent construct characterized previously (i.e., E1371S mutation in a construct without the R domain, or E1371S/R; see Bompadre et al., 2005). Login to comment 172 ABCC7 p.Glu1371Ser Again, the time constant of the current recovery for E1371S/R is 6 min (Fig. 6, C and D). Login to comment 173 ABCC7 p.Glu1371Ser Because the activity of E1371S/R-CFTR is independent of phosphorylation, in the following sections we will use Figure 6. Slow recovery from inhibition for the locked-open CFTR. Login to comment 175 ABCC7 p.Glu1371Ser (D) The diagram shows the average rate of recovery from inhibition by CFTRinh-172 for WT and E1371S CFTR locked in an open state. Login to comment 177 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser The mean rate constants of recovery are: 0.024 ± 0.003 s1 (n = 17) for ATP-gated WT-CFTR; 0.0042 ± 0.0011 s1 (n = 10) for WT-CFTR locked in an open state with ATP plus PPi; 0.0026 ± 0.0004 s1 (n = 18) for E1371S; and 0.0039 ± 0.0005 s1 (n = 18) for E1371S/R. Figure 5. Inhibition of locked-open CFTR by CFTRinh-172. Login to comment 178 ABCC7 p.Glu1371Ser CFTR channels were locked into an open state by using ATP plus PPi for WT-CFTR (A) or ATP for E1371S-CFTR (B). Login to comment 182 ABCC7 p.Glu1371Ser (C) Dose-response relationships of CFTRinh-172 for WT-CFTR locked open by PPi (green symbols) and E1371S-CFTR locked open with ATP (blue symbols). Login to comment 196 ABCC7 p.Glu1371Ser Again, E1371S-CFTR allows us to address this question. Login to comment 211 ABCC7 p.Glu1371Ser Once E1371S-CFTR channels were activated with PKA and ATP, the channels were subsequently inhibited by applying 5 µM CFTRinh-172. Login to comment 216 ABCC7 p.Glu1371Ser Some of those brief openings (Fig. 7, blue inset) probably represent ATP-independent events observed normally in the absence of ATP for E1371S-CFTR (Bompadre et al., 2005). Login to comment 218 ABCC7 p.Glu1371Ser Based on the idea that the open state represents an NBD dimer (Vergani et al., 2005; Zhou et al., 2006; Tsai et al., 2009), we conclude that CFTRinh-172 does not promote the Figure 7. Reopening of inhibited E1371S-CFTR channels upon removing CFTRinh-172. Login to comment 219 ABCC7 p.Glu1371Ser A continuous recording of E1371S-CFTR, a hydrolysis-deficient mutant with a prolonged open time, shows reopening of the inhibited channels in the absence of ATP upon removal of CFTRinh-172. Login to comment 245 ABCC7 p.Glu1371Ser However, unlike inhibitor-free E1371S channels that are locked open by ATP, the current from inhibitor-bound channels, first elicited by ATP, dropped by itself within a few seconds, as the inhibitor-bound open channels are now inactivated. Login to comment 246 ABCC7 p.Glu1371Ser Again, this observation is some- Figure 8. The binding and inhibition of CFTRinh-172 in the closed state of E1371S/R-CFTR channels. Login to comment 262 ABCC7 p.Glu1371Ser E1371S/R CFTR channels were opened with 1 mM ATP and then inhibited with 2 µM CFTRinh-172. Login to comment 284 ABCC7 p.Glu1371Ser (A) A continuous current trace of E1371S/R-CFTR channels showing that some of the inhibited channels recover from inhibition without reopening. Login to comment 286 ABCC7 p.Glu1371Ser (B) Faster recovery of inhibited E1371S/R-CFTR channels in the absence of ATP. Login to comment 346 ABCC7 p.Glu1371Ser In addition to the aforementioned differences between WT and E1371S channels regarding the mechanism of recovery from inactivation, Scheme 3 also predicts differences in inactivation per se. Login to comment 347 ABCC7 p.Glu1371Ser For E1371S-CFTR, because the closing rate is much slower than the rate of inactivation (i.e., Oinh→Cinh << Oinh→Iinh), the application of ATP to channels preexposed to CFTRinh-172 (Fig. 8) will generate a biphasic current response as a result of Cinh→Oinh→Iinh. Login to comment 386 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment 391 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel. Login to comment