ABCMdb

PMID: 22508846

Jih KY, Sohma Y, Li M, Hwang TC
Identification of a novel post-hydrolytic state in CFTR gating.
J Gen Physiol. 2012 May;139(5):359-70. doi: 10.1085/jgp.201210789. Epub 2012 Apr 16., [PubMed]

Sentences

No. Mutations Sentence Comment

89 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Fig. S3 shows the open dwell-time histograms of WT- and W401F-CFTR. Login to comment 118 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe On the other hand, mutating the residue Trp401 (W401), which forms ring-ring stacking interactions with ATP in ABP1 (Lewis et al., 2004; Zhou et al., 2006), to phenylalanine significantly increased the propensity of state X in response to PPi (i.e., a higher percentage of channels locked open from state X; WT, 49 ± 2% and n = 13; W401F, 59 ± 2% and n = 13; P < 0.05). Login to comment 120 ABCC7 p.Trp401Phe Therefore, opposite to P-ATP, the W401F mutation enhances the responsiveness of state X to PPi but may destabilize the C2 state. Login to comment 135 ABCC7 p.Trp401Phe It is noteworthy that the success rate of this line of experiments is lower for WT channels (see supplemental Discussion for more details), but similar observations were made for WT-CFTR (Fig. S1), indicating that such open X-locked-open transitions are not just idiosyncratic for W401F mutant channels. Login to comment 147 ABCC7 p.Trp401Phe However, the conserved W401F mutation (see below), which significantly prolongs the open time (Tsai et al., 2010a), makes such experiments more feasible. Login to comment 148 ABCC7 p.Trp401Phe In the presence of ATP, the W401F-CFTR channel exhibits similar behavior as WT-CFTR: the channel opens into bursts that last for hundreds of milliseconds to seconds, and each opening burst is separated by a long interburst that also closes in the range of hundreds of milliseconds (Fig. 6). Login to comment 157 ABCC7 p.Trp401Phe (B) A similar protocol was conducted with W401F-CFTR channels. Login to comment 163 ABCC7 p.Trp401Phe For reasons unclear at this moment, the W401F mutation could somehow improve the responsiveness of state X to PPi (Fig. 5) and therefore presumably to ATP as well. Login to comment 164 ABCC7 p.Trp401Phe We therefore quantified the open burst time of W401F-CFTR at different [ATP]. Login to comment 166 ABCC7 p.Trp401Phe Furthermore, the notion that a prolonged open burst time seen with the W401F mutation is caused by reentry of the channel from state X predicts a disparity between the mean open time estimated from microscopic analysis and the relaxation time constant obtained from macroscopic current decay upon ATP removal, which prohibits reentry to occur. Login to comment 167 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Fig. 7 (B and C) shows that when fitting the current relaxation of W401F mac roscopic current after washout of 10 mM ATP, the time constant is almost identical to the mean open time of W401F channels in the presence of micromolar ATP. Login to comment 168 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Figure 6. Single-channel ligand exchange for W401F-CFTR. Login to comment 169 ABCC7 p.Trp401Phe (A and C) A single W401F-CFTR was activated by ATP, and the ligand was switched to a 1-s PPi (A) or AMP-PNP (C) pulse in the open state. Login to comment 188 ABCC7 p.Trp401Phe As shown in Fig. 5, the W401F mutation and the high-affinity ATP analogue P-ATP, both acting on ABP1, affect state X and the C2 state very differently. Login to comment 193 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Figure 7. The mean open time of W401F-CFTR is [ATP] dependent. Login to comment 194 ABCC7 p.Trp401Phe (A) Microscopic current traces of W401F-CFTR in the presence of 10 mM (top), 1 mM (middle), or 100 µM (bottom) ATP. Login to comment 195 ABCC7 p.Trp401Phe (B) Mean open time of W401F-CFTR at different [ATP] (closed circles). Login to comment 197 ABCC7 p.Trp401Phe (C)Macroscopic current of W401F-CFTR was activated by 10 mM ATP to a steady state. Login to comment 205 ABCC7 p.Trp401Phe Furthermore, the prolonged open burst time in the presence of higher [ATP] (>1 mM; P < 0.05 when comparing 1 and 10 mM) for W401F-CFTR indicates that the lifetime of state X as a closed state must be indistinguishable from that of the flickers so that closed events corresponding to state X are excluded by the analysis. Login to comment 217 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser These flickering closings have been long thought to be ATP independent, as they can be easily discerned in a complete absence of ATP within an opening burst of hydrolysis-deficient mutants, such as E1371S or K1250A (Carson et al., 1995; Powe et al., 2002; Bompadre et al., 2005b; Vergani et al., 2005). Login to comment 223 ABCC7 p.Trp401Phe By comparing WT-CFTR and W401F-CFTR, the increasing reentry events (X→X→O1) results in differences only in the mean open time and the tail of the fitted curve (Fig. S3), which is supplanted with an increased number of long opening bursts (>1,000 ms). Login to comment 304 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