ABCMdb

PMID: 10102935

Zeltwanger S, Wang F, Wang GT, Gillis KD, Hwang TC
Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.
J Gen Physiol. 1999 Apr;113(4):541-54., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Lys1250Ala Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. Login to comment 34 ABCC7 p.Lys1250Ala We studied gating of CFTR in excised inside-out patches from NIH3T3 cells stably transfected with wild-type (wt) or K1250A-CFTR. Login to comment 36 ABCC7 p.Lys1250Ala This concentration dependence of the mean open time is more prominent in K1250A-CFTR, a mutant CFTR of which the conserved lysine residue in the Walker A motif of NBD2 is converted to alanine. Login to comment 38 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala m a t e r i a l s a n d m e t h o d s Cell Culture and Electrophysiology Both wt (Berger et al., 1991) and K1250A (lysine to alanine mutation) CFTR channels were stably expressed in NIH3T3 cells (NIH3T3-CFTR and NIH3T3-K1250A, respectively). Login to comment 39 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala NIH3T3-K1250A cells stably expressing K1250A-CFTR were established using the retroviral vector pLJ (a generous gift from Dr. Mitchell Drumm, Case Western Reserve University, Cleveland, OH). Login to comment 58 ABCC7 p.Lys1250Ala Curve fits of the time courses for deactivation by washout of AMP-PNP and ATP from patches containing wt-CFTR or the time courses for deactivation by washout of ATP from patches containing K1250A-CFTR were obtained by using the Igor software. Login to comment 153 ABCC7 p.Lys1250Ala due to ATP binding to NBD2, we next examined [ATP]-dependent gating of K1250A-CFTR, a mutant in which the conserved lysine in the Walker A motif of NBD2 is converted to alanine. Login to comment 155 ABCC7 p.Lys1250Ala Gating of K1250A-CFTR channels was examined in the presence of either 10 ␮M or 2.75 mM ATP. Login to comment 156 ABCC7 p.Lys1250Ala Fig. 6 A shows that a K1250A-CFTR channel, preactivated with PKA and ATP (not shown), was "locked" in an open state with 2.75 mM ATP and the channel closed ‫2ف‬ min after ATP washout. Login to comment 159 ABCC7 p.Lys1250Ala The single channel amplitude, obtained from the all-point histograms (not shown), of K1250A-CFTR channels opened with millimolar ATP is about the same as that for brief openings in the presence of 10 ␮M ATP (Fig. 6 B). Login to comment 162 ABCC7 p.Lys1250Ala To quantify the brief openings of K1250A-CFTR in the presence of 10 ␮M ATP, dwell time analysis of the cumulative open time from three different patches was performed. Login to comment 164 ABCC7 p.Lys1250Ala Since the "locked open" time of K1250A-CFTR is apparently very long (Fig. 6 A), it will be very difficult to collect enough events for dwell time analysis. Login to comment 165 ABCC7 p.Lys1250Ala Even if we obtain patches containing a single K1250A-CFTR channel, the flickering closures in locked open state may interfere with the analysis. Login to comment 166 ABCC7 p.Lys1250Ala Although excluding closing intervals Ͻ80 ms is appropriate for eliminating flickers in analysis of wt-CFTR, this exclusion is not sufficient to eliminate flickers from analysis of K1250A-CFTR because the ratio of flickers to "true" closings (gating) of CFTR is much higher. Login to comment 168 ABCC7 p.Lys1250Ala At this concentration, most of K1250A-CFTR channels are locked open, as can be judged from the small magnitude of macroscopic current fluctuations (Fig. 6 D). Login to comment 172 ABCC7 p.Lys1250Ala These results suggest that, at low micromolar [ATP], K1250A-CFTR can assume brief openings with a time constant close to that of wt-CFTR at equivalent [ATP], but this mutant CFTR Figure 6. Login to comment 173 ABCC7 p.Lys1250Ala ATP concentration dependence of the channel open time for K1250A-CFTR. Login to comment 174 ABCC7 p.Lys1250Ala (A) A continuous current trace of K1250A-CFTR in the presence or absence of ATP. Login to comment 178 ABCC7 p.Lys1250Ala (B) Single channel amplitudes of K1250A-CFTR (obtained from all point histograms of 30 s recordings) at 10 ␮M or 2.75 mM ATP. Login to comment 179 ABCC7 p.Lys1250Ala (C) The open time histogram of K1250A-CFTR at 10 ␮M ATP. Login to comment 182 ABCC7 p.Lys1250Ala (D) Slow closing of PKA-phosphorylated K1250A-CFTR. Login to comment 185 ABCC7 p.Lys1250Ala Macroscopic relaxation of the K1250A-CFTR channel current was constructed from multiple washouts of ATP from the same patch. Login to comment 188 ABCC7 p.Lys1250Ala Assuming ATP is not hydrolyzed by the NBD2 of K1250A-CFTR, the slow closing rate reflects a slow dissociation of ATP from CFTR (presumably from NBD2). Login to comment 189 ABCC7 p.Lys1250Ala If the brief openings of K1250A-CFTR at 10 ␮M ATP represent open channel conformations without NBD2 being occupied, this observation suggests that CFTR can close even when ATP acts exclusively on NBD1 (see Carson et al., 1995; also see discussion). Login to comment 205 ABCC7 p.Lys1250Ala This stabilizing effect is greatly magnified when hydrolysis at NBD2 is eliminated, either through chemical modification of the binding molecule (AMP-PNP) or molecular alteration of the CFTR protein itself (i.e., K1250A mutation). Login to comment 243 ABCC7 p.Lys1250Ala We clearly resolve two open times with K1250A-CFTR and demonstrate a dramatic [ATP] dependence of the channel open time for this mutant CFTR. Login to comment 244 ABCC7 p.Lys1250Ala One interesting observation is that at 10 ␮M ATP, the mean open time for K1250A-CFTR is ‫052ف‬ ms, a value very close to that for wt-CFTR at the equivalent [ATP]. Login to comment 247 ABCC7 p.Lys1250Ala Based on this interpretation, the brief opening with K1250A-CFTR is coupled to hydrolysis of one ATP molecule at NBD1 and subsequently the channel can close without ATP hydrolysis at NBD2. Login to comment 249 ABCC7 p.Lys1250Ala This same observation that the open time of K1250A-CFTR depends on [ATP] is also inconsistent with the proposal that ATP binding at NBD2 opens the channel (Gunderson and Kopito, 1995). Login to comment 250 ABCC7 p.Lys1250Ala According to this latter model, every opening of K1250A-CFTR should last for minutes. Login to comment 262 ABCC7 p.Lys1250Ala The fact that this short open time constant is very close to the mean open time of K1250A-CFTR at 10 ␮M ATP further supports this assignment. Login to comment 309 ABCC7 p.Lys1250Ala Carson et al. (1995), using 20 ms as a cutoff, reported a mean burst time of ‫1ف‬ s for K1250A-CFTR. Login to comment 310 ABCC7 p.Lys1250Ala This number is evidently an underestimation of the true ATP-coupled open time for K1250A-CFTR as a continuous burst of opening that lasts for minutes is observed even when ATP is removed (Fig. 6 A). Login to comment 342 ABCC7 p.Lys1250Ala Assuming the opening rate of K1250A-CFTR is the same as that of wt-CFTR, our kinetic data suggest a maximal ATP hydrolysis rate of ‫500.0ف‬ s-1, which is 1/200 of that for wt-CFTR. Login to comment 343 ABCC7 p.Lys1250Ala Recent preliminary data that K1250A-CFTR has a drastically reduced rate of ATP hydrolysis support this coupled ATP turnover hypothesis (Ramjeesingh et al., 1998). Login to comment