ABCMdb

PMID: 20421370

Tsai MF, Li M, Hwang TC
Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
J Gen Physiol. 2010 May;135(5):399-414., [PubMed]

Sentences

No. Mutations Sentence Comment

85 ABCC7 p.Gly551Asp Figs. S5 and S6 are control experiments for ATP/PATP exchange experiments with G551D-CFTR. Login to comment 98 ABCC7 p.Trp401Gly When the ligand exchange experiment was performed with a single W401G channel (Fig. 3 A; similar results were seen in five other single-channel recordings), PATP (red trace) induced longer openings without an obvious delay observed with WT channels (compare Fig. 3 B with Fig. 1 B). Login to comment 100 ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly We interpreted these results to mean that the W401G mutation in NBD1 significantly decreases the resident time of the stably bound ATP molecule (from 50 s for WT-CFTR to 2.5 s for W401G-CFTR) so that PATP can replace it more rapidly and exert its second effect: increasing channel open change in channel kinetics and shows that ligand switches from PATP back to ATP also cause an immediately changed closed time and a delayed alteration of the open time. Login to comment 117 ABCC7 p.Trp401Gly ABCC7 p.Ser1347Gly ABCC7 p.Ser1347Gly Indeed, single S1347G channels, like W401G-CFTR (Fig. 3 A), opened into long bursts without a delay after changing the ligand from ATP to PATP (red trace in Fig. 4 B; channel kinetics summarized in Fig. 4 C), suggesting that the S1347G mutation in NBD2 dramatically shortens the dwell time of ATP in NBD1. Login to comment 123 ABCC7 p.Ser1347Gly ABCC7 p.Ser1347Val As in the case for mutations at W401 (Fig. 3 E), more drastic mutations at S1347, e.g., S1347V and S1347G, shorten this time constant to a greater extent than the more conservative time. Login to comment 124 ABCC7 p.Trp401Tyr ABCC7 p.Trp401Ile Similar macroscopic experiments were conducted with W401I and W401Y mutations. Login to comment 125 ABCC7 p.Trp401Tyr ABCC7 p.Trp401Ile The shorter time constant was not significantly affected by either of the mutations (Fig. 3 D), whereas the second time constant (Fig. 3 E) was shortened by nonaromatic substitutions of W401 (W401I) but increased by the conservative W401Y mutation. Login to comment 132 ABCC7 p.Trp401Gly (A) The response of a single-ATP-gated W401G channel to a sudden exposure of PATP (red trace). Login to comment 134 ABCC7 p.Trp401Gly A similar observation was seen in five other patches containing a single W401G-CFTR channel. Login to comment 136 ABCC7 p.Trp401Gly (C) Macroscopic currents recorded from hundreds of W401G channels. Login to comment 145 ABCC7 p.Ser1347Thr In brief, compared with 2.75 mM ATP, 8-N3-ATP at a saturating concentration (100 µM) catalyzes the opening of WT-CFTR at an approximately two- to threefold slower rate but induces openings approximately fourfold longer, resulting in a maximal Po for channels mutation, such as S1347T (Fig. 5 B). Login to comment 150 ABCC7 p.His1348Gly Of particular note is the H1348G mutation, which increased the time constant of the second phase (Fig. 5 B), suggesting that this mutation actually prolongs the ATP dwell time in NBD1. Login to comment 152 ABCC7 p.Ser1347Gly Consistent with this idea, for the ABC protein TAP2 whose corresponding residue of H1348 is Figure 4. Effects of the S1347G mutation on ligand exchange. Login to comment 158 ABCC7 p.Ser1347Gly (B) A representative single S1347G-CFTR channel trace from seven similar recordings. Login to comment 160 ABCC7 p.Ser1347Gly (D) Macroscopic currents from S1347G channels. Login to comment 186 ABCC7 p.Gly551Asp Indeed, as shown and explained in more detail in Fig. S4, the lock-open time with 8-N3-ATP plus PPi is nearly identical as that with ATP plus PPi; in addition, just like ATP, 8-N3-ATP fails to increase the activity of G551D-CFTR by binding to NBD1. Login to comment 189 ABCC7 p.Ser1347Gly The current rising phase Figure 7. Changes of WT-CFTR and S1347G-CFTR currents upon 8-N3-ATP/PATP exchange. Login to comment 191 ABCC7 p.Ser1347Gly (B) The second-phase current increase seen in A was essentially abolished by the S1347G mutation. Login to comment 204 ABCC7 p.Ser1347Gly Because most channels will stay in the open state when exposed to ATP and MgPPi, the subsequent solution change to PATP plus MgPPi will allow us to test whether ligand exchange in NBD1 occurs from the open state. If ATP/PATP exchange does occur in the open state (Fig. 10 B), PATP will further prolong the lock-open time because it is known that the lock-open state with MgPPi bound in NBD2 and PATP in NBD1 is more stable demonstrated biochemically (Aleksandrov et al., 2008), the trapping of 8-N3-ATP in the current study is also dependent on the tail of NBD2 because the S1347G mutation essentially abolished the second phase of current increase elicited by PATP upon switching the ligand from 8-N3-ATP to PATP (Fig. 7 B). Login to comment 241 ABCC7 p.Gly551Asp In this scenario, the G551D mutation in the helical subdomain of NBD1 has to allosterically affect tight nucleotide binding observed in NBD1`s core subdomain in spite of >10-Å distance in between (Lewis et al., 2004, 2005). Login to comment 245 ABCC7 p.Gly551Asp G551D, a mutation in the signature sequence of the NBD1 tail, offers an opportunity to differentiate between these two possibilities. Login to comment 246 ABCC7 p.Gly551Asp Because this mutation eliminates ATP-dependent openings of CFTR with rare spontaneous openings left (Bompadre et al., 2007), the stable partial dimer state will not exist for G551D-CFTR if its formation requires prior ATP-induced openings (C1→C2`→O→C2 in Fig. 12 A). Login to comment 247 ABCC7 p.Gly551Asp Our previous studies have demonstrated that PATP increases the Po of G551D channels by binding to NBD1, whereas it exerts no effects when binding to NBD2 (Bompadre et al., 2008). Login to comment 248 ABCC7 p.Gly551Asp Thus, the time course of current rise upon ATP/PATP switch will reflect the dissociation rate of ATP from NBD1 of G551D-CFTR. Login to comment 249 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In the current trace shown in Fig. 11, after ligand changes from ATP to PATP, the rapid monophasic ( = 2.1 ± 0.3 s and n = 11) increase of G551D currents indicates that unlike WT channels, ATP is not stably trapped in the NBD1 of G551D-CFTR. Login to comment 250 ABCC7 p.Gly551Asp In other words, the binding of ATP in G551D-CFTR`s NBD1 does not lead to a stable partial dimer state. Login to comment 253 ABCC7 p.Gly551Asp The G551D mutant channels were activated by PKA and ATP to a steady state before the solution was changed to one containing PATP. Login to comment 254 ABCC7 p.Gly551Asp The macroscopic current increased monotonically (inset) by PATP, although G551D-CFTR did not respond to ATP. Login to comment 272 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly However, in Figs. S5 and S6, we show that this is unlikely the case because introducing the Y1219G mutation, which greatly disrupts ATP or PATP binding in NBD2 (Fig. S5), into the G551D background does not significantly (P = 0.78) alter the time constant of current increase upon ATP/PATP switch (Fig. S6). Login to comment 284 ABCC7 p.Gly551Asp The finding that G551D-CFTR, unlike WT channels, does not trap ATP in NBD1 (Fig. 11) was interpreted to mean that the disassembly of the stable partial NBD dimer (C2→C1 in Fig. 12 A) is a poorly reversible process. Login to comment 293 ABCC7 p.Gly551Asp If the aforementioned argument made for the G551D-CFTR is also valid for WT-CFTR, when WT channels occasionally exit from the primary gating cycle (C2↔O) through a rare separation of the partial dimer (C2→C1), the channel, now in the monomeric NBD state, will reenter the open state via a distinct opening pathway, during which ATP binding in NBD1 is relatively unstable. Login to comment 402 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 404 ABCC7 p.Gly551Asp Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog. 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