ABCMdb

PMID: 16966475

Zhou Z, Wang X, Liu HY, Zou X, Li M, Hwang TC
The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics.
J Gen Physiol. 2006 Oct;128(4):413-22. Epub 2006 Sep 11., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Trp401Gly Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. Login to comment 7 ABCC7 p.Trp401Gly The W401G mutation, however, shortens the open time constant. Login to comment 18 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser It is generally agreed that ATP hydrolysis at NBD2 precedes channel closing since mutations (e.g., K1250A and E1371S) that abolish ATP hydrolysis at the NBD2 site drastically prolong the open time (Carson et al., 1995; Gunderson and Kopito, 1995; Zeltwanger et al., 1999; Vergani et al., 2003; Bompadre et al., 2005b). Login to comment 35 ABCC7 p.Trp401Gly Although conversion of W401 to glycine (i.e., W401G) has little effect on the sensitivity of the channel opening rate to [ATP], this mutation shortens the mean open time. Login to comment 36 ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly The shortening of the open time by the W401G mutation is also seen with the hydrolysis-deficient mutant background (i.e., E1371S), suggesting that the effect of W401G mutation is not through a perturbation of ATP hydrolysis. Login to comment 61 ABCC7 p.Phe508Ala The crystal structure of human F508A NBD1-ATP complexes (pdb code: 1xmi, chain A) was used as a template. Login to comment 77 ABCC7 p.Trp401Gly For the W401G mutant, 0.1 mM ATP elicits a similar level of activity. Login to comment 78 ABCC7 p.Tyr1219Gly However, for the Y1219G mutant, 0.1 mM hardly induces any current different from the basal activity. Login to comment 79 ABCC7 p.Trp401Gly Unlike WT and W401G, 2.75 mM ATP does not saturate Figure 1. Login to comment 81 ABCC7 p.Phe508Ala (A) Interactions between ATP and key amino acids in the NBD1 binding pocket, adopted from the monomeric crystal structure of the human F508A NBD1-ATP complexes (pdb code: 1xmi, chain A) (left). Login to comment 94 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Current induced by various [ATP] was normalized to 2.75 mM ATP in the case of WT and W401G and to 20 mM ATP in the case of Y1219G. Login to comment 96 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly (D) ATP dose-response relationships of WT (black), W401G (red), and Y1219G (green). Login to comment 98 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly The K1/2 values are 0.09 ± 0.02 mM, 0.11 ± 0.02 mM, and 4.72 ± 1.12 mM for WT, W401G, and Y1219G, respectively. Login to comment 100 ABCC7 p.Tyr1219Gly the current for Y1219G (unpublished data). Login to comment 102 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Fig. 1 D summarizes normalized macroscopic ATP dose-response relationships of WT, W401G, and Y1219G. Login to comment 103 ABCC7 p.Trp401Gly Although the W401G mutation does not affect the apparent affinity, converting Y1219 to glycine causes a dramatic rightward shift of the ATP dose-response curve with a K1/2 of 4.72 ± 1.12 mM, >50-fold higher than that of WT (0.09 ± 0.02 mM). Login to comment 105 ABCC7 p.Tyr1219Trp A more conserved mutation (Y1219W) does not change the K1/2 value significantly. Login to comment 106 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe The ATP dose-response relationships of Y1219F and Y1219I mutants lie between those of WT and Y1219G. Login to comment 109 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly To test this hypothesis, we examined single-channel kinetics of WT, W401G, and Y1219G. Login to comment 111 ABCC7 p.Trp401Gly At 2.75 mM ATP, WT and W401G channels close for hundreds of milliseconds between opening bursts. Login to comment 112 ABCC7 p.Tyr1219Gly However, even at 20 mM ATP, most of the closed events for Y1219G last for several seconds. Login to comment 113 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile Since the opening rate of Y1219G is not saturated at 20 mM ATP, we also studied single-channel kinetics of Y1219I, which shows a smaller shift in the ATP dose-response relationship (Fig. 2 A). Login to comment 115 ABCC7 p.Tyr1219Ile The opening rate of Y1219I at 10 or 20 mM ATP is very similar to that of WT at 2.75 mM ATP (Fig. 2, B and C), indicating that mutations at the Y1219 residue likely affect the ATP binding step with minimal effect on the post-binding events. Login to comment 116 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile Fig. 2 C shows the relationship between the opening rate and [ATP] for WT, W401G, Y1219I, and Y1219G. Login to comment 122 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe (A) Normalized ATP dose-response relationships of WT (black line, Michaelis-Menten fit from Fig. 1 D), Y1219W (brown), Y1219F (pink), Y1219I (blue), and Y1219G (green line, Michaelis-Menten fit from Fig. 1 D). Login to comment 124 ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe K1/2 values are 0.13 ± 0.02 mM (Y1219W), 0.46 ± 0.06 mM (Y1219F), and 0.94 ± 0.20 mM (Y1219I), respectively. Login to comment 125 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile (B) Representative single-channel current traces of WT, W401G, Y1219G, and Y1219I in response to [ATP] as marked. Login to comment 126 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile (C) Relationships between channel opening rates and [ATP] for WT (black), W401G (red), Y1219I (blue), and Y1219G (green). Login to comment 127 ABCC7 p.Tyr1219Ile Solid lines are Michaelis-Menten fits to the data of WT (black) and Y1219I (blue). Login to comment 128 ABCC7 p.Tyr1219Ile The maximal opening rate and K1/2 values are 2.42 ± 0.11 s-1 and 0.11 ± 0.02 mM for WT, and 2.60 ± 0.11 s-1 and 1.73 ± 0.26 mM for Y1219I, respectively. Login to comment 129 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile (D) Relationships between channel opening rates and [ATP] for ∆R-CFTR (black), ∆R-Y1219I (blue), and ∆R-Y1219G (green). Login to comment 130 ABCC7 p.Tyr1219Ile K1/2 from Michaelis-Menten fits (solid lines) are 0.16 ± 0.04 mM and 1.27 ± 0.16 mM for ∆R-CFTR and ∆R-Y1219I, respectively. Login to comment 134 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile Fig. 2 D shows a similar rightward shift of the ATP dose-response relationships for Y1219I and Y1219G mutants under the ∆R-CFTR background. Login to comment 136 ABCC7 p.Trp401Gly Although mutations of the W401 residue at the NBD1 site had minimal effect on the relationship between [ATP] and the opening rate (Fig. 2, B and C), close inspection of the single-channel current trace reveals that the channel open time is shorter for the W401G mutant (Fig. 2 B). Login to comment 137 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Fig. 3 A summarizes the mean open time for WT, W401G, and Y1219G. Login to comment 138 ABCC7 p.Tyr1219Gly Although the Y1219G mutation causes a dramatic change of the relationship between [ATP] and the opening rate, it has negligible effect on the mean open time. Login to comment 139 ABCC7 p.Trp401Gly In contrast, W401G exhibits ‫%04ف‬ decrease of the mean open time, suggesting that mutations that decrease the ATP binding affinity at the NBD1 site destabilize the open channel conformation (i.e., increase the closing rate). Login to comment 140 ABCC7 p.Trp401Gly It should be noted that this shortening of the mean open time by W401G mutant is not readily reflected in the ATP dose-response relationship (Fig. 1 D). Login to comment 142 ABCC7 p.Trp401Gly We considered three possibilities for the shortened open time seen with the W401G mutation at NBD1. Login to comment 143 ABCC7 p.Trp401Gly First, since it is established that ATP hydrolysis at NBD2 leads to channel closing during the ATP hydrolysis-driven gating cycle, it is possible that the W401G mutation accelerates the ATP hydrolysis rate of NBD2. Login to comment 147 ABCC7 p.Trp401Gly Third, the shortened open time of the W401G could be due to an allosteric effect. Login to comment 148 ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly To examine the potential effect of mutation in NBD1 on ATP hydrolysis at NBD2, we introduced the W401G mutation into the E1371S background, a mutant CFTR whose ATP hydrolysis is abolished (Moody et al., 2002; Tombline et al., 2004; Vergani et al., 2005). Login to comment 149 ABCC7 p.Trp401Gly If the shortened open time seen with the W401G mutant is indeed due to an increased ATP hydrolysis rate, the effect on the open time should be at least reduced once the ATP hydrolysis is eliminated. Login to comment 150 ABCC7 p.Glu1371Ser Since the gating cycle of channels in the E1371S background is extremely long, it is technically difficult to do microscopic single-channel kinetic analysis. Login to comment 155 ABCC7 p.Trp401Gly (A) Mean open times of WT and W401G in the presence of 2.75 mM ATP are 441.3 ± 49.4 ms (n = 13) and 248.7 ± 11.3 ms (n = 22), respectively. Login to comment 156 ABCC7 p.Tyr1219Gly The mean open time of Y1219G in the presence of 20 mM ATP is 399.1 ± 40.4 ms (n = 5). Login to comment 157 ABCC7 p.Trp401Gly *** indicates P < 0.001 between WT and W401G. Login to comment 158 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly (B) Representative current relaxation traces upon withdrawal of 1 mM ATP plus PKA for E1371S, W401G/E1371S, triple/ E1371S, Y1219G/E1371S, and W401G/Y1219G/E1371S. Login to comment 161 ABCC7 p.Glu1371Ser ** indicates P < 0.01 and *** indicates P < 0.001 (compared with E1371S). Login to comment 164 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly Fig. 3 B shows experiments using current relaxation analysis to estimate the open time constants for E1371S and W401G/E1371S. Login to comment 165 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly Our results show that the relaxation time constant for W401G/E1371S (59.1 ± 4.6 s, n = 8) is shortened by ‫,%05ف‬ compared with that of E1371S (111.7 ± 12.1 s, n = 15) (Fig. 3 C), suggesting that the shorter open time of W401G is not secondary to an altered ATP hydrolysis rate. Login to comment 166 ABCC7 p.Glu1371Ser ABCC7 p.Tyr1219Gly In contrast, although the Y1219G mutation greatly decreases the apparent affinity of ATP (Fig. 2 C), introducing this mutation into the E1371S background has little effect on the relaxation time constant (107.6 ± 12.4 s, n = 7) (Fig. 3, B and C). Login to comment 167 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly In addition, W401G/Y1219G/E1371S has a relaxation time constant of 49.0 ± 5.3 s (Fig. 3, B and C), which is similar to that of W401G/E1371S, indicating that W401, but not Y1219, plays a dominant role in modulating the open time. Login to comment 172 ABCC7 p.Trp401Gly Third, the shortened relaxation time constant due to the W401G mutation can be prolonged by P-ATP (see below). Login to comment 178 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Phe430Gly ABCC7 p.Phe409Gly We converted all three aromatic amino acids, including W401, F409, and F430 to glycine in the E1371S background and examined current relaxations of the W401G/F409G/F430G/E1371S (or triple/E1371S). Login to comment 179 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly Compared with the current relaxation of W401G/ E1371S (Fig. 3 B), the triple/E1371S mutation further shortens the time course of current decay. Login to comment 186 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Fig. 4 B shows experiments examining current relaxations upon removal of ATP or P-ATP for E1371S, W401G/E1371S, Y1219G/ E1371S, and triple/E1371S. Login to comment 187 ABCC7 p.Glu1371Ser As demonstrated previously (Zhou et al., 2005), the relaxation time course upon washout of P-ATP for E1371S is approximately twofold longer than that with ATP. Login to comment 188 ABCC7 p.Glu1371Ser ABCC7 p.Tyr1219Gly P-ATP also increases the relaxation time constant of Y1219G/E1371S by approximately twofold. Login to comment 189 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly However, this prolongation effect of P-ATP is significantly larger for W401G/E1371S (2.7-fold) and triple/E1371S (greater than fourfold; Fig. 4 C). Login to comment 194 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Studies using different mutations that perturb ATP hydrolysis (e.g., K1250A, E1371S) indicate that ATP hydrolysis drives channel closure. Login to comment 198 ABCC7 p.Lys1250Ala Although these studies have provided significant insight into the role of ATP hydrolysis in CFTR gating, this kind of approach does not provide a distinct advantage in understanding the role of ATP binding since altering the ligand binding affinity with these mutations is often complicated by the mutational effect on ATP hydrolysis (e.g., K1250A in Vergani et al., 2003). Login to comment 205 ABCC7 p.Tyr1219Ile The Y1219I mutation lowers the sensitivity of the opening rate to [ATP] without altering the maximal opening rate (Fig. 2), indicating that this mutation indeed decreases the binding affinity for ATP at the NBD2 site. Login to comment 206 ABCC7 p.Tyr1219Gly Interestingly, although the Y1219G mutation causes a drastic shift of the ATP dose-response relationship (Figs. 1 and 2), it does not affect the mean open time. Login to comment 217 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly (B) Representative current relaxation traces of E1371S, W401G/E1371S, triple/ E1371S, and Y1219G/E1371S after withdrawal of 1 mM ATP plus PKA or 50 μM P-ATP plus PKA. Horizontal scale bars represent 200 s. Login to comment 218 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser (C) The ratio of the relaxation time constant upon withdrawal of 50 μM P-ATP plus PKA to that upon withdrawal of 1 mM ATP plus PKA from the same patch was calculated for E1371S and various mutants in the E1371S background. Login to comment 220 ABCC7 p.Glu1371Ser *** indicates P < 0.001 and **** indicates P < 0.0001 (compared with E1371S). Login to comment