ABCMdb

PMID: 19332621

Tsai MF, Shimizu H, Sohma Y, Li M, Hwang TC
State-dependent modulation of CFTR gating by pyrophosphate.
J Gen Physiol. 2009 Apr;133(4):405-19., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser The idea that ATP hydrolysis precedes channel closing is further supported by the observations that CFTR mutations whose ATPase activity is abrogated (e.g., K1250A and E1371S) (Ramjeesingh et al., 1999) can remain open for minutes (Gunderson and Kopito, 1995; Zeltwanger et al., 1999; Vergani et al., 2003; Bompadre et al., 2005b), and that channel closure is markedly delayed in the presence of nonhydrolyzable ATP analogue AMP-PNP (Hwang et al., 1994), or of inorganic phosphate analogue orthovanadate, which presumably forms a stable complex with the hydrolytic product ADP (Baukrowitz et al., 1994). Login to comment 19 ABCC7 p.Trp401Gly The stability of the C2 state is enhanced when the channel is initially opened by N6 -phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C2 state. Login to comment 20 ABCC7 p.Tyr1219Gly Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C2-equivalent state (C2*), which exists before the channel in the C1 state is opened by ATP. Login to comment 35 ABCC7 p.Glu1371Ser ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Ser1347Gly Electrophysiological recordings Before inside-out patch clamp recordings, glass chips containing CHO cells transfected with various CFTR constructs, W401G, Y1219G, S1347G, E1371S, and WT-CFTR, were transferred to a continuously perfused chamber located on the stage of an inverted microscope (Olympus). Login to comment 56 ABCC7 p.Gly551Ser Carson et al. (1995) confirmed and expanded this observation by showing that PPi also strongly potentiates cystic fibrosis-associated mutations ⌬F508 and G551S. Login to comment 65 ABCC7 p.Gly1349Asp Cai et al. (2006) showed that PPi cannot potentiate G1349D, a mutation at the signature sequence of NBD2, which forms the ATP-binding pocket with the nucleotide-interacting motifs of NBD1. Login to comment 105 ABCC7 p.Ser1347Gly Online supplemental material Fig. S1 shows that S1347G-CFTR has a weaker response to MgPPi than WT. Login to comment 106 ABCC7 p.Glu1371Ser The observation that MgPPi induces shorter opening events than ATP in E1371S channels is shown in Fig. S2. Login to comment 198 ABCC7 p.Tyr1219Gly (C) Effects of PPi on Y1219G-CFTR. Login to comment 207 ABCC7 p.Tyr1219Gly Fitting the data points (red curve) by a single-exponential function estimates the lifetime of the C2 state to be 27.4 s. the data for WT-CFTR (Fig. 5 A), the current relaxation upon the removal of ATP and MgPPi follows a monotonic decay with a time constant of ␶ = 30.7 ± 4.5 s (n = 5), indicating that almost all Y1219G-CFTR channels have been locked open under this experimental condition. Login to comment 208 ABCC7 p.Trp401Gly As a control, when a similar experiment was performed for W401G, ATP still out-competes MgPPi as demonstrated by a large fraction of the fast component during current relaxation (not depicted). Login to comment 232 ABCC7 p.Tyr1219Gly Fig. 5 C shows that 2 mM MgPPi, when added to 2 mM ATP solution, dramatically increased the Y1219G-CFTR macroscopic current. Login to comment 243 ABCC7 p.Trp401Gly Previously, Zhou et al. (2006) showed that P-ATP may assume a tighter binding than ATP at NBD1 of W401G-CFTR. Login to comment 244 ABCC7 p.Trp401Gly Indeed, opening of W401G-CFTR channels with P-ATP results in a higher fraction (24 ± 1.5%; n = 5) of lock-open channels (Fig. 7, B and C). Login to comment 246 ABCC7 p.Ser1347Gly Interestingly, we also found that S1347G, a mutation at NBD2 signature sequence, which presumably forms the ATP-binding pocket with NBD1`s Walker A domain upon NBD dimer formation, greatly attenuates the stability of the C2 state, and this reduced stability can also be partly reversed by P-ATP (Fig. S1). Login to comment 252 ABCC7 p.Tyr1219Gly To address this question, we used Y1219G-CFTR to test whether MgPPi has the same effect on these two account for the long-lasting memory assumed by the C2 state. Login to comment 265 ABCC7 p.Trp401Gly We then used the same protocol to test the effect of MgPPi on the W401G mutation, which likely decreases the Figure 7. Login to comment 268 ABCC7 p.Trp401Gly (B) The lock-open efficiency of MgPPi was reduced by the W401G mutation, and P-ATP can partially restore the effectiveness of MgPPi on W401g-CFTR. Login to comment 285 ABCC7 p.Trp401Gly Similar experiments as shown in Fig. 7 were performed for MgAMP-PNP with W401G-CFTR and P-ATP, and virtually identical results were obtained (not depicted). Login to comment 291 ABCC7 p.Tyr1219Gly Treating the channels that have been closed for a long time (thus in the C1 state) with a low concentration of ATP should favor an accumulation of the C2* state because the transition rate from C2* to O is significantly decreased by the Y1219G mutation, whereas a high concentration of ATP opens the channel more frequently and thus brings more channels to the C2 state. Login to comment 293 ABCC7 p.Tyr1219Gly After PKA and ATP activation and a 1-min washout, we first treated Y1219G channels with 500 μM ATP, which only elicited minimal current. Login to comment 302 ABCC7 p.Tyr1219Gly tition between ATP and MgAMP-PNP, suggesting that the Y1219G mutation decreases the binding affinity of ATP to a similar extent as it lowers the affinity for MgAMP-PNP. Login to comment 309 ABCC7 p.Tyr1219Gly As seen in Fig. 10 (B and C), when [ATP] = [MgAMP-PNP] = 2 mM, the fractional amplitudes of the slow component upon current relaxation are 73 ± 3% (n = 6) and 71 ± 4% (n = 6) for WT-CFTR and Y1219G-CFTR, respectively. Login to comment 310 ABCC7 p.Tyr1219Gly Thus, although the Y1219G mutation alters the competition between ATP and MgPPi for the NBD2 site (Fig. 5), the same mutation does not significantly affect the compe- Figure 9. Login to comment 312 ABCC7 p.Tyr1219Gly (A) A continuous current trace of Y1219G-CFTR. Login to comment 326 ABCC7 p.Tyr1219Gly (C) Y1219G channels opened by ATP plus PKA were locked open by 2 mM MgAMP-PNP plus 2 mM ATP. Login to comment 330 ABCC7 p.Gly551Asp Third, although G551D-CFTR cannot be opened by ATP, this mutant channel can be gated by Cd2+ . Login to comment 335 ABCC7 p.Tyr1219Gly Zhou et al. (2006) reported that Y1219G-CFTR, which presumably loses the ␲-electron- stacking interaction between the aromatic side chain of the tyrosine residue and the adenine ring of ATP, has a far lower ATP binding affinity compared with WT-CFTR. Login to comment 340 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly Although we cannot rule out the possibility that MgPPi has a low binding affinity simply because it does not bind to the Walker A domain as well as ATP, the observation that MgAMPPNP elicits a maximal effect on CFTR at low millimolar concentration (Vergani et al., 2003) (Fig. 10 A), and that the binding affinity of MgAMP-PNP is weakened by Y1219G mutation (Fig. 10 C), suggests that a lack of the ring-ring interaction may have a greater impact on the ligand binding affinity than a slight structural alteration of the phosphate group. Login to comment 358 ABCC7 p.Gly551Asp Second, the observation that the G551D mutation at the signature sequence completely abolishes ATP-dependent gating of CFTR (Bompadre et al., 2007, 2008) points to the functional significance of this tational effect on the stability of the C2 state can be at least partially overcome by P-ATP suggests that this mutational effect can be attributed to a lower binding affinity rather than some nonspecific allosteric effects. Login to comment 375 ABCC7 p.Glu1371Ser This result thus suggests that it is MgPPi dissociation from NBD2, but not ATP from NBD1, that is associated with E1371S-CFTR, a mutant whose ATPase activity is abolished (Moody et al., 2002; Tombline et al., 2004; Vergani et al., 2005; Zhou et al., 2006; Stratford et al., 2007). Login to comment 376 ABCC7 p.Glu1371Ser We reasoned that if MgPPi elicits longer open bursts due to a slower hydrolysis rate, E1371S mutation should further prolong the burst duration induced by MgPPi. Login to comment 377 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser However, as can be seen in Fig. S2, although the E1371S mutation dramatically increases the relaxation time constant of the ATP-gated channels (␶ = 126.1 ± 24.2 s; n = 5), the lifetime of MgPPi-induced openings for E1371S channels (1.65 s, ensemble current relaxation from five data) is very close to that of WT-CFTR (~1.5 s in Fig. 1 B). Login to comment 378 ABCC7 p.Glu1371Ser This result suggests that both MgPPi-opened WT-CFTR and E1371S-CFTR channels close through a nonhydrolytic pathway. Login to comment 380 ABCC7 p.Glu1371Ser Finally, the data also indicate that MgPPi is a poor ligand for CFTR channels because, unlike ATP, it fails to induce a stable open state with E1371S-CFTR. Login to comment 392 ABCC7 p.Trp401Gly Second, W401G mutation, which likely reduces ATP binding affinity in NBD1 (Zhou et al., 2006), decreases the stability of the C2 state. Login to comment 433 ABCC7 p.Lys464Ala ABCC7 p.Trp401Gly Echoing this observation, the lock-open duration of MgPPi is reduced by mutations that decrease the ATP binding affinity to NBD1, K464A, and W401G, but can be partially restored by a high affinity ATP analogue, P-ATP (unpublished data; compare Powe et al., 2002; Zhou et al., 2006). Login to comment 440 ABCC7 p.Trp401Gly ABCC7 p.Ser1347Gly Supporting this hypothesis, we demonstrated that the C2 state dissipates much faster when residues at either NBD1 (W401G; Fig. 7 B) or the signature sequence of NBD2 were mutated (S1347G; Fig. S1). Login to comment 442 ABCC7 p.Tyr1219Gly Here, using the Y1219G mutation to slow down the channel opening rate, we identify another closed state (C2*) that exists before the channel is opened by ATP from the C1 state (Figs. 8 B and 9). Login to comment 506 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 508 ABCC7 p.Gly551Asp Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog. Login to comment 511 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 2006. Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. J. Biol. Chem. 281:1970-1977. Login to comment