ABCMdb

PMID: 18957373

Muallem D, Vergani P
Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator.
Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):247-55., 2009-01-27 [PubMed]

Sentences

No. Mutations Sentence Comment

59 ABCC7 p.Asp1370Asn For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment 61 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment 63 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile The dose-response curve of channel opening rate as a function of [ATP] was seen to shift dramatically to the right when the consensus site 2 was mutated ( Y1219G or Y1219I) but not when the degenerate site 1 was altered (W401G). Login to comment 65 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile The dose-response curve of channel opening rate as a function of [ATP] was seen to shift dramatically to the right when the consensus site 2 was mutated ( Y1219G or Y1219I) but not when the degenerate site 1 was altered (W401G). Login to comment 66 ABCC7 p.Trp401Gly Possibly W401G channels open frequently as monoliganded, while WT, with its high-affinity site 1, only rarely would. Login to comment 68 ABCC7 p.Trp401Gly Possibly W401G channels open frequently as monoliganded, while WT, with its high-affinity site 1, only rarely would. Login to comment 80 ABCC7 p.Trp401Gly For example, the W401G mutant, with a weakened contact with the adenine of the ATP molecule bound at site 1, presented a less than twofold reduction in mean open time, interpreted by the 10-2 0 0 2 4 6 relativeopeningrate closingrate(s-1) 0.5 1.0 10-1 1 10 10-2 10-1 1 10 [MgATP] (mM)[MgATP] (mM) (b) (a) 10s 5mM 5mM [ATP] 50µM2s0.4 pA WT CFTR (c) Figure 2. Login to comment 82 ABCC7 p.Trp401Gly For example, the W401G mutant, with a weakened contact with the adenine of the ATP molecule bound at site 1, presented a less than twofold reduction in mean open time, interpreted by the 10-2 0 0 2 4 6 relativeopeningrate closingrate(s-1) 0.5 1.0 10-1 1 10 10-2 10-1 1 10 [MgATP] (mM)[MgATP] (mM) (b) (a) 10s 5mM 5mM [ATP] 50µM2s0.4 pA WT CFTR (c) Figure 2. Login to comment 84 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment 86 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment 97 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn The WT, the two single mutants (R555K and T1246N) and the double mutant (R555K T1246N) form the corners of a thermodynamic cycle (figure 4a). Login to comment 99 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn The WT, the two single mutants (R555K and T1246N) and the double mutant (R555K T1246N) form the corners of a thermodynamic cycle (figure 4a). Login to comment 113 ABCC7 p.Thr1246Asn ABCC7 p.Lys1250Arg Introducing the T1246N mutation in a non-hydrolytic background (mutated at a crucial lysine, K1250R; Lerner-Marmarosh et al. 1999) strongly destabilized the open state with respect to the closed one. Login to comment 114 ABCC7 p.Arg555Lys ABCC7 p.Lys1250Arg However, the same T to N mutation, when introduced in the R555K K1250R non-hydrolytic background, did not significantly alter the closed- to-open equilibrium. Login to comment 115 ABCC7 p.Thr1246Asn ABCC7 p.Lys1250Arg Introducing the T1246N mutation in a non-hydrolytic background (mutated at a crucial lysine, K1250R; Lerner-Marmarosh et al. 1999) strongly destabilized the open state with respect to the closed one. Login to comment 116 ABCC7 p.Arg555Lys ABCC7 p.Lys1250Arg However, the same T to N mutation, when introduced in the R555K K1250R non-hydrolytic background, did not significantly alter the closed- to-open equilibrium. Login to comment 117 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn The apparent dissociation constant obtained from [ATP] dependence of opening rate is, for CFTR (Vergani et al. 2005), a reasonable estimate for the real dissociation constant for the ATP binding reaction WT CFTR R555K R555K T1246N T1246N 0.4 pA 20 s ∆∆int = ∆3-∆1 = ∆4-∆2 WT ∆1 ∆3 ∆2 ∆4 T1246N R555K R555K T1246N (a) (c) OH T H2N H2NNH2 + COOH NH R H2NCOOH WT CFTR H2NCOOH NH3 + K NH2 H2NCOOH O N R555K T1246N (b) Figure 4. Login to comment 119 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn The apparent dissociation constant obtained from [ATP] dependence of opening rate is, for CFTR (Vergani et al. 2005), a reasonable estimate for the real dissociation constant for the ATP binding reaction WT CFTR R555K R555K T1246N T1246N 0.4 pA 20 s ∆∆int = ∆3-∆1 = ∆4-∆2 WT ∆1 ∆3 ∆2 ∆4 T1246N R555K R555K T1246N (a) (c) OH T H2N H2NNH2 + COOH NH R H2NCOOH WT CFTR H2NCOOH NH3 + K NH2 H2NCOOH O N R555K T1246N (b) Figure 4. Login to comment 137 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn The R555K mutation did not significantly affect apparent affinity, while the T1246N mutation reduced it to the same degree whether the residue at position 555 was WT R or mutant K. The effects of the T to N mutation in WT and mutant background are thus similar, yielding a negligible energetic coupling between the two target residues (DDGint(unbound- bound)Z0.3G0.5 kT). Login to comment 139 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn The R555K mutation did not significantly affect apparent affinity, while the T1246N mutation reduced it to the same degree whether the residue at position 555 was WT R or mutant K. The effects of the T to N mutation in WT and mutant background are thus similar, yielding a negligible energetic coupling between the two target residues (DDGint(unbound-bound)Z0.3G0.5 kT). Login to comment