ABCMdb

ABCC7 p.Thr1246Asn

ClinVar:	c.3737C>T , p.Thr1246Ile ? , not provided
CF databases:	c.3737C>T , p.Thr1246Ile (CFTR1) ? , A novel mutation was identified by DGGE and direct sequencing; the nucleotide change C->T at position 3869leads to T1246I in exon 20. This mutation was identified on a CF chromosome of an Irish patient, in collaboration with Dr. Watson. This was also discovered by Malone, Haworth, and Schwarz. The mutation was identified by direct DNA sequencing. It is the substitution of a single base (C to T) at position 3869, which results in the replacement of a threonine residue by an isoleucine residue at codon 1246. The patient's other CF mutation is [delta]F508, but we have yet been unable to show that it is on his other CF chromosome. We note that there are a number of other isoleucine substitutions (two with threonine: at 1059 and at 1220) which have been classified as polymorphins, and for this reason we are uncertain of the status of T1246I. We have seen this sequence change once in approximately 60 non-[delta]F508 CF chromosomes so far analysed.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, V: D, W: D, Y: D,

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[hide] CFTR channel opening by ATP-driven tight dimerizat... Nature. 2005 Feb 24;433(7028):876-80. Vergani P, Lockless SW, Nairn AC, Gadsby DC
CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.
Nature. 2005 Feb 24;433(7028):876-80., 2005-02-24 [PMID:15729345]

Abstract [show]
ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.

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90 b, Representative records from WT, single mutants R555K and T1246N, and double mutant R555K T1246N. Login to comment
100 To quantify this suspected interaction between Arg 555 and Thr 1246 side chains (Fig. 2a), we applied double mutant-cycle analysis6,21 (see Supplementary Information), after mutating Arg 555 to Lys and Thr 1246 to Asn, both individually and jointly. Login to comment
102 If the two residues do not interact, the effects of mutating Arg 555 to Lys should be the same in a Thr 1246 background as in a T1246N background (and vice versa); that is, the effects of the single mutations should be independent and hence additive, and mutation-linked changes on parallel sides of the cycles should thus be equal. Login to comment
112 Current levels of the triple mutant R555K T1246N K1250R did not change when [ATP] was increased to 10 mM, indicating that 5 mM [ATP] was saturating. Login to comment
117 The apparent affinity for ATP was little influenced by the mutation R555K (R555K K0.5 ¼ 71 ^ 14 mM versus WT K0.5 ¼ 55 ^ 5 mM), but was reduced by the mutation T1246N (T1246N K0.5 ¼ 261 ^ 49 mM) by the same extent in the WT background as in the R555K background (R555K T1246N K0.5 ¼ 257 ^ 51 mM). Login to comment
125 The T1246N mutation also greatly slowed channel opening, increasing the energy barrier by 2.5 ^ 0.4kT. Login to comment
133 Introducing the T1246N mutation into the K1250R background decreased Po, corresponding to destabilization of the open burst state by 2.5 ^ 1.0kT with respect to the closed state. However, adding the R555K mutation to T1246N-K1250R channels restored high stability of the open state (Fig. 4e, f). Login to comment
157 For constructs with very low Po (R555K and T1246N), we could not exclude the presence of unseen channels in the patch (even though the records lasted on average 6-7 min). Login to comment
158 The prolonged tib values we extract for R555K and T1246N channels are therefore most probably underestimates, and the real effects of the mutations are more severe (and, hence, jDDG‡ int(opening)j is actually larger) than the values we report. Login to comment

[hide] Control of the CFTR channel's gates. Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7. Vergani P, Basso C, Mense M, Nairn AC, Gadsby DC
Control of the CFTR channel's gates.
Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7., [PMID:16246032]

Abstract [show]
Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1-NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

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62 The WT, the two single mutants (in our case R555K and T1246N) and the double mutant (R555K/T1246N) form the corners of a thermodynamic cycle (Figure 3, inset). Login to comment
68 The R555K mutation did not significantly affect the apparent affinity, while the T1246N mutation reduced it to the same degree whether the residue at position 555 was Arg or Lys. Login to comment
78 Introducing the T1246N mutation in a non-hydrolytic background (mutated at a crucial lysine, K1250R [39]) strongly destabilized the open state with respect to the 3 Mutant cycle analysis using activation free energies for the opening reaction Coupling between Arg555 and Thr1246 increases as the channel approaches the open state. Login to comment
79 (A) Representative records from WT, single mutants R555K and T1246N, and double mutant R555K/T1246N, activated with 300 nM cAMP-dependent protein kinase and 5 mM ATP. Login to comment

[hide] Review. ATP hydrolysis-driven gating in cystic fib... Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):247-55. 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 [PMID:18957373]

Abstract [show]
Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

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99 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
115 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
119 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
139 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
97 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 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
117 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 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

[hide] Mutant cycles at CFTR's non-canonical ATP-binding ... J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16. Szollosi A, Muallem DR, Csanady L, Vergani P
Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.
J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16., [PMID:21576373]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

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239 Site-1 mutations affect channel closing, likely by affecting the relative stability of open states In contrast to composite site-2 mutations R555K and T1246N (Vergani et al., 2005, but compare Teem et al., 1996), in positions equivalent to T460 and L1353, all site-1 mutations studied here had relatively small effects on channel gating, consistent with the notion that the gating cycle is driven by the catalytic cycle at composite site 2. Login to comment

[hide] Nonintegral stoichiometry in CFTR gating revealed ... J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10. Jih KY, Sohma Y, Hwang TC
Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.
J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10., [PMID:22966014]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR's gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating-a strict coupling between the ATP hydrolysis cycle and the gating cycle-is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1-->O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1-->O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

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254 Indeed, mutations of the amino acid residue that directly interacts with ATP have been reported to decrease the opening rate in an ATP-dependent manner (Zhou et al., 2006); further, mutations located at the NBD dimer interface have also been shown to lower the opening rate (e.g., T1246N in Vergani et al., 2005). Login to comment
256 Figure 7. R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment
257 Macroscopic current traces of E1371S-CFTR (A), R352C/E1371S-CFTR (B), T1246N/E1371S-CFTR (C), and R352C/T1246N/E1371S-CFTR (D). Login to comment
259 The current relaxation was fitted with a single-exponential function resulting in the relaxation time constant for each mutant: 65.6 ± 10.1 s (n = 8) for E1371S-CFTR, 4.9 ± 0.8 s (n = 12) for R352C/ E1371S-CFTR, 7.8 ± 1.6 s (n = 7) for T1246N/E1371S-CFTR, and 2.27 ± 0.27 s (n = 6) for R352C/T1246N/E1371S-CFTR. Login to comment
261 *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment
292 It follows that mutations that decrease the rate of NBD dimerization (CATP→CAD), e.g., T1246N in Vergani et al. (2005), or those that reduce the rate for CAD→O1 (presumably the R352C mutation), will accelerate the decay rate of macroscopic currents in hydrolysis-deficient mutants upon ATP washout. Login to comment
293 Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment
294 Notably, this result is somewhat different from that shown in Vergani et al. (2005) in which the current relaxation is prolonged in T1246N/K1250R- CFTR. Login to comment
296 Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. Login to comment
295 Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment
298 Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. Login to comment

[hide] Model of the cAMP activation of chloride transport... J Theor Biol. 2010 Jan 7;262(1):73-9. Epub 2009 Sep 17. Moran O
Model of the cAMP activation of chloride transport by CFTR channel and the mechanism of potentiators.
J Theor Biol. 2010 Jan 7;262(1):73-9. Epub 2009 Sep 17., [PMID:19766125]

Abstract [show]
Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis, a hereditary lethal disease. CFTR is a chloride channel expressed in the apical membrane of epithelia. It is activated by cAMP dependent phosphorylation and gated by the binding of ATP. The impaired chloride transport of some types of cystic fibrosis mutations could be pharmacologically solved by the use of chemical compounds called potentiators. Here it is undertaken the construction of a model of the CFTR activation pathways, and the possible modification produced by a potentiator application. The model yields a novel mechanism for the potentiator action, describing the activatory and inhibitory activities on two different positions in the CFTR activation pathway.

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No. Sentence Comment
57 Disruption of the binding site on NBD1 and NBD2 by mutations R555K and T1246N, respectively, yield an approximation of the independent equilibrium constants, KATP1 ¼ 71 mM and KATP2 ¼ 261 mM (Vergani et al., 2005). Login to comment
59 Disruption of the binding site on NBD1 and NBD2 by mutations R555K and T1246N, respectively, yield an approximation of the independent equilibrium constants, KATP1 &#bc; 71 mM and KATP2 &#bc; 261 mM (Vergani et al., 2005). Login to comment

[hide] Timing of CFTR Pore Opening and Structure of Its T... Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22. Sorum B, Czege D, Csanady L
Timing of CFTR Pore Opening and Structure of Its Transition State.
Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22., [PMID:26496611]

Abstract [show]
In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation DeltaF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the DeltaF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in DeltaF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.

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56 Timing of Motion at Position 1246 of the NBD1-NBD2 Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations T1246V, T1246P, T1246C, T1246N, and T1246A, respectively, in the same background. Login to comment