ABCC7 p.Tyr1219Trp
| Predicted by SNAP2: | A: D (85%), C: D (85%), D: D (95%), E: D (91%), F: D (80%), G: D (91%), H: D (85%), I: D (85%), K: D (91%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (85%), W: D (80%), |
| 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, T: D, V: D, W: D, |
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[hide] The two ATP binding sites of cystic fibrosis trans... J Gen Physiol. 2006 Oct;128(4):413-22. Epub 2006 Sep 11. 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., [PMID:16966475]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. 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. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.
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No. Sentence Comment
105 A more conserved mutation (Y1219W) does not change the K1/2 value significantly.
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ABCC7 p.Tyr1219Trp 16966475:105:27
status: NEW122 (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).
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ABCC7 p.Tyr1219Trp 16966475:122:103
status: NEW124 K1/2 values are 0.13 ± 0.02 mM (Y1219W), 0.46 ± 0.06 mM (Y1219F), and 0.94 ± 0.20 mM (Y1219I), respectively.
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ABCC7 p.Tyr1219Trp 16966475:124:37
status: NEW[hide] CLC-0 and CFTR: chloride channels evolved from tra... Physiol Rev. 2008 Apr;88(2):351-87. Chen TY, Hwang TC
CLC-0 and CFTR: chloride channels evolved from transporters.
Physiol Rev. 2008 Apr;88(2):351-87., [PMID:18391167]
Abstract [show]
CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level.
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No. Sentence Comment
786 A more conservative mutation (Y1219W), however, did not change the K0.5 value significantly.
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ABCC7 p.Tyr1219Trp 18391167:786:30
status: NEW[hide] A stable ATP binding to the nucleotide binding dom... J Physiol Sci. 2010 Sep;60(5):353-62. doi: 10.1007/s12576-010-0102-2. Epub 2010 Jul 14. Shimizu H, Yu YC, Kono K, Kubota T, Yasui M, Li M, Hwang TC, Sohma Y
A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.
J Physiol Sci. 2010 Sep;60(5):353-62. doi: 10.1007/s12576-010-0102-2. Epub 2010 Jul 14., [PMID:20628841]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2.
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No. Sentence Comment
19 The degree of dose-response shift, a descending order of Y1218G, Y1219I, Y1219F and Y1219W, seemed to depend on the similarity of chemical properties, e.g., hydrophobicity and p bond by aromatic ring, of the side chains introduced by the mutations to tyrosine.
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ABCC7 p.Tyr1219Trp 20628841:19:84
status: NEW59 Figure 2b compares macroscopic current relaxations for Y1219G, Y1219I, Y1219F and Y1219W mutants.
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ABCC7 p.Tyr1219Trp 20628841:59:82
status: NEW60 Although a slow phase of current decay can be seen with Y1219G and Y1219I, this second phase is hardly discernable for the Y1219W mutant.
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ABCC7 p.Tyr1219Trp 20628841:60:123
status: NEW65 b A representative current trace of WT-CFTR channel showing immediate closing upon a rapid removal of ATP somewhere between Y1219W and Y1219I.
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ABCC7 p.Tyr1219Trp 20628841:65:125
status: NEW67 The time constants of the initial fast phase for all the Y1219 mutants are within hundreds of milliseconds (sfast: 504 &#b1; 76 ms, n = 5 for Y1219G; 450 &#b1; 44 ms, n = 7 for Y1219I; 571 &#b1; 142 ms, n = 4 for Y1219F; 513 &#b1; 66 ms, n = 5 for Y1219W) and there is not significant difference in the fast time constant among these Y1219 mutants (Fig. 2b).
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ABCC7 p.Tyr1219Trp 20628841:67:248
status: NEW68 On the other hand, the time constants of the slow component for Y1219F and Y1219W are shorter than those of Y1219G and Y1219I (Fig. 3a).
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ABCC7 p.Tyr1219Trp 20628841:68:75
status: NEW69 More importantly, the fraction of the slow component (Fig. 3b) shows a gradual decrease as the mutation becomes less 'preserved` (Y1219G [ Y1219I [ Y1219F [ Y1219W).
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ABCC7 p.Tyr1219Trp 20628841:69:157
status: NEW70 It should be noted that, for technical reasons (detailed in ''Discussion``), our calculation of the fraction of the slow component is somewhat overestimated for Y1219F and Y1219W.
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ABCC7 p.Tyr1219Trp 20628841:70:172
status: NEW71 The actual values for Y1219F and Y1219W are lower than those shown in Fig. 3b.
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ABCC7 p.Tyr1219Trp 20628841:71:33
status: NEW80 b Macroscopic current relaxations upon ATP wash-out for Y1219G, Y1219I, Y1219F and Y1219W.
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ABCC7 p.Tyr1219Trp 20628841:80:83
status: NEW82 The time constant and the fraction of the slow component are summarized in Fig. 3. c Single channel recording of Y1219G-CFTR showing a long opening even after ATP removal 35 30 25 20 15 10 5 0 Y1219G Y1219I Y1219F Y1219W 0.5 0.4 0.3 0.2 0.1 0.0 Y1219G Y1219I Y1219F Y1219W A slow / (A slow + A fast ) c4; slow (s) A B Fig. 3 Kinetic parameters of macroscopic current relaxations for Y1219G, Y1219I, Y1219F and Y1219W.
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ABCC7 p.Tyr1219Trp 20628841:82:214
status: NEWX
ABCC7 p.Tyr1219Trp 20628841:82:266
status: NEWX
ABCC7 p.Tyr1219Trp 20628841:82:413
status: NEW83 a Time constants of the slow component of the current relaxation for the Y1219 mutants. Y1219G: n = 5; Y1219I: n = 7; Y1219F: n = 4; and Y1219W: n = 5. b Fraction of the slow component (Aslow/Aslow ?
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ABCC7 p.Tyr1219Trp 20628841:83:137
status: NEW84 Afast) for the Y1219 mutants. Y1219G: n = 5; Y1219I: n = 7; Y1219F: n = 4; and Y1219W: n = 5 ATP because of their similar rate-limiting hydrolyzing rates [11].
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ABCC7 p.Tyr1219Trp 20628841:84:79
status: NEW132 Although the results with Y1219G and Y1219I mutations are quantified accurately, this may not be the case with the data for Y1219F and Y1219W.
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ABCC7 p.Tyr1219Trp 20628841:132:135
status: NEW133 First, like WT-CFTR, the slow component for Y1219W is too obscure for reliable curve fitting.
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ABCC7 p.Tyr1219Trp 20628841:133:44
status: NEW162 If different mutations at Y1219 can affect the entry rate and the exit rate differently as shown in Table 2, this hypothesis may explain most of the results Table 1 Parameter sets (s-1 ) for the ''kiss and run`` hypothesis kCO1 kO1C kO1O2 kO2C Y1219G 3 or 0a 3 2 9 10-2 3.8 9 10-2 Y1219I : : 1.3 9 10-2 : Y1219F : : 1.1 9 10-2 : Y1219W : : 4 9 10-3 : a kCO1 was set to 3 s-1 for simulating the ''with ATP`` condition and 0 s-1 for simulating the ''after the ATP washout`` condition.
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ABCC7 p.Tyr1219Trp 20628841:162:329
status: NEW166 b Representative reproduced macroscopic current relaxations mimicking those in Y1219G (red), Y1219I (green), Y1219F (cyan) and Y1219W (blue).
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ABCC7 p.Tyr1219Trp 20628841:166:127
status: NEW