ABCMdb

ABCC7 p.Leu1353Met

Predicted by SNAP2:	A: D (63%), C: D (59%), D: D (91%), E: D (85%), F: D (80%), G: D (85%), H: D (80%), I: D (63%), K: D (80%), M: D (66%), N: D (80%), P: D (91%), Q: D (75%), R: D (75%), S: D (80%), T: D (80%), V: D (63%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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Publications
[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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No. Sentence Comment
24 Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Login to comment
36 M AT E R I A L S A N D M E T H O D S Molecular biology pGEMHE-WT (Chan et al., 2000), carrying the coding sequence of human WT CFTR, was used as a template for mutants T460S, L1353M, H1348A, H1375A, T460S/L1353M, T460S/H1348A, and T460S/H1375A. Login to comment
79 Fig. S2 illustrates experiments to assay the rate of unlocking from the pyrophosphate-induced locked-open state for WT, T460S, L1353M, and T460S/L1353M channels. Login to comment
112 Effects of mutations at positions 460 and 1353 on ATP-dependent (hydrolytic) gating We first tested changes in energetic coupling between positions 460 and 1353 by perturbing these positions using mutations T460S and L1353M. Login to comment
115 To determine if the mutations T460S and L1353M, individually or together, had any effect on channel gating in saturating 2 mM ATP, burst durations were determined from patches containing 1-10 channels (Fig. 2 A). Login to comment
116 There was a small increase in closing rate (defined as inverse of the mean burst duration; Fig. 2 B) for T460S (3.6 ± 0.3 s1 ; n = 20; Fig. 2 B, red bar) and L1353M (3.3 ± 0.4 s1 ; n = 8; Fig. 2 B, blue bar) compared with WT (2.6 ± 0.3 s1 ; n = 13; Fig. 2 B, black bar), whereas there was no significant change for T460S/L1353M (n = 9; Fig. 2 B, green bar). Login to comment
134 We confirmed this was also the case for T460S and L1353M using multichannel analysis on patches containing <10 channels (not depicted), which showed that when [ATP] was reduced from 2 mM to 50 µM, burst duration was not significantly affected, and the fractional Po supported by 50 µM ATP (0.39 ± 0.07 and n = 6 for T460S, and 0.51 ± 0.08 and n = 5 for L1353M) could be accounted for by the fractional opening rate observed under the same conditions (0.39 ± 0.06 and n = 6 for T460S, and 0.46 ± 0.07 and n = 5 for L1353M). Login to comment
152 To determine if the mutations T460S, L1353M, and T460S/L1353M increased the rate of nonhydrolytic closure from an open state with ATP bound at both composite sites, we introduced the above site-1 mutations in a K1250R background. Login to comment
154 The fitted time constant for current decay, relaxation (Fig. 5 A, inset), provided an estimate for the average lifetime of the open state, which was 5.9 ± 0.5 s (n = 13) for K1250R (black bar) and unchanged in L1353M/ K1250R (7.2 ± 0.8 s; n = 10; P = 0.11; blue bar), but significantly reduced in T460S/K1250R (4.2 ± 0.3 s; n = 13; P < 0.01; red bar). Login to comment
155 Because relaxation was additively affected in T460S/L1353M/K1250R (4.5 ± 0.6 s; n = 10; P < 0.05; green bar), G‡ int(closing) was not significantly different from zero (Fig. 5 B), indicating that the coupling between the two residues on opposite sides of composite site 1 was not changed along the nonhydrolytic closure pathway between the ATP-bound open state and the transition state. Login to comment
178 Therefore, as an alternative means to study nonhydrolytic channel closing rates, we also determined the effect of mutations T460S, L1353M, and T460S/L1353M on the closing of channels locked open by ATP plus PPi (Fig. S2). Login to comment
185 Consistent with changes in closing rate, Po was significantly reduced for T460S/K1250R (0.28 ± 0.06; n = 6; P < 0.01; Fig. 5 C, red bar) and T460S/L1353M/ K1250R (0.26 ± 0.03; n = 8; P < 0.01; green bar) compared with K1250R (0.55 ± 0.07; n = 9; black bar), but not for L1353M/K1250R (0.55 ± 0.05; n = 8; blue bar). Login to comment