ABCMdb

ABCC7 p.His1375Ala

ClinVar:	c.4123C>A , p.His1375Asn ? , not provided c.4124A>C , p.His1375Pro ? , not provided
CF databases:	c.4123C>A , p.His1375Asn (CFTR1) ? , Exon 22 c.4124A>C , p.His1375Pro (CFTR1) ? , This mutation was identified on 3 Italian CF chromosomes, applying a protocol of extended mutational search (5?-flanking region, all the exons and adjacent intronic regions) by DHPLC and direct sequencing. No other mutations were found on the same alleles. The H1375P mutation was not found in 232 alleles from the general population. In two of the subjects carrying this mutation and sharing the same regional origin, the 2789+5GtoA was found on both other alleles. In the other subject the G85E was found.
Predicted by SNAP2:	A: N (61%), C: D (53%), D: D (66%), E: D (66%), F: N (57%), G: N (53%), I: N (53%), K: D (59%), L: N (53%), M: D (53%), N: N (93%), P: D (75%), Q: N (53%), R: D (59%), S: N (72%), T: N (57%), V: N (53%), W: D (66%), Y: N (72%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N,

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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
27 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. 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
102 Fitting the relaxation time course after ATP removal for the nonhydrolytic H1375A/K1250R and T460S/H1375A/K1250R constructs consistently required a double exponential with two slow time constants (each in the seconds range), suggesting two populations of open-channel bursts (see Fig. 9 A). Login to comment
195 Energetic coupling between positions 460 and 1375 is little changed during gating To study interactions between positions 460 and 1375, we compared the effects of mutation T460S in H1375A and WT backgrounds. For single channels gating under normal hydrolytic conditions (Fig. 8 A, top), mutation H1375A also slowed closure (to 1.3 ± 0.1 s1 ; n = 6; Fig. 8 B, left, blue bar), similarly to what was observed for H1348A (see Fig. 6 B, left, blue bar). Login to comment
197 The increased open probability of T460S/H1375A relative to that of H1375A (Fig. 8 C, in Fig. 6 B, left), just as that of T460S relative to WT (compare red and black bar in Fig. 2 B). Login to comment
215 Because for both H1375A/K1250R green vs. blue bar) attested to an increased opening rate in the double mutant (Fig. 8 D, left, green vs. blue bar). Login to comment
225 (A) Representative single-channel current traces from prephosphorylated H1375A and T460S/H1375A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. Login to comment
226 (B; left) Closing rates of H1375A (blue bar) and T460S/H1375A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). Login to comment
229 (C) Noise analysis was used to estimate Po for H1375A (blue bar) and T460S/H1375A (green bar). Login to comment
230 (D; left) Opening rates of H1375A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and closing rate (see B). Login to comment
241 An alteration and T460S/H1375A/K1250R adequate fitting of the relaxation time course after ATP removal consistently required a double exponential with two slow time constants (each in the seconds range; Fig. 9 A), average steady-state closing rate was estimated from a double-exponential fit as described in Materials and methods. Login to comment
242 Unexpectedly, when the H1375A mutation was introduced into the K1250R background, average nonhydrolytic closing rate was not slowed, but rather slightly accelerated (Fig. 9 A, blue bar). Login to comment
244 Finally, by noise analysis (Fig. 9 C), mutation T460S reduced Po in the H1375A/K1250R background (compare green vs. blue bars in Fig. 9 D, left) to a similar extent as it did in the single-mutant K1250R background (compare red vs. black bars in Fig. 5 C), yielding a Gint(open-closed) not significantly different from zero (Fig. 9 D; P = 0.15). Login to comment
248 (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Login to comment
249 Fitted parameters were 1 = 2.8 s, 2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/ K1250R trace, and 1 = 2.8 s, 2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/ K1250R trace. Login to comment
253 (C) Noise analysis for estimation of Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. Login to comment
254 (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). Login to comment
277 In contrast to the simple interpretation of the effects of the above two mutations, the functional consequences of the H1375A mutation are more complex. Login to comment
281 Although the H1375A phenotypes are not easily interpretable, altogether our findings are generally consistent with evidence from other studies of mutations in site 1, which suggest that protein-nucleotide and protein-protein interactions at composite site 1 contribute to the stability of the open-state dimer (Vergani et al., 2003; Bompadre et al., 2005; Cai et al., 2006; Zhou et al., 2006; Tsai et al., 2009). Login to comment