ABCMdb

ABCC7 p.Thr460Ser

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 (91%), 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 (75%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: N, 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
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
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
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
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
165 (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Login to comment
172 Energetic coupling between positions 460 and 1348 is little changed during gating Following a similar methodology, we proceeded to study changes in coupling between positions 460 and 1348 during gating, using perturbations T460S and H1348A. Login to comment
173 In these mutant cycles, two of the corners (WT and single-mutant T460S) are identical to the corresponding corners of the respective T460-L1353 mutant cycle. Login to comment
174 To rigorously compare the effects of the H1348A mutation onto the T460S versus WT backgrounds, the gating parameters for the latter two constructs should have been repeatedly measured in experiments side by side with those conducted on H1348A and T460S/H1348A. Login to comment
175 However, because Gint can be calculated using any two parallel sides of a mutant cycle, we did not repeat experiments for WT and T460S; instead, we calculated Gint using the two horizontal sides of each cycle, i.e., by comparing the effects of the T460S mutation onto the H1348A versus WT backgrounds. For this reason, we refrain from providing absolute G values for the vertical sides of the T460-H1348 mutant cycles (Figs. 6, B and D, and 7, B and D); and the same applies for the T460-H1375 mutant cycles (see below; Figs. 8, B and D, and 9, B and D). Login to comment
176 We first studied the single-channel gating pattern of H1348A and T460S/H1348A under normal hydrolytic conditions (Fig. 6 A) and extracted single-channel closing rates (Fig. 6 B, left). Login to comment
177 Although the H1348A mutation dramatically slowed closure (to 1.1 ± 0.2 s1 ; n = 8), the closing rate for T460S/H1348A was slightly accelerated relative to that of H1348A (compare green and blue bar (Gunderson and Kopito, 1994; Carson et al., 1995; Tsai et al., 2009), likely by inhibiting hydrolytic closure (Scott-Ward et al., 2007; Tsai et al., 2009). 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
186 Here too, the coupling energy, Gint(open-closed), was not significantly different from zero (Fig. 5 D), indicating that there was Figure 5. The T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. Login to comment
194 nonhydrolytic relaxation nor the reduction in nonhydrolytic Po by the T460S mutation (compare red vs. black bars in Fig. 5, A and C) was apparent when this mutation was introduced into an H1348A/K1250R background (compare green vs. blue bars in Fig. 7, A and D, left), these deviations from additivity resulted in a small change in T460-H1348 interaction energy only between the transition state for nonhydrolytic closure and the open ground state (Gint(closing) = 0.43 ± 0.14 kT; P = 0.01; Fig. 7 B), but not between open and closed ground states (Fig. 7 D, right; P = 0.1). 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
196 A slight tendency of mutation T460S to accelerate channel closure was also observed in this background (see Fig. 8 B, left, green bar), such that a mutant cycle built on closing rates (Fig. 8 B, right) did not reveal significant nonadditivity (Gint ≈ 0). 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
199 The slight difference in closing rates between T460S/H1348A and H1348A was mirrored by the slightly lower Po value of the double mutant (Fig. 6 C; compare green and blue bar). Login to comment
200 Consequently, for the calculated opening rates (Fig. 6 D, left), we did not detect the slight acceleration by the T460S mutation, which was observed when this mutation was introduced into a WT background (compare red and black bars in Fig. 3 C). Login to comment
202 To look for changes in interactions between positions 460 and 1348 during nonhydrolytic closure, we created nonhydrolytic H1348A/K1250R and T460S/H1348A/ K1250R channels and compared their closing rates by studying macroscopic current relaxations after ATP removal (Fig. 7 A). Login to comment
205 (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. Login to comment
206 (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). Login to comment
208 The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Login to comment
209 Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute G values are not printed for the vertical sides of the cycle. Login to comment
210 (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). Login to comment
211 (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). Login to comment
217 (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/ H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Login to comment
221 (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. Login to comment
222 (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/ H1348A (green 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
228 The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. 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
240 The most significant phenotypes were observed for T460S and H1348A, which, respectively, increased and decreased not only normal hydrolytic channel closing rate (Figs. 2 B and 6 B) but also the rate of nonhydrolytic closure (Figs. 5 A and 7 A; compare Fig. S2 B), suggesting that these mutations simultaneously affect the energy barriers for both closing pathways. 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
243 Although in this double-mutant background the T460S mutation did not noticeably affect the rate of nonhydrolytic closure (Fig. 9 A, green bar; compare with Fig. 5 A, red vs. black bar), this small deviation from additivity did not result in any significant coupling energy (Fig. 9 B; P = 0.2). 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
274 Our results on T460S/K1250R and H1348A/K1250R indeed show a respective decrease and increase in Po (Figs. 5 C and 7 D), confirming that the open ground state is destabilized in T460S/K1250R, but stabilized in H1348A/K1250R, with respect to the closed ground state. Login to comment
275 The simplest interpretation is that the T460S and H1348A mutations specifically affect the stability of the open state. Login to comment