ABCMdb

ABCC7 p.His1348Ala

None has been submitted yet.

Publications

PMID: 21576373 [PubMed] Szollosi A et al: "Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating."

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
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
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
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
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
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
203 Interestingly, mutation H1348A prolonged the time constant of the current relaxation (to 20 ± 2 s; n = 8; Fig. 7 A, blue bar) to a similar extent as it did normal burst durations; and noise analysis (Fig. 7 C) attested to the fact that the prolonged open time of H1348A/K1250R is associated with an unusually high open probability (Po = 0.83 ± 0.03 s; n = 6; Fig. 7 D, left, blue bar). 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
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
216 However, because a similar tendency (although to a lesser extent) was also apparent in the WT background (see Fig. 3 C, red vs. black bar), the mutant cycle built Figure 7. The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. 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
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
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

PMID: 25886173 [PubMed] Golin J et al: "The multidrug transporter Pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric ABC transporters."

No. Sentence Comment

282 Recently, however, Csanady et al. [63] studied the properties of the degenerate site C-loop substitution H1348A. Login to comment

PMID: 23752332 [PubMed] Csanady L et al: "Conformational changes in the catalytically inactive nucleotide-binding site of CFTR."

No. Sentence Comment

18 Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N 6 -(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Login to comment
20 Mutation H1348A also slowed (&#e07a;3&#d7;) the O1࢐O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (&#e07a;3&#d7;) and E1371S (&#e07a;3&#d7;) background mutants. Login to comment
21 Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. Login to comment
30 In addition, the H1348A mutation in the NBD2 signature sequence, which perturbs the NBD2 side of site 1, was found to slow channel closure (Szollosi et al., 2011). Login to comment
32 However, the exact mechanisms by which the H1348A mutation, or P-ATP bound at site 1, affects gating have not yet been elucidated. Login to comment
35 M A T E R I A L S A N D M E T H O D S Molecular biology Human WT CFTR and CFTR segment 433-1480 in the pGEMHE plasmid (Chan et al., 2000) served as templates for mutants H1348A, K1250A, E1371S, K1250A/H1348A, E1371S/H1348A, E1371S/K464A, and 433-1480(K1250A), which were created using the QuikChange kit (Agilent Technologies). Login to comment
93 [ATP] does not affect the prolongation of steady-state burst duration by the H1348A mutation In an earlier study, we found that the H1348A mutation prolongs burst durations of CFTR channels gating in 2 mM ATP by approximately threefold (Szollosi et al., 2011). Login to comment
96 These results confirm Figure 2.ߓ Steady-state mean burst duration of H1348A CFTR is insensitive to elevation of [ATP]. Login to comment
97 (A) Steady-state current recording from a patch containing two active H1348A CFTR channels gating in 2 mM (green segment) or 10 mM (dark blue segment) ATP. Login to comment
98 (B-D) Steady-state open probabilities (B), mean burst durations (C), and mean interburst durations (D) of H1348A CFTR in the presence of 2 mM (green bars) or 10 mM (dark blue bars) ATP, extracted from records with ࣘ5 active channels, as described in Materials and methods. Login to comment
111 Channels opened by Because for another site-1 mutant (W401F) mean burst durations were shown to increase at high millimolar ATP concentrations (Jih et al., 2012b), we tested whether that was the case for H1348A CFTR. Login to comment
113 Both P-ATP and the H1348A mutation slow nonhydrolytic channel closure To quantitatively compare the effects of our site-1 perturbations on the slow rate of nonhydrolytic closure, we studied macroscopic closing rates after nucleotide removal for channels in which nucleotide hydrolysis at site 2 is abrogated by mutagenesis. Login to comment
116 Both P-ATP (Fig. 3, A and D, red fit lines and time constants) and the H1348A Figure 3.ߓ P-ATP and the H1348A mutation slow nonhydrolytic CFTR closure. Login to comment
119 (B and E) Macroscopic currents of prephosphorylated K1250A/ H1348A (B) and E1371S/ H1348A (E) CFTR channels elicited by transient exposure (bars) to either 10 mM (B) or 2 mM (E) ATP. Login to comment
122 (C and F) Nonhydrolytic closing rates of channels opened by ATP (blue bars) or P-ATP (red bars), or of channels bearing the H1348A mutation opened by ATP (green bars), measured in the K1250A (C) or E1371S (F) background. Login to comment
125 We first characterized the effect of P-ATP on hydrolytic closing rate in these two mutant backgrounds (Fig. 4) by recording single-channel activity of H1348A (Fig. 4 A), or of &#e044;RI channels (Fig. 4 D), alternately exposed to either 2 mM ATP (Fig. 4, A and D, green segments) or 10 &#b5;M P-ATP (Fig. 4, A and D, brown segments). Login to comment
126 Consistent with previous work, the H1348A and &#e044;RI perturbations differentially affected hydrolytic closure: the closing rate in ATP was unchanged for &#e044;RI (Fig. 4 E; compare green bar with blue bar; cf. Csan&#e1;dy et al., 2005), but two- to threefold slowed for H1348A (Fig. 4 B; compare green bar with blue bar; cf. Fig. 2 C). Login to comment
132 On the NBD2 side of site 1 we chose mutation H1348A, because this perturbation causes a large effect on closing rate in ATP, which we had already characterized (Fig. 2). Login to comment
134 This "&#e044;RI" perturbation leaves ATP-dependent Figure 4.ߓ Mutation H1348A and deletion of segment 415-432 (&#e044;RI) abolish the effect of P-ATP on hydrolytic channel closure. Login to comment
135 (A and D) Steady-state recordings of single-channel currents in the presence of 2 mM ATP (green segments) or 10 &#b5;M P-ATP (brown segments) for H1348A (A) or &#e044;RI CFTR (D). Login to comment
138 (B and E) Closing rates, obtained as the inverses of the steady-state mean burst duration, for H1348A (B) and &#e044;RI (E) CFTR channels gating in ATP (green bars) or P-ATP (brown bars). Login to comment
143 The H1348A and &#e044;RI perturbations affected nonhydrolytic closing rate in opposite ways; whereas mutation H1348A slowed it by approximately threefold (Fig. 5 B; compare green bar with blue bar; cf. Fig. 3 C), the &#e044;RI perturbation accelerated it by approximately threefold (Fig. 5 E; compare green bar with blue bar; cf. Csan&#e1;dy et al., 2005). Login to comment
145 Thus, whereas P-ATP slowed nonhydrolytic closure by approximately fourfold for K1250A channels with an intact site 1 (Fig. 3 C; compare red with blue bar; replotted in Fig. 5, B and E), this effect increased to greater than sixfold and to approximately ninefold, respectively, in the presence of the site-1 perturbations H1348A and &#e044;RI (Fig. 5, B and E; compare brown with green bars), again suggesting nonadditivity. Login to comment
146 Indeed, mutant cycles built on nonhydrolytic closing rates (Fig. 5, C and F) yielded interaction free energies between the P group and residue 1348 (Fig. 5 C), as well as between the P group and the RI region (Fig. 5 F), significantly different from zero (P < 0.05 hydrolytic closure when applied in the H1348A or &#e044;RI mutant background (Fig. 4, B and E; compare brown with green bars). Login to comment
149 Nonadditive effects of P-ATP and site-1 mutations on nonhydrolytic closure support the slowing of this gating step by P-ATP bound in site 1 Additivity of effects on nonhydrolytic closure of the same site-1 perturbations with those of P-ATP was tested in the K1250A nonhydrolytic background (Fig. 5) by measuring the macroscopic closing rate of K1250A/H1348A channels (Fig. 5 A), and of channels obtained by co-expression of segments 1-414 and 433-1480(K1250A) (K1250A/&#e044;RI; Fig. 5 D) upon nucleotide removal. Login to comment
150 Figure 5.ߓ Mutation H1348A and deletion of segment 415-432 (&#e044;RI) potentiate the effect of P-ATP on nonhydrolytic channel closure. Login to comment
151 (A and D) Macroscopic currents from K1250A/H1348A (A) and K1250A/&#e044;RI (D) CFTR channels elicited by exposures to 10 mM ATP or 50 &#b5;M P-ATP (bars). Login to comment
153 (B and E) Nonhydrolytic closing rates for K1250A/ H1348A (B) and K1250A/&#e044;RI (E) CFTR channels, obtained as the inverses of the relaxation time constants upon removal of ATP (green bars) or P-ATP (brown bars); closing rates of K1250A CFTR upon removal of ATP (blue bars) and P-ATP (red bars) were replotted from Fig. 3 C. Login to comment
160 Because the W401F mutation also resides in site 1, we asked whether a reentry mechanism might explain the longer burst durations in P-ATP or in the presence of the H1348A mutation. Login to comment
161 To this end, we measured macroscopic closing rates after nucleotide removal for WT and &#e044;RI channels opened by either 2 mM ATP or by 10 &#b5;M P-ATP (Fig. 6, A and C), and for H1348A channels opened by 2 or 10 mM ATP (Fig. 6 B, top) or by 10 &#b5;M P-ATP (Fig. 6 B, bottom). Login to comment
163 Neither P-ATP nor the H1348A mutation disrupts near 1:1 stoichiometry between ATP occlusion and channel burst events Both P-ATP and the H1348A mutation prolong steady-state mean burst durations (Figs. 1 F and 2 C). Login to comment
165 Such a "reentry" mechanism should allow for more than one ATP hydrolysis cycle to happen within a single Figure 6.ߓ Relaxation time courses of macroscopic WT, H1348A, and &#e044;RI CFTR currents upon sudden nucleotide removal. Login to comment
166 (A-C) Macroscopic currents of prephosphorylated WT (A), H1348A (B), and &#e044;RI (C) CFTR channels elicited by exposure (bars) to either 2 mM ATP alternating with 10 &#b5;M P-ATP (A and C), or 2 mM ATP alternating with either 10 mM ATP (B, top) or 10 &#b5;M P-ATP (B, bottom). Login to comment
168 (D) Closing time constants of WT CFTR currents upon removal of 2 mM ATP (blue bar) or 10 &#b5;M P-ATP (red bar), of H1348A CFTR currents upon removal of 2 (left green bar) or 10 mM ATP (dark blue bar) or 10 &#b5;M P-ATP (left brown bar), and of &#e044;RI CFTR currents upon removal of 2 mM ATP (right green bar) or 10 &#b5;M P-ATP (right brown bar). Login to comment
170 Both P-ATP and the H1348A mutation prolong bursts by slowing the O1࢐O2 transition Thus, under the conditions studied here, the duration of each open burst includes some time spent in a prehydrolytic open state (O1; Fig. 7 C) in which a nucleoside triphosphate is occluded at site 2, followed by a shorter time interval in a less stable post-hydrolytic open state (O2; Fig. 7 C). Login to comment
173 To determine which of these two rates is affected by P-ATP and the H1348A mutation, respectively, we recorded currents from patches containing a single active channel (Fig. 7, A and B, insets). Login to comment
175 The histograms of burst durations were distinctly peaked for both WT channels gating in 10 &#b5;M P-ATP (Fig. 7 A) and H1348A channels gating in 2 mM ATP (Fig. 7 B), consistent with a non-equilibrium gating cycle that involves nucleotide lines). Login to comment
178 This suggests that both for WT (and &#e044;RI) channels gating in P-ATP, and for H1348A channels gating in 2 or 10 mM ATP or 10 &#b5;M P-ATP, each steady-state burst involves occlusion of a single nucleotide at site 2, just as suggested for WT channels gating in ATP (Csan&#e1;dy et al., 2010). Login to comment
182 Figure 7.ߓ P-ATP and the H1348A mutation slow the O1࢐O2 transition of CFTR channels. Login to comment
183 (A and B) Histograms of open burst durations compiled from 621 open burst events of single WT CFTR channels gating in 10 &#b5;M P-ATP (A) and from 908 open burst events of single H1348A CFTR channels gating in 2 mM ATP (B). Login to comment
190 (D) Summary of rates k1 and k2 obtained from the fits in A and B for WT CFTR gating in 10 &#b5;M P-ATP (red bars) and H1348A CFTR gating in 2 mM ATP (green bars); as a comparison, the values measured for WT CFTR gating in 2 mM ATP (blue bars) are replotted from Csan&#e1;dy et al. (2010). Login to comment
194 Because one such mutation (W401F) resides in site 1, we have evaluated the possibility that the H1348A mutation, or P-ATP bound in site 1, might also act by such a mechanism. Login to comment
195 However, the lack of effect of increasing [ATP] on steady-state burst durations of H1348A CFTR (Fig. 2 C) and the close agreement of macroscopic closing time constants upon nucleotide removal with steady-state mean burst durations (Fig. 6) ruled out this possibility and confirmed that the two site-1 perturbations studied here do not disrupt the near 1:1 stoichiometry between ATP occlusion events and pore opening events, characteristic to WT CFTR gating in ATP (Csan&#e1;dy et al., 2010). Login to comment
196 Although comparison of macroscopic and steady-state closing rates (Fig. 6) might not be sensitive enough to rule out slight deviations from 1:1 stoichiometry, it is clear that small effects would not be sufficient to explain the two- to threefold prolongation of burst durations by the H1348A mutation and P-ATP. Login to comment
202 Interestingly, this is exactly what we observed for the H1348A mutant, the opening rate of which is approximately twofold increased relative to WT (compare Fig. 2 D, green bar, with Fig. 1 G, blue bar). Login to comment
208 Accordingly, the maximum likelihood fit yielded two- to threefold slower k1 values (Fig. 7 D, left) in the presence of P-ATP (red bar) or the H1348A mutation (green bar) compared with the earlier estimate for WT channels gating in ATP (blue bar; replotted from Csan&#e1;dy et al., 2010). Login to comment
209 In contrast, neither P-ATP nor the H1348A mutation seemed to dramatically affect the faster rate k2 (Fig. 7 D, right). Login to comment
211 The H1348A mutation removes a histidine side chain from the signature sequence of NBD2, thereby perturbing the NBD2 side of site 1, whereas the use of P-ATP introduces a phenylethyl group into this composite binding site. Login to comment
217 They further proposed that mutations or pharmacological modulation might prolong CFTR channel bursts by increasing the frequency of such "reentry" events (Jih et al., 2012a,b; Jih and Furthermore, the selective energetic stabilization of state O1 relative to O2 (Fig. 8) by both perturbations implies that in a WT channel, the physico-chemical environment of the H1348A side chain, or of the phenylethyl group of a P-ATP molecule bound at site 1, experiences a significant change also during the O1࢐O2 transition, i.e., upon ATP hydrolysis at the active composite site 2. Login to comment
233 Figure 8.ߓ Energetic interpretation of the gating effects of P-ATP and the H1348A mutation. Login to comment