ABCMdb

PMID: 23752332

Csanady L, Mihalyi C, Szollosi A, Torocsik B, Vergani P
Conformational changes in the catalytically inactive nucleotide-binding site of CFTR.
J Gen Physiol. 2013 Jul;142(1):61-73. doi: 10.1085/jgp.201210954. Epub 2013 Jun 10., [PubMed]

Sentences

No. Mutations Sentence Comment

18 ABCC7 p.His1348Ala 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 19 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2&#d7;) transition O1࢐O2 and decreases the nonhydrolytic closing rate (transition O1࢐C) of CFTR mutants K1250A (&#e07a;4&#d7;) and E1371S (&#e07a;3&#d7;). Login to comment 20 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 29 ABCC7 p.Lys464Ala For instance, the K464A mutation, which-by removing the side chain of the conserved NBD1 Walker A lysine-perturbs the NBD1 side of site 1, dramatically alters the mechanism of gating (Csan&#e1;dy et al., 2010). Login to comment 30 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala ABCC7 p.His1348Ala 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 49 ABCC7 p.Trp401Phe In contrast, [ATP]-dependent prolongation of open burst durations for the W401F CFTR mutant (Jih et al., 2012b), or for WT CFTR gating in the presence of the potentiator compound Vx-770 (Jih and Hwang, 2013), has led to the suggestion that post-hydrolytic ADP/ATP exchange in site 2 might occur without intervening pore closure, thereby increasing the coupling ratio by allowing more than one ATP to be hydrolyzed in site 2 within a single gating cycle (Jih et al., 2012a). Login to comment 57 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser For the nonhydrolytic mutants (K1250A, E1371S, and double mutants), the decay time courses after nucleotide removal often required a double-exponential function-of the form I(t) = I0(A1exp(&#e032;t/&#e074;1) + (1 &#e032; A1)exp(&#e032;t/&#e074;2))-with two slow time constants for a satisfactory fit (e.g., Fig. 3, B and E), suggesting the presence of two populations of open-channel bursts. Login to comment 66 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser Fig. S2 shows the effect of the K464A mutation on nonhydrolytic closing rate measured in the E1371S background. Login to comment 72 ABCC7 p.Lys1250Ala MgATP (Sigma-Aldrich) was added from a 400-mM aqueous stock solution (pH 7.1 with NMDG) to achieve a final concentration of 2 mM (or 10 mM for channels bearing the K1250A mutation). Login to comment 73 ABCC7 p.Lys1250Ala A 10-mM aqueous stock solution of P-ATP Na+ salt (BIOLOG Life Science Institute) was stored at &#e032;80&#b0;C and diluted into the bath solution immediately before recording to achieve a final concentration of 10 &#b5;M (or 50 &#b5;M for K1250A mutants). Login to comment 93 ABCC7 p.His1348Ala ABCC7 p.His1348Ala [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 ABCC7 p.His1348Ala These results confirm Figure 2.ߓ Steady-state mean burst duration of H1348A CFTR is insensitive to elevation of [ATP]. Login to comment 97 ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala (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 107 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Thus, the nonhydrolytic closing rate (Fig. 3, C and F, bars; calculated from the fitted relaxation time constants as described in Materials and methods) was similarly affected by both site-1 perturbations, and this was true regardless of whether the K1250A (Fig. 3 C) or the E1371S (Fig. 3 F) mutant was chosen as the nonhydrolytic model; i.e., both site-1 perturbations decreased this rate by two- to threefold. Login to comment 111 ABCC7 p.Trp401Phe ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 115 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser We therefore compared the effects of our site-1 perturbations on the closing rates of two nonhydrolytic mutants, NBD2 Walker A mutant K1250A (Fig. 3, A-C) and NBD2 Walker B mutant E1371S (Fig. 3, D-F). Login to comment 116 ABCC7 p.His1348Ala ABCC7 p.His1348Ala 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 117 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser (A and D) Macroscopic currents of prephosphorylated K1250A (A) and E1371S (D) CFTR channels elicited by exposure (bars) to either 10 mM ATP alternating with 50 &#b5;M P-ATP (A) or 2 mM ATP alternating with 10 &#b5;M P-ATP (D); the fivefold higher nucleotide concentrations for the K1250A constructs were used to compensate for the large decrease in apparent ATP affinity caused by this mutation (Vergani et al., 2003). Login to comment 119 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (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 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala ABCC7 p.His1348Ala 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 ABCC7 p.Lys1250Ala ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.His1348Ala (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 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.His1348Ala (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 ABCC7 p.Trp401Phe ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala ABCC7 p.His1348Ala 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 164 ABCC7 p.Trp401Phe Recent studies suggested a novel mechanism for a prolongation of steady-state CFTR burst durations for NBD1 mutant W401F (Jih et al., 2012a,b), by proposing the presence of a short time window at the end of each burst during which the hydrolysis products ADP and phosphate at site 2 can be replaced by a new ATP molecule without intervening channel gate closure. Login to comment 165 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala Figure 7.ߓ P-ATP and the H1348A mutation slow the O1࢐O2 transition of CFTR channels. Login to comment 183 ABCC7 p.His1348Ala (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 ABCC7 p.His1348Ala (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 ABCC7 p.Trp401Phe ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 ABCC7 p.His1348Ala 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 219 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser Interestingly, the K464A mutation, which perturbs site 1 by removing the conserved Walker A lysine, was also shown to affect the energetics of both of the C1࢒O1 and O1࢐O2 gating steps (Csan&#e1;dy et al., 2010), although in a different way: in this mutant, rate k1 decreased approximately fourfold, whereas the rate of nonhydrolytic closure, in a K1250A mutant background, increased by &#e07a;10-fold (this is also replicated in the E1371S background; Fig. S2). Login to comment 220 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Thus, although the distortion of the energetic profile by the K464A mutation differs from that seen for the two perturbations studied here, the K464A result is again consistent with movements in site 1 occurring in both of the above gating steps. Login to comment 233 ABCC7 p.His1348Ala Figure 8.ߓ Energetic interpretation of the gating effects of P-ATP and the H1348A mutation. Login to comment