ABCMdb

PMID: 20876359

Szollosi A, Vergani P, Csanady L
Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2.
J Gen Physiol. 2010 Oct;136(4):407-23., [PubMed]

Sentences

No. Mutations Sentence Comment

18 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Phe1296Ser We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Login to comment 19 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Login to comment 28 ABCC7 p.Lys1250Arg M AT E R I A L S A N D M E T H O D S Molecular biology CFTR mutants were constructed by using either pGEMHE-WT (Chan et al., 2000) or pGEMHE-K1250R (Vergani et al., 2005) as template. Login to comment 72 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser In this study, the average durations of stationary segments of record used for estimating Po;max were 40-50 s for the wild-type (WT), F1296S, N1303Q, and F1296S/N1303Q constructs (estimated single-channel cycle times 1.25 s in saturating ATP; Fig. 8 A), but 100-130 s for K1250R, F1296S/K1250R, and N1303Q/K1250R, and 220 s for F1296S/ N1303Q/K1250R (estimated single-channel cycle times 13 s in saturating ATP; Fig. 8 A). Login to comment 105 ABCC7 p.Asn1303Gln By elongating the side chain at site 2, the substitution N1303Q might interfere with potential H bonds linking residue 1303 to either 1296 or 1358. Login to comment 109 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Figs. S3 and S4 show verification of Po;max estimates in single-channel patches for WT, F1296S, N1303Q, and F1296S/N1303Q (Fig. S3), as well as for K1250R, F1296S/K1250R, N1303Q/K1250R, and F1296S/N1303Q/K1250R (Fig. S4). Login to comment 111 ABCC7 p.Lys1250Arg Fig. S6 illustrates apparent affinities for ATP in the K1250R background. Login to comment 112 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala Fig. S7 shows example macroscopic current traces to illustrate the apparent affinities of R1358A and R1358A/N1303Q for ATP. Login to comment 113 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Fig. S8 depicts predicted Po time courses in response to the addition/removal of ATP for WT, F1296S/N1303Q, K1250R, and F1296S/N1303Q/K1250R, calculated using Scheme 2. Login to comment 118 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (A-D) Inward chloride currents recorded in patches excised from resting oocytes expressing WT (A), F1296S (B), N1303Q (C), and F1296S/N1303Q (D) CFTR. Login to comment 122 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Phosphorylation dependence of channel activity is largely unchanged for F1296S, N1303Q, and F1296S/N1303Q CFTR mutants, whereas the double mutant displays significantly increased ATP-independent activity Similarly to WT CFTR (Fig. 2 A), in patches excised from resting oocytes expressing F1296S (Fig. 2 B), N1303Q (Fig. 2 C), or double mutant F1296S/N1303Q (Fig. 2 D) CFTR channels, we observed very low channel activity upon the application of 2 mM ATP, but robust macroscopic currents could be activated for all three constructs by the subsequent addition of 300 nM PKA to the bath solution. Login to comment 127 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser This pattern of coevolution between sites 1 and 2 suggests that possible functional interactions between the two residues, perturbed by single mutations F1296S and N1303Q, might be restored in a double mutant F1296S/N1303Q. Login to comment 129 ABCC7 p.Arg1358Ala To perturb site 3, we chose mutation R1358A, which is likely to prevent a possible hydrogen-bonding interaction between the R1358 and N1303 side chains. Login to comment 131 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (A) Representative traces of WT, F1296S, N1303Q, and F1296S/N1303Q currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level. Login to comment 132 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (B) Estimation of Po;max for WT (black), F1296S (red), N1303Q (blue), and F1296S/N1303Q (green) by stationary noise analysis. Login to comment 138 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser maximal open probabilities were little changed in all three mutant constructs (0.22 ± 0.02, 0.32 ± 0.04, and 0.33 ± 0.03, respectively, for F1296S, N1303Q, and F1296S/ N1303Q) compared with WT (0.35 ± 0.03) (Fig. 3 B), open probability in zero ATP was 10-fold higher for the F1296S/N1303Q double mutant than for WT or the two single mutants (Fig. 3 C). Login to comment 140 ABCC7 p.Lys1250Arg Because openings in the absence of ATP must necessarily be nonhydrolytic, the NBD2 Walker A mutation K1250R, known to abolish ATP hydrolysis in ABC proteins (Lerner-Marmarosh et al., 1999; Payen et al., 2005), is not expected to affect this spontaneous gating. Login to comment 141 ABCC7 p.Lys1250Arg To test this idea, we studied the same mutant cycle also in a K1250R background (Fig. 4). Login to comment 142 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser Indeed, although K1250R, F1296S/K1250R, and N1303Q/K1250R ATP removal rapidly abolished currents for both single mutants just as for WT (Fig. 2, A-C), in the case of the double mutant, a constitutive basal activity persisted even after ATP removal (Fig. 2 D, magnified in inset) and did not vanish even over the time course of several minutes. Login to comment 146 ABCC7 p.Lys1250Arg Although Figure 4. The stabilizing site-1-site-2 interaction that facilitates channel opening in the absence of ATP is preserved in the ATP hydrolysis-deficient K1250R mutant. Login to comment 147 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (A) Representative traces of K1250R, F1296S/K1250R, N1303Q/K1250R, and F1296S/N1303Q/K1250R currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level (determined for the triple mutant similarly to that in Fig. S2). Login to comment 148 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (B) Estimation of Po;max for K1250R (black), F1296S/K1250R (red), N1303Q/K1250R (blue), and F1296S/N1303Q/K1250R (green) by stationary noise analysis. Login to comment 150 ABCC7 p.Lys1250Arg (D) Thermodynamic mutant cycle built on Po;bas/(1Po;bas) values; notation as in Fig. 3 D. analogous constructs in the K1250R background (Fig. S4, A and B). Login to comment 152 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ala ABCC7 p.Phe1296Ala Again, Po;bas of the double mutant F1296A/N1303Q was 10-fold larger than that of WT or the single mutants F1296A and N1303Q (Fig. S5, A-C), producing a negative Gint for the mutant cycle built on Po;bas/(1Po;bas) values (Fig. S5 D), even though in this case the overall free energy change (1.18 ± 0.84 kT) could not be claimed significantly different from zero because of the smaller Po;bas values obtained for WT in this set of experiments (Fig. S5 C, black). Login to comment 155 ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser constructs showed hardly detectable basal activity, a markedly elevated spontaneous activity was observed for the F1296S/N1303Q/K1250R triple mutant (Fig. 4 A), persisting even minutes after ATP washout. Login to comment 157 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Similarly to their hy-drolytic counterparts, Po;bas was 10-fold higher in F1296S/N1303Q/K1250R compared with the other three constructs, and the mutant cycle built on the closed-open equilibrium constant Po;bas/(1Po;bas) yielded a Gint of 2.36 ± 0.58 kT (Fig. 4 D)-again, significantly different from zero (P < 0.01). Login to comment 160 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Conventional dwell-time analysis of such records yielded Po;max values similar to those obtained by noise analysis both for the WT, F1296S, N1303Q, and F1296S/ N1303Q constructs (Fig. S3, A and B), and for the Figure 5. Energetic coupling between sites 1 and 2 changes between ATP-bound open and ATP-free closed states, but not between ATP-bound closed and open states. Login to comment 161 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (A) Summary of Po;max values for K1250R (black), F1296S/K1250R (red), N1303Q/K1250R (blue), and F1296S/N1303Q/K1250R (green) obtained from the data presented in Fig. 4 B. Login to comment 168 ABCC7 p.Lys1250Arg Interestingly, Po;max of K1250R (0.68 ± 0.06; n = 6) was only slightly affected by the substitutions at sites 1 and 2, and even these small changes were mostly additive (Fig. 5 A; compare Fig. S4 B), such that a mutant cycle built on Po;max/(1Po;max) yielded a Gint of zero (0.03 ± 0.14 kT; Fig. 5 B; compare Fig. S4 C). Login to comment 171 ABCC7 p.Lys1250Arg The K1250R mutation itself is known to prolong open-channel burst durations by 20-30-fold (Vergani et al., 2005; Csanády et al., 2006) due to the slow rate of dissociation of the ATP-bound NBD dimer in the absence of ATP hydrolysis. Login to comment 172 ABCC7 p.Lys1250Arg Consistent with those reports, upon the sudden removal of ATP, we saw macroscopic K1250R currents decline with a time constant of 8 s (Fig. 5 C, black single-exponential fit In a nonhydrolytic background, interaction between sites 1 and 2 is similar for ATP-bound closed and open states, but changes after ATP removal Because the interaction between sites 1 and 2 changes during ATP-independent spontaneous openings (Figs. 3 D and 4 D), we wondered whether the same interaction also plays a role in normal, ATP-dependent channel opening. Login to comment 175 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Thus, to test for a possible change in interaction between sites 1 and 2 during ATP-driven reversible opening and closure, we repeated the mutant cycle analysis in the nonhydrolytic K1250R background, comparing Po;max values for K1250R, F1296S/K1250R, N1303Q/ K1250R, and F1296S/N1303Q/K1250R (Fig. 5 A), Figure 6. ATP binding affects energetic coupling between sites 1 and 2 in closed channels. Login to comment 176 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (A) [ATP] dependence of macroscopic currents was assayed for WT (top left), F1296S (top right), N1303Q (bottom left), and F1296S/N1303Q (bottom right) channels by exposure to various test [ATP] bracketed by exposures to 2 mM ATP. Login to comment 177 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (B) ATP-dependent current fractions (II0)/(ImaxI0) plotted as a function of [ATP] for WT (black), F1296S (red), N1303Q (blue), and F1296S/N1303Q (green). Login to comment 184 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Although neither the F1296S nor the N1303Q mutation, when introduced one at a time, affected the time constant of current relaxation of K1250R upon ATP removal (Fig. 5 C, red and blue fit lines and bars), this relaxation time constant (relax) was prolonged by approximately fourfold, to 31 ± 5 s (n = 10), in the triple mutant F1296S/ N1303Q/K1250R (Fig. 5 C, green fit line and bar). Login to comment 186 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala (A) Representative traces of R1358A and R1358A/N1303Q currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level. Login to comment 187 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala (B) Estimation of Po;max for WT (black), R1358A (red), N1303Q (blue), and R1358A/N1303Q (green) by stationary noise analysis. Login to comment 188 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala Estimated Po;max was 0.62 ± 0.05 for R1358A and 0.36 ± 0.04 for R1358A/N1303Q. Login to comment 191 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala (E) ATP-dependent current fractions (II0)/(ImaxI0) plotted as a function of [ATP] for WT (black), R1358A (red), N1303Q (blue), and R1358A/N1303Q (green). Login to comment 195 ABCC7 p.Asn1303Gln channels retained substantial basal activity after ATP removal (Fig. 7 A; traces for WT and N1303Q are illustrated in Fig. 3 A). Login to comment 196 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala Combining Po;bas/Po;max, obtained from current segments in 0 mM and bracketing periods in 2 mM ATP with Po;max estimated for the 2-mM ATP segments using stationary noise analysis (Fig. 7 B), provided Po;bas estimates (Fig. 7 C) that were higher in both R1358A and R1358A/N1303Q compared with WT or N1303Q. Login to comment 199 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala We also investigated a possible change in coupling between sites 2 and 3 upon ATP binding by studying [ATP] dependence of macroscopic currents (sample current traces for R1358A and R1358A/N1303Q are shown in Fig. S7, A and B). Login to comment 200 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala Fitting the [ATP] dose-response curve of the ATP-sensitive current fractions (Fig. 7 E) yielded a slightly increased KPo value for R1358A/N1303Q (inset), but for the calculated KrCO values (Fig. 7 F), a similar trend was apparent even for R1358A. Login to comment 205 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser For instance, in TAP1, the site-1 phenylalanine backbone forms an H bond with the asparagine at site 2 (corresponding to N1303 in CFTR`s NBD2) when ATP is bound to the protein (Procko et al., 2006), whereas in the ADP-bound form Right-shift in ATP dependence of opening rate for F1296S/ N1303Q indicates a change in interaction between sites 1 and 2 upon ATP binding to closed channels To test whether the site-1-site-2 interaction was affected by ATP binding, we evaluated the apparent affinity for ATP to open WT, F1296S, N1303Q, and F1296S/ N1303Q channels. Login to comment 209 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Just as for KPo, the resulting KrCO values were slightly lower for F1296S and N1303Q than for WT, but approximately twofold increased for the double mutant F1296S/N1303Q (Fig. 6 C). Login to comment 213 ABCC7 p.Lys1250Arg Thus, in the K1250R background, apparent ATP affinities were three- to fourfold decreased (Fig. S6, A and B), corresponding to six- to ninefold increased values of KrCO (Fig. S6 C), but a mutant cycle built on the latter values yielded a Gint not significantly different from zero (Fig. S6 D). Login to comment 214 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala Truncation of the site-3 arginine side chain promotes spontaneous, ATP-independent opening regardless of the side chain at site 2 To determine the functional importance of site 3 within our triad of target residues (Fig. 1), we investigated functional coupling between sites 2 (position 1303) and 3 (position 1358) by comparing the effects of removal of the R1358 side chain (R1358A) in either a WT or an N1303Q background. Login to comment 215 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala ABCC7 p.Arg1358Ala Interestingly, after prephosphorylation by 300 nM PKA, both R1358A and R1358A/N1303Q result from formation of a stabilizing interaction in state B (or breaking of a destabilizing interaction present in state A). Login to comment 227 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Phenotypic pattern of single and double mutants at site 1 and 2 suggests toggle switch mechanism Using mutants F1296S, N1303Q, and F1296S/N1303Q, we studied the interaction between sites 1 and 2 in the form of mutant cycles built on (a) ATP-independent spontaneous activity (i.e., Po;bas/(1Po;bas)) both in hydrolysis-competent (Fig. 3 D) and (b) in catalytically inactive (Fig. 4 D) background; (c) ATP-dependent maximal activity (i.e., Po;max/(1Po;max)) (Fig. 5 B) and (d) de- activationrateuponATPremoval(Fig.5D)ofcatalytically incompetent constructs; and (e) apparent affinity for ATP to open the channels (Fig. 6 D). Login to comment 228 ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Interestingly, although four of the above five mutant cycles (Figs. 3 D, 4 D, 5 D, and 6 D) yielded Gint significantly different from zero, in each case this was due to a significantly altered phenotype in the double mutant F1296S/N1303Q, whereas the single mutations F1296S and N1303Q did not measurably affect gating (Figs. 3-6). Login to comment 242 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Gln ABCC7 p.Lys1250Arg Although for WT CFTR and for the nonhydrolytic mutant D1370N these two parameters are in rough agreement (Csanády et al., 2010), such comparisons have not yet been done for several other NBD2mutantsdefectiveinATPhydrolysis(e.g.,K1250R, K1250A, E1371S, and E1371Q). Login to comment 268 ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser The two rates assumed to be changed by the F1296S/N1303Q double mutation, and by the K1250R mutation, are shown in red and magenta, respectively, belowtheWTrates. Login to comment 269 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser (B)Tablesummarizingparam- eters Po;bas and KPo predicted by Scheme 2 for WT (using the rates in black in A) and F1296S/ N1303Q (using the two rates in red in A), as well as Po;max and relax for K1250R and F1296S/ N1303Q/K1250R (using the rates printed in magenta for steps C4→O2 and O2→C1). Login to comment 272 ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser in an unchanged Po;max (Fig. 8 B), just as we have observed for F1296S/N1303Q/K1250R (Fig. 5 A). Login to comment 274 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser ABCC7 p.Phe1296Ser Such predicted time courses are summarized in Fig. S8 for WT, F1296S/N1303Q, K1250R, and F1296S/N1303Q/K1250R. Login to comment 275 ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser Thus, this alteration of only two analogous rates in the double mutant F1296S/N1303Q is sufficient to account for all of our data on site-1-site-2 interactions (Figs. 1-6). Login to comment 276 ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser Because S-Q (and A-Q) pairings are also quite frequent alternatives of the most typical F-N pairing at these two sites (Fig. 1 A), it is conceivable that the site-1-site-2 interaction postulated for states C2, C4, O1, and O2 in WT can also be formed to some extent in F1296S/N1303Q. Login to comment 281 ABCC7 p.Arg1358Ala The observed facilitation of spontaneous channel openings by the R1358A mutation (Fig. 7 C) therefore likely reflects loss of a stabilizing interaction in the C1 state between R1358 and a residue other than N1303. Login to comment 286 ABCC7 p.Lys1250Arg nonhydrolytic mutant K1250R (Fig. 5 C, black). Login to comment 287 ABCC7 p.Lys1250Arg The Po;max values of 0.6 measured for the long-burst nonhydrolytic mutants (Fig. 5 A) suggest that the K1250R mutation, in addition to abrogating ATP hydrolysis, also slows maximal opening rate. Login to comment 288 ABCC7 p.Lys1250Arg We therefore modeled the effect of the K1250R mutation by simultaneously setting rate O2→C1 to zero and rate C4→O2 to 0.2 s1 (Fig. 8 A, magenta). Login to comment 291 ABCC7 p.Lys1250Arg Parameters Po;bas and KPo calculated for Scheme 2 using the rates plotted in black in Fig. 8 A, as well as Po;max and relax calculated using the two rates adjusted for K1250R (Fig. 8 A, magenta), are in good agreement with the measured values (Fig. 8 B, left column, measured values are shown in parentheses below each calculated parameter). Login to comment 293 ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser The lack of phenotype in the single mutants N1303Q and F1296S can be accounted for by assuming stabilizing F-X and N-Y interactions in states C1 and C3 replaced by a stabilizing F-N interaction in states C2 and C4 (retained in O1 and O2) in WT CFTR (illustrated in the cartoon in Fig. 8 A). Login to comment 295 ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser In contrast, the double mutation F1296S/N1303Q removes two stabilizing interactions (F-X and N-Y) in states C1 and C3, but only one such interaction (F-N) in states C2, C4, O1, and O2. Login to comment 297 ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser Indeed, a 160-fold decrease in rate C2→C1 (Fig. 8 A, red) increases Po;bas by 10-fold (Fig. 8 B) to the level we have observed for F1296S/N1303Q (Fig. 3 C). Login to comment 298 ABCC7 p.Lys1250Arg ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser A comparable (100-fold) decrease in rate C4→C3 (Fig. 8 A, red) reproduces the approximately fourfold prolonged relax (Fig. 8 B) we have observed for F1296S/N1303Q/K1250R (Fig. 5 C). Login to comment 300 ABCC7 p.Asn1303Gln ABCC7 p.Phe1296Ser Moreover, the reduction in rate C4→C3 has no impact on maximal opening rate, and hence on Po;max, consistent with our observation for the double mutant F1296S/N1303Q (Fig. 3 B). Login to comment