ABCMdb

PMID: 17043148

Csanady L, Nairn AC, Gadsby DC
Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle.
J Gen Physiol. 2006 Nov;128(5):523-33. Epub 2006 Oct 16., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg ∆H‡ for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 ± 2, 39 ± 4, and 37 ± 6 kJ/mol, respectively. Login to comment 41 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg M AT E R I A L S A N D M E T H O D S Molecular Biology pGEMHE-WT was constructed as previously described (Chan et al., 2000), and the K1250R and K1250A mutations introduced using QuikChange (Stratagene) as previously described (Vergani et al., 2003, 2005). Login to comment 88 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg S2-S4 show parallel macroscopic current and temperature records illustrating temperature dependence of closure of partially phosphorylated K1250R and K1250A, and of AMPPNP-locked WT, CFTR, respectively, recorded at -80 to -20 mV between 25°C and 31°C. Fig. S5 demonstrates that exposure to millimolar levels of the hydrolysis products ADP+Pi does not cause opening of prephosphorylated WT CFTR channels. Login to comment 89 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg Fig. S6 shows the predicted energetic profile of CFTR gating obtained using K1250A, not K1250R (as in Fig. 6), as a model for nonhydrolytic channel closure. Login to comment 105 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg Temperature Dependence of Closing Rate of Hydrolysis-deficient Mutant K1250R and K1250A CFTR Channels To estimate ∆H‡ for channel closing when the normal route for channel closure via ATP hydrolysis was unavailable, we studied the temperature dependence of the closing rate of two channel constructs in which the composite NBD2 site was made catalytically inactive by mutation of the conserved NBD2 Walker A lysine, K1250. Login to comment 106 ABCC7 p.Lys1250Ala The mutation K1250A has been shown to abolish ATPase activity of purified CFTR (Ramjeesingh et al., 1999). Login to comment 107 ABCC7 p.Lys1250Arg In another ABC ATPase, P-glycoprotein, the Lys-to-Arg mutation of either Walker A lysine abolishes ATP hydrolysis (Lerner-Marmarosh et al., 1999), and in CFTR, the K1250R mutation prolongs open burst durations by >20-fold (compare Vergani et al., 2005), consistent with abolished, or greatly diminished, ATP hydrolysis. Login to comment 108 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg Closure of K1250R or of K1250A mutant CFTR channels is too slow to allow kinetic analysis of individual gating events and so it was assayed as current decay after sudden removal of ATP. Login to comment 110 ABCC7 p.Lys1250Arg Prephosphorylated K1250R CFTR channels in macropatches were repeatedly opened by brief exposures to 2 mM MgATP at temperatures alternating between 25°C and ‫°51ف‬C (Fig. 3 A), or 25°C and ‫°13ف‬C (Fig. S2). Login to comment 113 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Because the K1250A mutation greatly diminishes the affinity for ATP binding (Vergani et al., 2003), 10 mM MgATP was used to repeatedly activate macroscopic K1250A currents at temperatures alternating between 25°C and either ‫°51ف‬C (Fig. 4 A) or ‫°13ف‬C (Fig. S3). Login to comment 114 ABCC7 p.Lys1250Arg The Eyring plot (Fig. 4 B) of the normalized closing rates obtained from single exponential fits (Fig. 4 A, smooth lines) to the decaying currents after ATP removal yielded a ∆H‡ for nonhydrolytic closure of 39 ± 4 kJ/mol, similar to that obtained for K1250R channels (Fig. 3 B). Login to comment 115 ABCC7 p.Lys1250Arg Interestingly, whereas open burst duration of WT channels was greater than twofold longer in the presence of PKA than shortly after its withdrawal, the average closing time constant of K1250R at 25°C, equivalent to its mean open burst duration, was only slightly longer (Fig. 3 A; Fig. S2) upon removal of PKA+ATP (7.3 ± 0.8 s, n = 12) than upon removal of just ATP (5.7 ± 0.4 s, n = 30). Login to comment 116 ABCC7 p.Lys1250Arg But, because the fall in Po that signals partial dephosphorylation of WT channels upon PKA removal occurs so rapidly (i.e., in 2-3 s; Csanády et al., 2000), the phosphorylation status of K1250R channels after removal of ATP+PKA, during their gradual closure, which takes tens of seconds (compare Fig. 3 A, current segments fitted by magenta lines), is uncertain. Login to comment 117 ABCC7 p.Lys1250Ala For K1250A channels, the closing time constant after simultaneous removal of ATP and PKA was rarely assessed (τ = 38 ± 4 s, n = 3), and is not easily compared with that after removal of just ATP (τ = 25 ± 2 s, n = 29), as the latter usually progressively shortened during an experiment (τ = 29 ± 3 s, n = 18, for the first decay from each Figure 1. Login to comment 130 ABCC7 p.Lys1250Arg Temperature dependence of gating of partially phosphorylated K1250R CFTR. Login to comment 131 ABCC7 p.Lys1250Arg (A) Macroscopic current trace (top) from ‫000,2ف‬ K1250R CFTR channels at -20 mV, with simultaneously recorded temperature (bottom). Login to comment 135 ABCC7 p.Lys1250Arg (B) Eyring plot of normalized closing rates ( ˆk ) of K1250R CFTR channels upon ATP removal, fitted by a straight line to obtain ∆H‡ value shown; closing rates, obtained as 1/τ from single-exponential fits, as in A, were normalized to their average values in bracketing control segments at 25°C. Figure 4. Login to comment 136 ABCC7 p.Lys1250Ala Temperature dependence of gating of partially phosphorylated K1250A CFTR. Login to comment 137 ABCC7 p.Lys1250Ala (A) Macroscopic current recording (top) from K1250A CFTR channels with simultaneously recorded temperature (bottom). Login to comment 140 ABCC7 p.Lys1250Ala (B) Eyring plot of normalized closing rates ( ˆk ) of K1250A CFTR channels upon ATP removal, fitted by a straight line to obtain ∆H‡; closing rates, obtained as 1/τ from single-exponential fits, as in A, were normalized to their average values in bracketing control segments at 25°C. Login to comment 145 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg From an Eyring plot of normalized unlocking rates (Fig. 5 B), the rough estimate of ∆H‡ for unlocking from AMPPNP of partially phosphorylated WT CFTR was 37 ± 6 kJ/ mol, similar to the value obtained above for closure of partially phosphorylated K1250R and K1250A channels opened by just ATP (Fig. 3 B and Fig. 4 B). Login to comment 166 ABCC7 p.Lys1250Arg We chose NBD2 catalytic site mutant K1250R as one model for nonhydrolytic closure because the mutation conserves charge in the anticipated catalytic interface, and, in P-glycoprotein, the corresponding K-to-R mutation essentially abolishes ATP hydrolysis (Lerner-Marmarosh et al., 1999). Login to comment 167 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg We selected the mutant K1250A as a second model for nonhydrolytic closure because it displays much slower closure than K1250R (e.g., Vergani et al., 2003, 2005) and because the K1250A mutation abolishes ATP hydrolysis by CFTR (Ramjeesingh et al., 1999). Login to comment 179 ABCC7 p.Lys1250Arg Second, because ∆H‡ is high (117 kJ/mol) for opening, but small for nonhydrolytic closure (e.g., 43 kJ/mol using K1250R as a model), α Δ O-CH must be large (+74 kJ/mol; Fig. 6 A, red line). Login to comment 180 ABCC7 p.Lys1250Arg Third, in contrast, α Δ O-CG is estimated to be rather small (e.g., -1 kJ/mol using K1250R as a model; Fig. 6 A, blue line), which in turn suggests that α Δ O-CS is large ( α Δ O-CT S ≈ 75 kJ/mol; Fig. 6 A, green line). Login to comment 182 ABCC7 p.Lys1250Arg Also, independent evidence that α Δ O-CG ≈ 0 is provided by the observation that steady-state Po ≈ 0.5 for the nonhydrolytic K1250R mutant, which is presumed to gate at thermodynamic equilibrium (Vergani et al., 2005). Login to comment 187 ABCC7 p.Lys1250Arg The paucity of ATPase measurements for CFTR mutants means that we cannot be certain that the charge-sparing mutation K1250R abolishes ATP hydrolysis in CFTR, even though the equivalent mutation in P-glycoprotein abolishes ATP hydrolysis (Lerner-Marmarosh et al., 1999). Login to comment 188 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg The charge-neutralizing mutation K1250A, on the other hand,doesabrogateATPhydrolysisinCFTR(Ramjeesingh et al., 1999) and yields an open burst state more stable than that of K1250R (Vergani et al., 2003, 2005). Login to comment 189 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg But using the closing rate of K1250A (instead of K1250R) channels as a model for nonhydrolytic closure, and hence for reversal of channel opening, yields barrier values for this step (∆H‡ = 39 kJ/mol and Δ maxG‡ = 81 kJ/mol) that are onlyslightlydifferentfromthoseestimatedusingK1250R. Login to comment 191 ABCC7 p.Glu1371Gln Even for the Walker B Glu mutant E1371Q CFTR channels, which display the most stable open-burst state observed for CFTR (closing time constant ‫004ف‬ s; Vergani et al., 2005), corresponding to Δ maxG‡ = 88 kJ/mol, if ∆H‡ is assumed ‫04ف‬ kJ/mol (its experimental determination would be a formidable task), the model predicts α Δ O-CG = -12 kJ/ mol, and again a similar overall energy profile. Login to comment 202 ABCC7 p.Lys1250Arg Forward (left to right) ∆H‡ values were obtained from slopes of Eyring plots for WT opening and closing rates (Fig. 1 C), ∆H‡ for the reversal of opening reflects the slope of the Eyring plot for K1250R closing rate (Fig. 3B). Login to comment 203 ABCC7 p.Lys1250Arg Corresponding Δ maxG‡ values were computed as RT ln(kBT/(kh)), by substituting the rates of WT opening (0.3 s-1) and closure (3.9 s-1), and of K1250R closure (0.2 s-1), for k. Login to comment