High affinity ATP/ADP analogues as new tools for studying CFTR gating

Abstract

Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating.

Figure 1. Chemical structures of ATP and N⁶-modified ATP analogues

The three ATP analogues tested were N⁶-(2-phenylethyl)-ATP (P-ATP), N⁶-benzyl-ATP (B-ATP) and N⁶-(2-methylbutyl)-ATP (M-ATP).

Figure 2. N⁶-modified ATP analogues are more potent than ATP

A, after patch excision, wild-type cystic fibrosis transmembrane conductance regulator (WT-CFTR) channels were activated by protein kinase A (PKA) and 1 mm ATP. ATP (2.75 mm) was then applied before and after the application of each ATP analogue (5 μm). B, mean current responses to 2.75 mm ATP and 5 μm ATP analogues of WT-CFTR channels were compared. The ratios of the mean current induced by ATP analogue (5 μm) to that by 2.75 mm ATP for WT-CFTR channels are 1.12 ± 0.09 (n = 10), 0.54 ± 0.05 (n = 10) and 0.25 ± 0.04 (n = 8) for P-ATP, B-ATP and M-ATP, respectively. This ratio is 0.07 ± 0.04 (n = 8) for 5 μm ATP (data from Zeltwanger et al. 1999). For ΔR-CFTR channels, this ratio is 1.09 ± 0.06 (n = 9) for 5 μm P-ATP.

Figure 3. Concentration dependence of ΔR-CFTR activity in response to P-ATP or ATP

A, ΔR-CFTR single-channel current traces in the presence of 2.75 mm ATP, 30 μm P-ATP and 0.1 μm P-ATP. B, open probability (P_o) dose–response relationships of P-ATP (•) and that of ATP (○; from Bompadre et al. 2005a). Continuous lines are Hill equation fits to the data. The n values for the fits are 1.05 ± 0.30 for P-ATP and 0.66 ± 0.23 for ATP.

Figure 4. Single-channel kinetic parameters of ΔR-CFTR in the presence of P-ATP

The dose–response relationship between P-ATP and the opening rate (A) or the closing rate (B). Continuous lines are Hill equation fits to the data. The n values for the fits are 0.75 ± 0.30 for the opening rates, and 1.30 ± 0.90 for the closing rates. The dashed line in A shows the maximal opening rate in the presence of saturating [ATP] (2.6 ± 0.2 s⁻¹, n = 20). The dashed line in B shows the mean closing rate at various [ATP] (3.3 ± 0.1 s⁻¹, n = 8) (data from Bompadre et al. 2005a). *P < 0.05 compared with the closing rate at 0.1 μm P-ATP.

Figure 5. P-ADP is more potent than ADP in inhibiting CFTR

A, WT-CFTR current traces in the presence of 0.5 mm ADP or 50 μm P-ADP, both with 0.5 mm ATP. B, concentration dependence of P-ADP inhibition of WT-CFTR currents in the presence of 0.5 mm ATP. The continuous line is the Michaelis-Menten fit to the data for 0.5 mm P-ADP plus 0.5 mm ATP. The r² value for the fit is 0.998. The dashed line is the Michaelis-Menten fit to the data for 0.5 mm ADP plus 0.5 mm ATP (data from Bompadre et al. 2005a). The r² value for the fit is 0.993. K_½ of ADP in the presence of 0.5 mm ATP is 180.1 ± 38.6 μm.

Figure 6. Effects of [ATP] and [P-ADP] on P-ADP-dependent inhibition

A, inhibition of WT-CFTR currents by 50 μm P-ADP in the presence of two different [ATP]: 0.5 and 2 mm. B, mean data from four experiments. When [ATP] is increased from 0.5 to 2 mm, inhibition of the current by 50 μm P-ADP decreases from 74.1 ± 2.6 to 43.4 ± 5.1% (n = 4). ***P < 0.005. C, single-channel traces of ΔR-CFTR in the presence of 0.5 mm ATP alone, 0.5 mm ATP plus 25 μm P-ADP, and 0.5 mm ATP plus 50 μm P-ADP.

Figure 7. Effect of P-ATP on CFTR locked open state

A, macroscopic E1371S-CFTR current relaxations upon removal of 50 μm P-ATP or 1 mm ATP. For this particular experiment, the relaxation time constants are 255.1 s (P-ATP) and 150.8 s (ATP), respectively. B, mean data of the current relaxation experiments of E1371S-CFTR. The current relaxation time constants (τ_relaxation) for E1371S-CFTR channels are 297.6 ± 34.0 s (n = 5) upon the removal of 50 μm P-ATP, and 118.8 ± 9.4 s (n = 5) upon the removal of 1 mm ATP. ****P < 0.001. C, macroscopic WT-CFTR current relaxations upon washout of 10 μm P-ATP plus 2 mm AMP-PNP, or 0.5 mm ATP plus 2 mm AMP-PNP. Note that > 95% of the current decays 5 s after an initial washout of PKA and ATP. For this particular experiment, the relaxation time constants are 178.6 s (P-ATP plus AMP-PNP) and 66.7 s (ATP plus AMP-PNP), respectively. D, mean data of the current relaxation experiments of WT-CFTR. τ_relaxation values are 188.1 ± 32.7 s (n = 12) upon removal of 10 μm P-ATP plus 2 mm AMP-PNP, and 73.5 ± 9.3 s (n = 9) upon removal of 0.5 mm ATP plus 2 mm AMP-PNP. **P < 0.01. Dashed lines in A and C are single exponential fits to the data. PKA is present in all nucleotide-containing solutions to minimize potential effects of dephosphorylation by membrane-associated protein phosphatases.