CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR)

Abstract

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

Figure 1.

Effects of PKA activators and inhibitors on ΔR-CFTR. (A) A cell-attached recording showing that addition of 10 μM forskolin (Fsk) + 100 μM CPT-cAMP failed to increase the basal current of ΔR-CFTR. However, addition of 20 μM genistein (Gen) could potentiate the current (n = 4). (B and C) Effects of PKI, a peptide inhibitor of PKA, on whole-cell currents from either WT- or ΔR-CFTR. For WT-CFTR, in the presence of forskolin, a very brief outward current was obtained immediately after the whole-cell configuration was formed (arrow). Then, the currents decayed rapidly. Subsequent application of genistein did not potentiate the currents. For ΔR-CFTR, basal currents were seen without any cAMP stimulant after the whole-cell configurations are formed (arrow). Subsequent application of forskolin did not alter the currents. But, genistein could potentiate the channel activity. Similar results were obtained from seven cells. Ramp I–V curves (right) were taken as marked in the raw current traces.

Figure 2.

Effect of PKC on ΔR-CFTR. (A) A representative whole-cell ΔR-CFTR current trace showing lack of effects of BIM, a PKC inhibitor, on the basal current. The inset shows the I–V relationships at different conditions as marked. Similar results were obtained from seven cells. (B) A continuous ΔR-CFTR current trace in a cell-attached mode. PMA, a PKC activator, did not alter the ΔR-CFTR currents. Summary of the mean currents in the presence or absence of PMA. Values were normalized by mean currents with genistein (n = 6).

Figure 3.

PKA did not alter the ATP-induced ΔR-CFTR chloride channel currents. (A) Single-channel currents were induced by ATP application 2 min after patch excision. Subsequent addition of PKA did not alter the channel activity. (B) Expanded traces of the recording in A. (C) Summary of the measured Po with 2.75 mM ATP alone and with both 2.75 mM ATP and PKA (25 U/ml) (n = 6).

Figure 4.

Effect of different concentrations of ATP on single-channel kinetics. (A) Representative single-channel ΔR-CFTR traces with different concentrations of ATP in excised inside-out patches. Each trace represents a 60-s recording. ATP concentration dependence of the mean open time (B) and the mean closed time (C). All values are represented by mean ± SEM. Overlaid open circles (○) represent the corresponding values measured for WT-CFTR channels (from Zeltwanger et al., 1999).

Figure 5.

Mode shifts of ΔR-CFTR. (A) A single-channel recording of the ΔR-CFTR channel in an excised inside-out patch. The slow gating mode, i.e., long openings and closings of the channel, is often observed immediately after excision of the membrane patch. The slow gating mode usually switches to the fast gating mode spontaneously within a few minutes after patch excision. (B) A sample trace showing a spontaneous mode switch from the fast gating mode to the high Po mode. The mode switch occurred 4 min after the excision of membrane, and lasts for >6 min. (C) Summary of Po of ΔR-CFTR in different modes. *, P < 0.01.

Figure 6.

Inhibition of ATP-induced ΔR-CFTR currents by ADP. (A) Inhibitory effects of ADP on the ΔR-CFTR currents induced with different ATP concentrations. (B) The dose–response relationship between [ADP] and the magnitude of inhibition in the presence of four different ATP concentrations: (□) 75 μM ATP, (▪) 200 μM ATP, (○) 500 μM ATP, and (•) 1 mM ATP. All data points are presented as mean ± SEM of at least four values obtained from different patches. Data are fitted with the Michaelis-Menten equation.

Figure 7.

Effect of ADP on ΔR-CFTR single-channel current. (A) A representative single-channel current trace in an excised inside-out patch in the presence or absence of ADP. (B) Expanded traces with ATP alone (top) and with both ATP and ADP (bottom). (C) Summary of single-channel kinetic parameters (n = 11). *, P < 0.05.

Figure 8.

Single-channel dwell time histograms. Events from several single-channel recordings were pulled together to obtain these dwell time histograms to determine the open and closed times distributions in the presence of 1 mM ATP, 1 mM ATP + 1 mM ADP, and 1 mM ATP + 2 mM ADP. (A) The closed time distributions can be fitted with a double exponential function in the presence of ADP, indicating the presence of a new, longer closed time constant. The data could be fitted with a single exponential (τ = ∼2 s), but in the case of 2 mM ADP, the fitted curve fails to capture nearly all the closed events >10 s (not depicted). (B) The open time dwell time histograms show that the mean open time decreases as the concentration of ADP increases.

Figure 9.

Effect of ADP on the mean open time in the slow gating mode. (A) A representative single-channel ΔR-CFTR current trace in slow gating mode. (B) Expanded traces with ATP alone and with both ATP and ADP from the recording shown in A. (C) Reversible shortening of the mean open time by ADP from six patches where ΔR-CFTR channels are in slow gating mode.

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