Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme

Abstract

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent.

Figure 8

CFTR gating schemes. Scheme 1 is a linear model based on traditional ligand-gated channel gating schemes. Scheme 2 is a cyclic model for CFTR channel gating, but does not take into account the stabilization of the open state via ATP action at NBD2. Scheme 3 is a cyclic model for CFTR gating with two coordinating ATP binding sites. The cartoon depicts a hypothetical graphic presentation of the kinetic Scheme 3 (see text for details).

Figure 4

ATP concentration dependence of the mean open time and the mean closed time. Both open (τo) and closed (τc) time constants were obtained from at least five patches in the presence of different concentrations of ATP. (A) ATP concentration dependent of the closed time constant. The smooth curve represents fit of the data points to the approximate relationship from Eq. 1 (see text for details). The squares represent exact solutions using Q matrix techniques to Fig. 8, Scheme 3, with the parameters listed in Table I. (B) ATP concentration dependence of the open time constant.

Figure 7

Slow closing of AMP-PNP locked open wt-CFTR. A continuous current trace showing the effects of AMP-PNP on ATP-opened wt-CFTR. Ensemble currents were constructed from the slow component of the decay from multiple washouts in the same patch. The ensemble current decay could be fitted with a single exponential function yielding a time constant for deactivation of 32.33 ± 0.43 and 29.36 ± 0.07 s for the washout of 0.5 mM and 2.75 mM AMP-PNP, respectively. Multiple ensemble deactivations from different patches yielded an average time constant for the slow deactivation phase of 34.9 ± 13.7 s (n = 6) for 2.75 mM AMP-PNP and 33.2 ± 14.4 s (n = 6) for 0.5 mM AMP-PNP.

Figure 2

The ATP concentration dependence of CFTR channel activity. (A) A continuous current trace showing the effects of different ATP concentrations on CFTR channel activity. (B) The dose–response relationship between [ATP] and macroscopic CFTR channel current. For each patch (containing a different number of CFTR), current was normalized to the average of the current achieved with 2.75 mM ATP immediately before and after the tested concentration. (C) The dose–response relationship between [ATP] and the single-channel P _o. The smooth curve represents the fit of the data points to Eq. 2 (see discussion for details). (D) Overlaid plots of B and C. All data points are mean ± SEM of four or more values obtained from different patches at each concentration of ATP.

Figure 1

ATP is required to open PKA-phosphorylated CFTR in excised in side-out patches. (A) A continuous current trace showing activation of CFTR channels with PKA and ATP, and lack of channel openings in the absence of ATP. (B) A semicontinuous recording of a single CFTR channel in the presence of PKA plus ATP or ATP alone. Each stretch of single-channel recording is 45 s.

Figure 3

Effects of [ATP] on CFTR gating. (A) Single-channel traces of PKA-phosphorylated CFTR at different concentrations of ATP as marked. (B) Cumulative dwell time analysis of the traces shown in A.

Figure 5

Probability density function of the closed time distributions at 0.1 (A) or 0.5 (B) mM ATP. The smooth curves represent the predicted pdf using kinetic parameters derived for Fig. 8, Scheme 3 (see text for details). Insets show the expanded graphs that contain the first 20 bins.

Figure 6

ATP concentration dependence of the channel open time for K1250A-CFTR. (A) A continuous current trace of K1250A-CFTR in the presence or absence of ATP. The channel had been activated with PKA and ATP before the start of the trace. This mutant channel remains open for 2 min after removal of ATP. However, the same channel shows brief openings at 10 μM ATP. (B) Single channel amplitudes of K1250A-CFTR (obtained from all point histograms of 30 s recordings) at 10 μM or 2.75 mM ATP. (C) The open time histogram of K1250A-CFTR at 10 μM ATP. Pooled open times from four patches were analyzed. This distribution was fitted with a single exponential function. (D) Slow closing of PKA-phosphorylated K1250A-CFTR. The expanded trace shows stepwise closing of individual channels after ATP is removed. (E) Relaxation of the ensemble current. Macroscopic relaxation of the K1250A-CFTR channel current was constructed from multiple washouts of ATP from the same patch. A single exponential fit of the current relaxation yields a time constant of 181.28 ± 0.25 s for deactivation in the experiment shown.