ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore

Abstract

Despite substantial efforts, the entire cystic fibrosis transmembrane conductance regulator (CFTR) protein proved to be difficult for structural analysis at high resolution, and little is still known about the actual dimensions of the anion-transporting pathway of CFTR channel. In the present study, we therefore gauged geometrical features of the CFTR Cl(-) channel pore by a nonelectrolyte exclusion technique. Polyethylene glycols with a hydrodynamic radius (R (h)) smaller than 0.95 nm (PEG 300-1,000) added from the intracellular side greatly suppressed the inward unitary anionic conductance, whereas only molecules with R (h) ≤ 0.62 nm (PEG 200-400) applied extracellularly were able to affect the outward unitary anionic currents. Larger molecules with R (h) = 1.16-1.84 nm (PEG 1,540-3,400) added from either side were completely excluded from the pore and had no significant effect on the single-channel conductance. The cut-off radius of the inner entrance of CFTR channel pore was assessed to be 1.19 ± 0.02 nm. The outer entrance was narrower with its cut-off radius of 0.70 ± 0.16 nm and was dilated to 0.93 ± 0.23 nm when a non-hydrolyzable ATP analog, 5'-adenylylimidodiphosphate (AMP-PNP), was added to the intracellular solution. Thus, it is concluded that the structure of CFTR channel pore is highly asymmetric with a narrower extracellular entrance and that a dilating conformational change of the extracellular entrance is associated with the channel transition to a non-hydrolytic, locked-open state.

Fig. 1

Single CFTR Cl⁻ channel currents in the open and locked-open states recorded from HEK293T cells transiently transfected with CFTR gene. Representative current traces (a) and corresponding unitary current–voltage relationships (b) recorded from a cell-attached patch (left traces), inside-out patch (center traces) in the presence of 2 mM ATP and inside-out patch in the presence of 2 mM ATP + 5 mM AMP-PNP (right traces) in the bath (intracellular) solution with standard pipette solution at different membrane voltages as indicated on the left of each trace. Arrowheads on the right of each trace indicate zero current level. Each data point represents the mean ± SEM of 8–32 measurements from 5–7 different patches. Solid lines are linear fits with slopes corresponding to the unitary outward and inward conductances

Fig. 2

Effects of polyethylene glycols on the single CFTR Cl⁻ channel currents. Representative current traces recorded from excised inside-out patches at +60 and at −60 mV in control conditions in the absence of nonelectrolytes (a) and in the presence of PEG 300 (b) and PEG 2,000 (c) in the bath solution. C denotes the closed state, O corresponds to the open channel level, respectively. The respective all-point histograms are shown at the right of each record; the histograms (shadowed) were fitted with Gaussians (solid lines) to give the peak-to-peak single-channel amplitudes (i) as indicated. Single-channel current–voltage relationships (d) were obtained in control conditions (filled circles) and in the presence of PEG 300 (open circles) and PEG 2,000 (open triangles) in the bath solution. Each data point represents the mean ± SEM of 5–29 measurements from 5–9 patches. Solid lines are linear fits with slopes corresponding to the unitary outward and inward conductances given in the text. The slope for PEG 300 is significantly different from the control slope at P < 0.05

Fig. 3

Effects of polymers on the single-channel conductance of the CFTR Cl⁻ channel in the absence of AMP-PNP. a Relative changes in the unitary single-channel conductance (left axis) and bulk conductivity (right axis) as a function of the hydrodynamic radius (R _h) of PEG molecules (R _h = 0.45, 0.53, 0.62, 0.75, 0.95, 1.16, 1.39 and 1.84 nm for PEG 200, 300, 400, 600, 1,000, 1,540, 2,000 and 3,400, respectively). Filled circles represent the inward slope conductance obtained in the inside-out mode with polymers added to the bath solution (configuration 3). Open circles and open triangles represent the outward slope conductance obtained with polymers added to the pipette solution in the inside-out mode (configuration 1) and the cell-attached mode (configuration 2), respectively. Data were collected from 5–9 different patches for each polymer. Open squares represent the relative decrease in the bulk conductivity of the solutions used in these experiments (n = 5). *Significantly different from the slope conductance obtained in control conditions without polymers at P < 0.05. ^#Significantly different from the respective slope conductance obtained with polymers added to the pipette solution in the inside-out mode at P < 0.05. Inset Schematic illustrations of configurations 1–3 used in these experiments. b Filling coefficients calculated according to Eq. 1 as a function of the hydrodynamic radius of PEG molecules. Symbols are the same as in (a). Solid lines are linear fits to the descending parts of the curves with correlation coefficient of −0.90 and −0.99 for extracellular and intracellular PEG application experiments, respectively. A horizontal dashed line corresponds to zero filling. R1 and R2 denote the radius of the extracellular and intracellular entrance of the CFTR Cl⁻ channel pore, respectively

Fig. 4

Effects of polymers on the single-channel conductance of the CFTR Cl⁻ channel in the presence of AMP-PNP. a Relative changes in the unitary single-channel conductance as a function of the hydrodynamic radius of PEG molecules in the presence of AMP-PNP in the bath (intracellular) solution in the inside-out mode. Open diamonds represent the outward slope conductance obtained with polymers added to the pipette solution (configuration 4). Filled diamonds represent the inward slope conductance obtained with polymers added to the bath solution (configuration 5). Data were collected from 5–9 different patches for each polymer. *Significantly different from the slope conductance obtained in control conditions without polymers at P < 0.05. The slope conductances obtained in the presence of AMP-PNP for PEG 300, 400, 600 and 1,000 (filled diamonds) are significantly different from those in the absence of AMP-PNP (open triangles in Fig. 3a). Upper inset Schematic illustrations of configurations 4 and 5 used in this experiment. Lower inset Representative current traces recorded at +100 mV in the absence (upper trace) and presence (lower trace) of AMP-PNP in the bath (intracellular) solution. b Filling coefficients calculated according to Eq. 1 as a function of the hydrodynamic radius of PEG molecules. Symbols are the same as in (a). Solid lines are linear fits to the descending parts of the curves with correlation coefficient of −0.92 and −0.99 for extracellular and intracellular PEG application experiments, respectively. A horizontal dashed line corresponds to zero filling. R3 and R4 denote the radius of the extracellular and intracellular entrance, respectively, of the CFTR Cl⁻ channel in the presence of AMP-PNP in the bath solution

Fig. 5

AMP-PMP-dependent structural transitions from the open state (O1) to the locked-open (O*) state of CFTR Cl⁻ channel. Left A hypothetical drawing depicts the solute-accessible interior of a highly asymmetric pore with narrower extracellular (the radius of R1 = 0.70 nm) and wider intracellular (R2 = 1.19 nm) entrances in the presence of ATP and in the absence of AMP-PNP. The size of the narrowest constriction inside the pore (smallest filled circle, R5 = 0.27 nm) is based on the permeability data reported by Linsdell et al. [66]. Right A hypothetical drawing illustrates AMP-PNP-induced dilatation of the extracellular entrance (R3 = 0.93 nm) but not of the intracellular entrance (R4 = 1.15 nm) of the CFTR Cl⁻ channel pore. Smaller hatched circle in the middle corresponds to an AMP-PNP-dependent widening of the narrowest constriction which may represent the selectivity filter. For the radius of the narrowest constriction in the presence of AMP-PNP, the value of R6 = 0.69 nm reported by Linsdell and Hanrahan [43] is adopted, and its localization in the proximity to the extracellular entrance of the channel is based on the present PEG partition study. NBD1 and NBD2 denote two nucleotide-binding domains of CFTR channel protein. The transmembrane and regulatory domains are not depicted for the simplicity