Localizing a gate in CFTR

Abstract

Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR's gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2](-), we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338-341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2](-) in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2](-) in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2](-) when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2](-) and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.

Keywords: ABC transporters; anion channels; cystic fibrosis; gating.

Fig. 1.

Current understanding of CFTR’s pore architecture and thiol-specificity of [Au(CN)₂]⁻. (A) A cartoon depicting the essence of the pore of CFTR based on previous cysteine scanning studies (, , –21). TM1 and TM6 are shown, as these two segments likely span the entire pore axis. Labeled positions mark the residues that actually contribute to pore-lining. (B) Reversible block of cysless WT-CFTR by 1 mM [Au(CN)₂]⁻. (C) Reaction between I344C–CFTR and 1 mM [Au(CN)₂]⁻ in the presence or absence of ATP (see Results for details). Note a two-step current decay when [Au(CN)₂]⁻ was applied with ATP. (D) State-independent reactivity of I344C–CFTR to [Au(CN)₂]⁻. Data extracted from C. Red squares represent normalized current amplitudes obtained from the first part of the trace shown in C, where [Au(CN)₂]⁻ was applied without ATP, and the gray trace is the second phase (ligand exchange phase) of the current decay, when [Au(CN)₂]⁻ was applied in the presence of ATP. Fitting these data with a single exponential function resulted in similar secondary reaction rates.

Fig. 2.

State-dependent reactivity of T338C–CFTR to internal [Au(CN)₂]⁻. (A) In the presence of ATP, internal [Au(CN)₂]⁻ irreversibly decreases T338C–CFTR currents. The time constant τ was obtained by fitting the current decay with a single exponential function. (B) Contrary to that seen in A, T338C–CFTR currents were not significantly altered when the same concentration [Au(CN)₂]⁻ was applied in the absence of ATP for a total of 36 s.

Fig. 3.

Reaction of cysteines placed at positions 334 and 335 with external [Au(CN)₂]⁻. (A) External application of 20 µM [Au(CN)₂]⁻ diminished over 80% of forskolin (Fsk)-activated R334C–CFTR currents. After DTT reversed the effect brought by [Au(CN)₂]⁻, the second application of the reagent abolished the current in a similar manner. The residual current after [Au(CN)₂]⁻ is sensitive to a specific CFTR inhibitor (Inh-172). (B) [Au(CN)₂]⁻ reacted with R334C/G1349D at a faster rate than that with R334C. Note a small current increase immediately after the removal of DTT. As reported by Liu et al. (36), this may be due to reactions of the thiol in a small fraction of the channels with trace metals, or nonspecific oxidation. (C) Reaction of K335C–CFTR channels with [Au(CN)₂]⁻. For this construct, 1 mM [Au(CN)₂]⁻ was applied because of a much slower reaction rate. The addition of 1 mM [Au(CN)₂]⁻ led to a biphasic decay of forskolin-activated K335C–CFTR currents: a faster phase of blockade and a slower phase of ligand exchange reaction. (D) Reaction of K335C/G1349D–CFTR with [Au(CN)₂]⁻. Fifty micromolar [Au(CN)₂]⁻ was used, as the reaction rate for this double mutant is fast (see Results for details). Current decays in A and D were fitted with a single exponential function, whereas those in B and C were fitted with a double exponential function (red solid lines). Reaction time constants were indicated in each panel. (E) Comparisons of the mean current amplitude between R334C–CFTR and R334C/G1349D–CFTR and between K335C–CFTR and K335C/G1349D–CFTR in excised inside-out patches. Note that a ∼10-fold difference in the mean current amplitude—hence a ∼10-fold change in P_o—was seen with the introduction of the G1349D mutation, as shown previously for WT-CFTR (see Results for details). Number of patches is indicated above each bar.