Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl − channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)- o -phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl − conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)- o -phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl − conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO 2 ) 4 2− in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl − channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl − transport through a combination of failure to increase Cl − conductance and increased susceptibility to channel block by cytosolic substances.

Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel pore. While wild-type CFTR was associated with a linear current–voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl − concentrations, suggesting that these mutants had a reduced ability to concentrate Cl − ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl − concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl − ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl − ion permeation through the pore.

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO 2 ) 4 2− acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent K d 556 μM), kinetically fast, and weakened by extracellular Cl − ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO 2 ) 4 2− ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO 2 ) 4 2− increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO 2 ) 4 2− ions to the extracellular solution. This “punchthrough” was prevented by extracellular Cl − ions, reminiscent of a “lock-in” effect. Relief of block in F337A by Pt(NO 2 ) 4 2− permeation was only observed for blocker concentrations above 300 μM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl − current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO 2 ) 4 2− in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO 2 ) 4 2− to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO 2 ) 4 2− ions within the pore promotes their permeation to the extracellular solution.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl − ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions ( P X / P Cl = 0.048–0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that “lock” CFTR channels in the open state (pyrophosphate, 5′-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P X /P Cl ) sequence NO 3 − > Cl − > HCO 3 − > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P X /P Cl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl − pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl − activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P Na /P Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P I (1.8) > P Br (1.3) > P Cl (1.0) > P F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F − flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I − /Cl − permeability ratio (P I /P Cl < 0.4). The switch to low I − permeability was enhanced at potentials that favored Cl − entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl − and I − ions may influence I − permeation and be responsible for the wide range of P I /P Cl ratios that have been reported for the CFTR channel. The low P I /P Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.

The mechanism of Cl − ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl − channels by binding to a site ∼40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl − concentration. Increasing extracellular Cl − concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl − and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl − is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl − concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl − and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl − permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl − (but not gluconate) ions and the dependence of channel conductance on Cl − concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN − ) ions and anomalous mole fraction behavior seen in Cl − /SCN − mixtures.