Identification of a region of strong discrimination in the pore of CFTR

Nael A. McCarty and Zhi-Ren Zhang

Departments of Physiology and Pediatrics, Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl- channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl- and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl- and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl- and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

cystic fibrosis transmembrane conductance regulator; chloride channel; selectivity; anion permeation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE 11 years since the cloning of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, progress has been made in defining portions of the protein comprising the pore-lining domains and residues within those domains that play important roles in establishing the biophysical character of open CFTR Cl- channels (38). Several approaches have been applied to this study, including identification of transmembrane (TM) domains and residues therein that contribute to features such as open channel block by organic drugs, single-channel conductance in Cl-, accessibility to chemical modification, and selectivity between monovalent anions. Although some portions of the CFTR peptide have been studied extensively, we do not yet have a clear picture of how these TM domains and amino acids work together to determine the electrostatic profile and physical dimensions of the permeation pathway.

It is presently not known how many TM domains contribute to the conduction pathway of CFTR. Indeed, the oligomeric structure of the functional channel is unclear. Despite several recent studies (10, 12, 28, 35, 43, 48), it is unclear whether a single CFTR channel is formed by a single CFTR peptide (see Ref. 29). The possibility exists that the functional CFTR channel is constructed from a dimer of CFTR peptides. A dual-pore model has also been suggested (46) wherein the amino-terminal and carboxy-terminal halves of the protein each confer Cl--conducting pores with different characteristics. For the purposes of the present study, we assumed that functional channels are made from a single CFTR peptide and confer only a single pore (one-channel, one-pore hypothesis), although we also interpret our data in light of the dual-pore configuration. Based on previous work (29) in TM6, TM11, and TM12 and preliminary data in TM5, we predicted that these four TMs contribute to the pore. This notion suggests a similar architecture to that of the substrate-binding domain of the related P-glycoprotein (25). TM6 is clearly the best studied of the TM domains in CFTR, having received so much attention because it is the helix that includes a greater number of charged amino acids than any other helix. However, because it is unlikely that the conduction pathway of this channel protein is constructed from only one or two TM helices, other domains must also contribute amino acids to the pore. It is not yet possible to identify homologous positions in any two helices that may lie across the pore from each other.

One of the parameters that describes a channel to a particularly high degree of detail is its ability to select between ions of similar charge. This functional distinction arises from a structural arrangement that is finely tuned to provide this specification (11). In contrast to other investigators (19), we do not believe that CFTR exhibits a well-defined selectivity filter similar to that found in voltage-gated ion channels but rather that selectivity arises from the generalized character of the amino acids lining the pore (39) in combination with a region that discriminates between anions of various sizes as described herein. It is generally accepted that CFTR exhibits only weak selectivity (9), especially compared with the strong selectivity between monovalent ions exhibited by the well-studied voltage-gated ion channels. However, the CFTR channel is capable of some degree of discrimination between halides and between Cl- and larger polyatomic anions (e.g., Ref. 23).

From what portions of the protein does anion selectivity arise? The determinants of anion selectivity in CFTR have been studied by several investigators (29), again emphasizing primarily amino acids in TM6. Because most studies (e.g., Refs. 3, 6, 19, 28) have investigated either the effects of only a single substitution at a few positions or the effects of several substitutions at only one position, it has been difficult to assemble the data into a structural framework. To gauge the importance of mutations studied previously and new mutations presented as included here, we have begun to compare the results of a common substitution at several positions in TM6. Furthermore, because the anions that permeate CFTR are three-dimensional entities, the structures that confer selectivity (and anion binding sites) should be considered in three dimensions as well. Hence, it is important to compare the effects of mutations at positions in one TM domain with mutations at similar or homologous positions in other TM domains that may line the pore. Only this broad-scale approach will allow comparisons of the magnitude of effects at various positions and in more than one TM domain, making it possible to look for patterns in the data.

In the present work, we describe the initial steps in this study. We used macroscopic recording of CFTR currents in Xenopus oocytes to study selectivity between Cl- and a wide range of test anions in wild-type (WT) CFTR and 12 CFTR variants. Our first objective was to add to the two-dimensional view of the pore by comparing the effects of equivalent substitutions at multiple positions along the length of TM6. To this end, we assayed the effects of mutations at K335, T338, and T339 (22-24, 26) and provide new information for mutations at S341. The second objective was to build toward a three-dimensional view by studying the results from mutations at comparable positions in TM6 and TM12. Our results suggest that the determinants of selectivity are distributed along the length of the pore, arguing against the presence of a classic selectivity filter in this channel. However, we also show that the region of strong discrimination between monovalent anions near the middle of TM6 extends to S341. Selectivity between monovalent and divalent anions was affected most strongly by mutations at the cytoplasmic end of this region. These data are consistent with a narrowing of the pore from the extracellular vestibule toward the middle of TM6. Mutations at one position in TM12 match the pattern reflected by similar mutations in TM6, suggesting strongly that this TM domain also contributes to the pore of CFTR. Portions of these data have been presented in abstract form (30-32).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Preparation of oocytes and cRNA. WT CFTR was subcloned into the pAlter vector (Promega, Madison, WI). Mutants K335E, K335F, T338A, T339A, S341A, S341T, T1134A, and T1134F were prepared as previously described (33). Mutants K335A, T338E, and T1134E were prepared with the QuickChange protocol (Stratagene, La Jolla, CA). S341E CFTR was a generous gift from D. Dawson (Oregon Health Sciences University, Portland, OR). All mutant constructs were verified by sequencing across the entire open reading frame before use. Stage V-VI oocytes were isolated from female Xenopus and were incubated at 18°C in a modified Liebovitz L-15 medium with the addition of HEPES (pH 7.5), gentamicin, and penicillin-streptomycin. Capped transcripts (mMessage mMachine, Ambion, Austin, TX) for WT CFTR and for each of the variants (2-35 ng for most and 340 ng for T1134E CFTR) along with 0.6 ng of beta 2-adrenergic receptor cRNA were injected into each oocyte. Recordings were made at room temperature 42-96 h after injection.

Electrophysiology. Standard two-electrode voltage-clamp techniques were used to study whole cell currents. Electrodes were pulled in four stages from borosilicate glass (Sutter Instrument, Novato, CA) and filled with 3 M KCl. Pipette resistances measured 0.5-1.4 MOmega in standard ND96 bath solution that contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.5). Currents were acquired with a GeneClamp 500 amplifier and pCLAMP software (Axon Instruments, Foster City, CA); the corner frequency was 500 Hz. For selectivity experiments, NaCl was replaced with the Na+ salt of each of the anions studied. This resulted in the retention of a residual 4 mM Cl- from the K+ and Mg2+ salts. Data were corrected for junction potentials at the ground bridge (3 M KCl in 3% agar), which ranged from 0.2 to 2.4 mV as determined with a free-flowing KCl electrode. CFTR channels in oocytes were activated by exposure to isoproterenol (Iso) and alternately assayed in the presence of a Cl--containing bath or a substitute anion. Substitutions were always made in the same order and for a 1-min duration. Monovalent test anions, in order of use, included acetate, bromide (Br-), gluconate, glutamate, iodide (I-), nitrate (NO<UP><SUB>3</SUB><SUP>−</SUP></UP>), isethionate, perchlorate (ClO<UP><SUB>4</SUB><SUP>−</SUP></UP>), and thiocyanate (SCN-). Fluoride was excluded from this list due to the low solubility of MgF2. We also assayed selectivity between Cl- and one divalent anion, thiosulfate (S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>). Exposure to these test anions was kept brief to avoid alteration of cytoplasmic Cl- concentration (49). Data for each substitute anion were bracketed with the data for Cl- plus Iso before and after the substitute anion (e.g., Cl- plus Iso; acetate; Cl- plus Iso; Br-; Cl- plus Iso). Relative permeability [anion x-to-Cl- permeability (Px/PCl)] and relative conductance [anion x-to-Cl- conductance (Gx/GCl)] for each substitute anion (anion x) were calculated with the average data for the preceding and subsequent exposures to Cl-. This procedure allowed us to compare the Gx/GCl values for several anions by controlling for the changes in activation during the experiment.

CFTR currents were generated with three different protocols, each of which included a holding potential of -30 mV. For most of the data described here, the membrane potential was ramped between +60 and -80 mV over the course of 200 ms as shown in Fig. 1. We used both a depolarizing ramp (hold at -80 mV for 50 ms, then ramp to +60 mV) and a hyperpolarizing ramp (hold at +60 mV for 50 ms, then ramp to -80 mV) to test for protocol-dependent effects. Each ramp protocol was run in triplicate, and the data were averaged. For some experiments, we relied on a step protocol where the membrane potential was stepped for 75 ms to a range of potentials between -140 and +80 mV. Unless otherwise noted, all data presented in Tables 1-6 and Figs. 1-8 are from a hyperpolarizing ramp.


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Fig. 1.   Selectivity of the wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR) channel in oocytes. Macroscopic current-voltage (I-V) relationships were generated from currents obtained with hyperpolarizing voltage ramps between +60 and -80 mV. Raw currents are shown before correction for junction potentials. All data are from a single representative oocyte; currents in substitute anions were normalized to the initial current in Cl- to account for changes in activation level during the experiment (see MATERIALS AND METHODS). A: currents in the presence of Cl- and small monovalent anions. B: the same data for Cl- and currents in the presence of large monovalent anions and the divalent anion thiosulfate (S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>). Glut, glutamate; Acet, acetate; Gluc, gluconate; Iseth, isethionate; Vm, membrane potential. Substitution solutions contained 96 mM substitute anion plus 4 mM Cl-.


                              
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Table 1.   Ionic radii and hydration energies for the monovalent anions studied


                              
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Table 2.   Relative permeabilities for WT and mutant CFTRs for monovalent anions


                              
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Table 3.   Selectivity sequences for WT and mutant CFTRs


                              
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Table 4.   Relative conductances for WT and mutant CFTRs for monovalent anions


                              
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Table 5.   V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP> in ND96 bath solution for WT and mutant CFTRs


                              
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Table 6.   Selectivity between Cl- and the divalent anion S2O<UP><SUB>3</SUB><SUP>2<UP>−</UP></SUP></UP>



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Fig. 2.   Lyotropic permselectivity between monovalent anions in WT CFTR. A: relative permeabilities for most anions were determined by the change in Gibbs free energy on hydration [hydration energy (Delta hydrGo)]. Relative permeability (Px/PCl) values for I- and ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> were lower than expected from this relationship. B: relative conductance (Gx/GCl) values did not exhibit a clear dependence on hydration energy.



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Fig. 3.   Gx/GCl data identify anions that bind. The relationship between Gx/GCl and anion radius is plotted for all monovalent anions studied and for the divalent anion S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>. The substitute anions fall into three categories: ions that are highly conductive (on the dashed line), ions that are too large to traverse the pore (on the solid line), and ions that bind in the pore to inhibit conductance of the residual Cl- (below the lines). The baseline conductance due to the 4 mM residual Cl- in all solutions lies at the level of the solid line. The intersection of the solid and dashed lines suggests a pore diameter of ~5 Å.



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Fig. 4.   Dependence of macroscopic current on extracellular Cl- concentration ([Cl-]). Oocytes expressing WT CFTR were studied in ND96 control solution (100 mM Cl-) and after partial or complete substitution of Cl- with either gluconate or S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>. Fractional current was calculated after a step to +80 mV. Values are means ± SD; n = 5 oocytes. The data in gluconate, which does not block the pore from the extracellular side, suggest that the half-maximal inhibitory concentration for Cl- under these conditions is ~5 mM. The decreased conductance in S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>-containing solutions indicates that this anion blocks the current carried by the residual Cl-.



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Fig. 5.   Pore-domain mutations affect the shape of macroscopic I-V curves. A: data from WT CFTR, indicating mild outward rectification under these conditions. B-D: I-V relationships from oocytes expressing mutants at K335, T338 or T339, or S341, respectively, in TM6. E: I-V relationships from oocytes expressing mutants at T1134 in TM12. In each case, data were generated with a hyperpolarizing ramp from 1 representative oocyte in ND96 bath solution.



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Fig. 6.   Normalized Px/PCl (A) and Gx/GCl (B) for all anions in the alanine-substituted mutants. Px/PCl and Gx/GCl values for each anion x in each mutant were calculated for T1134A, K335A, T338A, T339A, and S341A CFTRs and normalized to Px/PCl and Gx/GCl values for WT CFTR. Solid lines at unity, Px/PCl and Gx/GCl in WT CFTR; dashed lines, ±2 SD of the WT data. Data points were plotted as a function of distance down the pore from the extracellular end at left to the middle of the pore at the right. Points that fall outside the dashed lines represent a large change in Px/PCl or Gx/GCl for that ion. Ions are listed from top to bottom in order of increasing ionic radius.



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Fig. 7.   Mutations that affect binding of small anions also affect binding of anionic pore blockers. Introduction of an acidic amino acid (glutamic acid) near the binding site for small anions, at S341 in TM6, also destabilizes binding of diphenylamine-2-carboxylate (DPC). Currents carried by WT CFTR or S341E CFTR were measured in the absence of drug and in the presence of 100 (for WT CFTR) or 500 (for S341E CFTR) µM DPC applied to the bathing solution. Shown are fractional currents remaining after 7-min exposure to DPC compared with currents measured in the absence of DPC (I/Io) after steps to membrane potentials between -140 and +80 mV. Values are means ± SD; n = 6 oocytes. WT CFTR exhibited voltage-dependent block by DPC at hyperpolarizing potentials. S341E CFTR was not blocked by DPC, even at high concentrations.



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Fig. 8.   Relative affinity [(Px/PCl)/(Gx/GCl)] for small monovalent anions for the alanine- and glutamic acid-substituted mutants. Data for each variant were plotted as a function of distance down the pore, with the extracellular end on the left and the middle of the pore on the right. Nos. in parentheses, range of relative affinity values for each variant calculated as the ratio of the highest to the lowest. A: alanine substitutions. B: glutamic acid substitutions. , Acetate; , ClO<UP><SUB>4</SUB><SUP>−</SUP></UP>; black-lozenge , Cl-; , I-; black-triangle, Br-; black-down-triangle , NO3-; hexagon, SCN-. WT CFTR was very effective at discriminating between monovalent anions. Both alanine and glutamic acid mutations at T338 and S341 diminished the discriminating power of the pore.

Analysis. Reversal potentials (Vrev) for Cl- (V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP>) and for each test anion were used to calculate Px/PCl values according to the Goldman-Hodgkin-Katz equation in the following form
<FR><NU>P<SUB>x</SUB></NU><DE>P<SUB>Cl</SUB></DE></FR><IT>=</IT><FR><NU>[Cl<SUP>−</SUP>]<SUB>r</SUB>10<SUP><IT>zF&Dgr;V</IT><SUB>r</SUB><IT>/RT</IT></SUP><IT>−</IT>[Cl<SUP>−</SUP>]<SUB>t</SUB></NU><DE>[<IT>x<SUP>−</SUP></IT>]<SUB>t</SUB></DE></FR>
where [Cl-]r and [Cl-]t are the concentrations of Cl- in the reference and test solutions, respectively; [x-]t is the concentration of anion x in the test solution (96 mM); Delta Vr is the change in Vrev; z is the valence; R is the gas constant; T is the absolute temperature; and F is the Faraday constant (49). Relative chord conductances for anion entry were calculated from the change in current over a voltage range from Vrev to Vrev+25 mV. By analyzing only the outward currents, we isolated the data for currents carried by the mixture of Cl- and each substitute anion under known conditions.

All data are background subtracted, with the currents measured in the absence of Iso in the same oocyte as background, with the exception of experiments with S341E CFTR. Oocytes expressing this construct exhibited spontaneously active currents in the absence of Iso that were much greater in amplitude than WT CFTR-expressing oocytes in the same batch, even after a prolonged wash in ND96 solution. To correct for the small currents carried by endogenous channels in the absence of bath Ca2+, we used uninjected oocytes for background subtraction for this mutant only. However, currents in S341E CFTR-expressing oocytes were increased by ~30% on further stimulation with Iso and exhibited time-independent behavior in response to steps in membrane potential, indicating that these currents reflect activity of CFTR. S341E CFTR expressed well in oocytes so that the signal-to-noise ratio (compared to a background of an uninjected oocyte) was at least 40 for each experiment. Furthermore, the selectivity pattern described in Characterization of the WT CFTR pore: lyotropic selectivity and anion binding for S341E CFTR differed considerably from the recently described pattern for the endogenous Ca2+-activated Cl- channel of oocytes (34). Hence, we felt confident in ascribing anion currents in oocytes expressing this cRNA to the S341E CFTR mutant channels rather than to the endogenous Ca2+-activated Cl- channel.

Statistical comparisons. Unless otherwise noted, all values are means ± SE. Statistical analysis was performed with the Wilcoxon rank sum test after appropriate tests for normality and variance (SigmaStat, Jandel Scientific, San Rafael, CA). In Tables 1-6, significance is indicated as P <=  0.001.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of the WT CFTR pore: lyotropic selectivity and anion binding. We studied CFTR currents in the presence of Cl- and nine substitute monovalent anions selected to span a wide range of radii (Table 1). Figure 1 compares currents generated from a hyperpolarizing voltage protocol in solutions containing Cl- as the sole anion or solutions containing 4 mM Cl- and 96 mM substitute anion for a representative oocyte expressing WT CFTR. Vrev values for currents in the presence of Br-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and SCN- were more negative than those for currents in the presence of Cl-, whereas all other ions studied reversed at potentials depolarized from the V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP>. Vrev data (summarized in Table 1) were used to calculate relative permeabilities for each anion (Table 2). Our results indicate that for most anions, Px/PCl values in WT CFTR were determined according to hydration energies (Fig. 2, Table 3), as expected for a channel with a lyotropic selectivity sequence (39, 41, 45). Two anions, I- and ClO<UP><SUB>4</SUB><SUP>−</SUP></UP>, exhibited Px/PCl values lower than expected from this relationship. This behavior was absent in similar studies of the oocyte endogenous Ca2+-activated Cl- channels (34), indicating that there may be distinctive interactions of these anions with the pore of CFTR. Relative permeabilities to isethionate, glutamate, and gluconate are very low, as if these ions are too large to traverse the pore.

Gx/GCl values were determined by measuring the slope conductance over a range of 25 mV depolarized from the Vrev (Table 4). Figure 3 shows the relative conductance for anion entry for all anions as a function of ionic radius. In WT CFTR, no anion exhibited a conductance for anion entry greater than that of Cl-. In no case did we measure a zero conductance in the presence of the substitute anion solutions; WT channels exhibited a baseline conductance in the presence of the substitute anion because all substitution solutions contained 4 mM residual Cl- (see below). This allowed us to separate the substitute anions into three classes with respect to the conductance for anion entry in WT CFTR: 1) anions that exhibit significant conductance and have Gx/GCl values that fall on the dashed line in Fig. 3 (Cl-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, Br-, and acetate); 2) anions that are too large to fit easily into the pore and have Gx/GCl values that fall on the solid line in Fig. 3 (glutamate, gluconate, and isethionate); and 3) anions that are small enough to fit in but bind so tightly that they block the current generated by the residual Cl-, resulting in Gx/GCl values that fall below the solid and dashed lines in Fig. 3 (SCN-, I-, and ClO<UP><SUB>4</SUB><SUP>−</SUP></UP>). The intersection of the solid and dashed lines in Fig. 3 provides an estimate of the narrowest diameter of the pore.

It is interesting to note that the value for the pore diameter, slightly >5 Å, determined according to Gx/GCl is very similar to the value determined according to the dependence of Px/PCl on anion size by other investigators (23). On the basis of the permeability of C(CN)<UP><SUB>3</SUB><SUP>−</SUP></UP>, which has an equivalent radius of 3 Å, Smith et al. (39) estimated the minimum pore diameter to be somewhat larger. The apparent size dependence for relative permeability reflects, at least partially, the impact of hydration energy. However, hydration energy and anion radius are inversely related (Fig. 2) such that larger anions have a higher Px/PCl, up to a point at which large anions with irregular charge distribution exhibit large hydration energies and low Px/PCl values (34, 39). In contrast, the relationship between Gx/GCl and anion size is a smooth function and does not depend on hydration energies except to the extent that for an ion to conduct, it must also permeate; no simple relationship exists between Gx/GCl and hydration energy (Fig. 2B). Gx/GCl decreases monotonically with increased size such that large anions that do not bind tightly have a reduced conductance. A test of this relationship and its ability to predict pore diameter would be to search for large anions with nonzero values for Px/PCl but a reduced conductance due to anion binding. Such a study may further refine the value for the minimum pore diameter.

We concluded that the conductance in the presence of the large anions is due to current carried by the residual Cl- rather than to the intrinsic conductance of the large anions based on the following observations. Ramp protocols run in the complete absence of extracellular Cl-, where all Cl- was replaced by gluconate, did not reverse at potentials up to +60 mV, indicating that the extracellular gluconate was not conductive. We also determined the dependence of conductance at +80 mV on extracellular Cl- concentration ([Cl-]) using mixtures of gluconate-containing and Cl--containing solutions (Fig. 4). In the complete absence of Cl-, current measured in response to a step to +80 mV was near zero. The dependence on bath [Cl-] suggests that under these conditions, the channel has an apparent affinity for Cl- of ~5 mM. However, the macroscopic conductance was not saturated at the maximal [Cl-] tested, so this value is likely to underestimate the true affinity; another study (41) has suggested a Michaelis-Menten constant of 37.6 mM determined over a broad range of symmetrical [Cl-] values in excised-patch experiments. Hence, in our studies, macroscopic conductance was ~50% of that in control conditions with the substitution of 96 mM gluconate for Cl-. Therefore, Gx/GCl for large anions arises from the inability of those anions to traverse the pore without blocking the conduction of the residual Cl-, and anions that exhibit Gx/GCl values below this baseline are capable of binding in the pore in such a way that they inhibit the conductance carried by the residual Cl-. These data indicate that Gx/GCl values in WT CFTR were determined according to anion size in combination with anion binding.

Rationale for the positions studied. For the purpose of this study, we assayed the effects of mutations at five positions chosen on the basis of 1) predictions made by comparing the sequence of TM6 and TM12 with the sequences of ligand-gated anion channels (29, 33), 2) the results of mutations at these positions on blockade by diphenylamine-2-carboxylate (DPC) and/or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (33, 50), and 3) previous studies by other investigators (24, 26). In TM6, we studied mutations at K335, T338, and S341, all of which were predicted to face into the pore based on our proposed alignments (29) and conservation of positioning of polar residues. However, on the basis of cysteine-scanning mutagenesis, Cheung and Akabas (5, 6) concluded that T338 does not face the pore. We also studied T339 as a control position; we do not believe that this amino acid faces into the pore based on the insensitivity of blockade by DPC to mutations at this site (33). In TM12, we studied mutations at T1134, predicted to lie at a position slightly more extracellular than K335. Interpretation of the results of these mutations is predicated on the assumption that their effects are limited to the site mutated without propagation to distant sites. However, we recognize the possibility of nonselective effects (see Refs. 1, 4). For instance, the R347D mutation has been shown to have nonspecific effects due to destruction of a salt bridge (8). It is also possible that mutation of a site in TM6 does not affect the interaction of anions with the pore at that site but affects the interaction of anions with the pore at a site in an adjacent TM domain.

Effects of alanine substitution on selectivity between monovalent anions. We have begun to use alanine substitution to determine the effect of the loss of side-chain functionality at each site listed in Rationale for the positions studied to identify the residues that contribute to selectivity in WT CFTR. Alanine-scanning mutagenesis avoids a priori bias regarding the location of residues that are "likely" to contribute to the pore. Because alanine is small and fairly unreactive, introduction of alanine is usually well tolerated in both soluble proteins and integral membrane proteins (e.g., Ref. 40). With alanine substitution, we expected to find a range in the magnitude of changes in Px/PCl. However, the changes should be largest for substitutions in regions of the pore that contribute the most to discrimination between anions.

Figure 5 shows current-voltage (I-V) relationships for WT and all mutant CFTRs described in this study in the presence of Cl- as the predominant bath anion. WT CFTR currents exhibited mild outward rectification in oocytes as previously described (33) and reversed at -21.24 ± 0.59 mV (Table 1). V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP> was affected by alanine substitution only at T338 and T1134 (Table 5). The shape of the I-V curve between -80 and +60 mV was not affected by the K335A, T338A, T339A, or T1134A mutations, whereas S341A CFTR exhibited less outward rectification than WT CFTR. Previous experiments by McDonough et al. (33) with a step protocol indicated pronounced inward rectification in this mutant when studied at more strongly hyperpolarizing potentials.

Alanine substitution at position T339 significantly affected only the Px/PCl for I- (Table 2). T339A CFTR exhibited altered Gx/GCl values only for acetate and NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Table 4). The limited effects of the T339A mutation are consistent with the hypothesis that this position is non-pore lining. Alanine substitution near the predicted extracellular ends of TM6 and TM12, at positions K335 and T1134, reduced Px/PCl values for all large anions and increased Px/PCl for SCN- and I-. K335A and T1134A CFTR exhibited decreased Gx/GCl values for most anions, with radii as large as or larger than acetate. However, neither Px/PCl nor Gx/GCl values for the smallest anions (NO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Br-) were altered in K335A CFTR or T1134A CFTR compared with WT CFTR. These results suggest that residues K335 and T1134 do not make important contributions to selectivity between small anions.

Because alanine substitution at the predicted extracellular end, at K335 and T1134, did not affect Px/PCl or Gx/GCl for small anions, we asked whether introduction of an amino acid with greater side-chain bulk (phenylalanine) would affect selectivity at these positions, as if the larger side chain was able to reduce the effective diameter of the pore at the external end, leading to enhanced interactions with even the smallest anions. Although the introduction of phenylalanine at K335 and T1134 resulted in several differences in Px/PCl and/or Gx/GCl compared with those in WT CFTR (Tables 2 and 4), we focus here only on comparisons between the alanine-substituted and phenylalanine-substituted variants. At K335, Gx/GCl values for all small anions were increased in K335F CFTR compared with K335A CFTR, whereas Px/PCl values for these anions were not affected by the bulky side chain. T1134F CFTR exhibited a similar pattern except that GNO3/GCl and GBr/GCl were decreased and GSCN/GCl was increased compared with T1134A CFTR. The greater impact of phenylalanine substitution at these two positions compared with alanine substitution supports the conclusion that the pore is relatively wide at its extracellular end.

Alanine substitution at T338 affected both Px/PCl and Gx/GCl for every anion tested (Tables 2 and 4), suggesting that this position may contribute to a region of high discrimination between monovalent anions. Px/PCl values for small anions were increased in T338A CFTR, whereas Px/PCl values for large anions were decreased. An exception to this trend was the very large (18-fold) increase in PClO4/PCl exhibited by this mutant. Although it seems surprising that Px/PCl values for the large anions should decrease rather than increase with mutations in this region of strong discrimination, the same phenomenon was observed for PTris/PNa in alanine mutants at the selectivity filter in the nicotinic ACh receptor (7). Interestingly, Px/PCl for SCN-, the most permeant anion tested in WT CFTR, was nearly doubled in T338A CFTR, whereas PI/PCl was increased sevenfold. Gx/GCl values for large anions were also decreased in T338A CFTR, whereas Gx/GCl values for small anions were increased (Table 4).

Alanine substitution at position S341 affected Px/PCl for all anions except ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> (Table 2). The pattern was similar to that for T338A CFTR in that Px/PCl values for large anions were decreased in S341A CFTR, whereas Px/PCl values for small anions were increased. Except for PNO3/PCl, the magnitude of the effect in S341A CFTR was less than that seen in T338A CFTR. The effects of alanine substitution at S341 on Gx/GCl were limited to only the anions of small and intermediate size. Gx/GCl values for small anions (NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, Br-, and SCN-) were increased in this mutant. Interestingly, although GClO4/GCl was increased in T338A CFTR, GClO4/GCl was decreased in S341A CFTR. The pattern in S341A CFTR was similarly inverted for Gacetate/GCl compared with that in T338A CFTR. These results suggest that T338 and S341 reside on opposite sides of a structure that determines selectivity between ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> and Cl- and between acetate and Cl-.

Previous results by McDonough et al. (33) indicated that the nature and orientation of the aliphatic hydroxyl side chain at S341 was critical to pore characteristics in that S341T was not identical to WT CFTR with respect to blockade by DPC. The side chain for serine is HOCH2, whereas that for threonine is CH3CH(OH). We asked whether side-chain orientation at S341 was important to anion selectivity by performing selectivity experiments in the S341T mutant. Relative permeabilities to large anions were greatly reduced in S341T CFTR. Gx/GCl values were less sensitive to this mutation because only GSCN/GCl was increased and only GClO4/GCl was decreased. These results suggest that the orientation of the hydroxyl side chain at S341 is, indeed, critical for some aspects of selectivity.

Figure 6 presents an attempt to look for patterns in the results of the alanine substitution mutations. In Fig. 6A, normalized Px/PCl values are shown for each anion for each alanine substitution studied. For example, PNO3/PCl for WT CFTR was set to unity, and PNO3/PCl for each alanine substitution was plotted relative to this value. Normalizing Px/PCl to the WT value for each anion removes the dependence of Px/PCl on anion character and facilitates comparisons between anions and between CFTR variants. Hence, in Fig. 6, the solid line in each box represents the WT data and the dashed lines delineate ±2 SD of the WT data. Data points are plotted as a function of the distance down the pore, with the extracellular end on the left and the middle of the pore on the right. Ions are listed from top to bottom in order of increasing ionic radius. This treatment allows comparison of the magnitude of change in Px/PCl (or Gx/GCl) with common substitutions and multiple sites.

The data show that 1) Px/PCl values for all anions are most sensitive to mutation at T338 and 2) mutations at the extracellular end of the pore do not dramatically affect Px/PCl values for small anions, yet 3) those mutations at the external end of the pore affect Px/PCl values for large anions (bottom of graph) to the same extent as do mutations at T338. In other words, small anions do not appear to sense the walls of the pore at the external end of this region, whereas large anions do. This is consistent with a narrowing of the pore from the external end toward the middle at T338 and S341.

Gx/GCl data treated in this manner show a similar pattern (Fig. 6B). The data suggest that only anions as small as or smaller than SCN- are able to reach through the pore to position S341. For the smallest anions (NO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Br-), alanine substitution at position S341 had a larger effect on Gx/GCl than did alanine substitution at position T338. For ions ranging in size between I- and isethionate, only alanine substitution at T338 dramatically affected Gx/GCl. The one exception to this pattern is the decreased GClO4/GCl in T1134A CFTR. For anions larger than isethionate (glutamate and gluconate), even the T338A mutation had only a very small effect on Gx/GCl. Gx/GCl values for the large anions were unaffected by alanine substitution at S341. These large anions may not reach down the pore of CFTR far enough to get to S341, as if a barrier to their permeation resides just extracellular to this position.

Effects of glutamic acid substitution on selectivity between monovalent anions. The alanine substitution experiments described in Effects of alanine substitution on selectivity between monovalent anions identified a region of strong discrimination between monovalent anions. Discrimination between anions could result from differences in energy barrier heights (reflected in Px/PCl) or from differences in well depths (reflected in Gx/GCl). Because CFTR exhibits a strongly lyotropic selectivity sequence, it may be appropriate to consider that energy barriers to permeation in this channel mostly reflect the energy of dehydration rather than a physical barrier in the pore walls. In contrast, we know that anion binding makes an important contribution to selectivity in CFTR (Fig. 3, Table 4) (26). We reasoned, therefore, that disruption of anion binding by the introduction of a negative charge at specific residues would identify the positions that may determine selectivity by contributing to anion binding sites. Hence we introduced glutamic acid residues at the same sites that we studied with alanine substitution and asked whether the mutant channels were affected in their ability to discriminate between monovalent anions. This model-dependent approach relies on the assumption that placement of a negative charge near anion binding sites would greatly destabilize anion binding, whereas placement of a negative charge at a distance from anion binding sites would have a smaller effect.

Glutamic acid substitution at K335 resulted in a nearly linear I-V relationship (Fig. 5) but did not alter V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP> (Table 5). In contrast, T1134E CFTR exhibited a leftward shift in V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP>. S341E CFTR exhibited a larger shift in V<UP><SUB>rev</SUB><SUP>Cl</SUP></UP> and pronounced outward rectification, whereas T338E CFTR exhibited pronounced inward rectification. The opposite patterns of macroscopic rectification in T338E CFTR and S341E CFTR suggest that these two positions may lie on opposite sides of a barrier in the pore. K335E CFTR exhibited decreased Px/PCl for large anions and increased Px/PCl for the "sticky" anions I- and ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> (Table 2). Px/PCl values for all anions except SCN- and ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> were affected by mutation T1134E. Gx/GCl values for most anions were altered in K335E CFTR, with decreases in Gx/GCl values for large anions and increases in Gx/GCl values for small anions (Table 4). T1134E CFTR exhibited a similar pattern, with changes in Gx/GCl values smaller in magnitude than those for K335E CFTR.

The effects of glutamic acid substitution on selectivity between monovalent anions were greatest at T338. Px/PCl was increased in T338E CFTR for all anions except acetate and gluconate. As in T338A CFTR, PClO4/PCl stands out as most sensitive to mutation at this position because PClO4/PCl was increased 18-fold in T338A CFTR and 11-fold in T338E CFTR. Mutation T338E resulted in increased Gx/GCl values for all anions smaller than acetate, whereas Gx/GCl values for acetate, glutamate, and gluconate were decreased. Introduction of a negative charge at S341 only affected Px/PCl values for anions smaller than acetate (Table 2). In contrast, Gx/GCl values for all anions were altered in S341E CFTR (Table 4). With the exception of acetate, all anions exhibited increased Gx/GCl values in this variant. Comparing S341E with S341A CFTR and T338E with T338A CFTR, we can see that the introduction of a negative charge at S341 more strongly destabilized the binding of SCN- (which is pronounced in WT CFTR) than did the equivalent mutation at T338. These data support the conclusion that T338 and S341 contribute to the region of high discrimination in the CFTR pore.

In a previous work, McDonough et al. (33) identified S341 as a probable anion binding site based on the reduction in single-channel conductance observed in S341A CFTR. S341 also appears to contribute the majority of the binding energy for blockade of CFTR by the anionic arylaminobenzoate drugs DPC and NPPB (33, 50). A prediction from the disruption of SCN- binding in S341E CFTR is that this mutation may result in disruption of DPC binding as well. To test this notion, we compared the blockade of CFTR macroscopic currents in oocytes expressing WT CFTR or S341E CFTR using methods established previously (50). Figure 7 shows that WT CFTR was blocked by 100 µM DPC in a voltage-dependent fashion, with an apparent dissociation constant of 201 µM at -100 mV (50). In contrast, S341E CFTR was insensitive to DPC at all voltages, even at high concentration; in fact, 0.5 mM DPC led to a slight increase in current at all potentials. Introduction of a negative charge at distant sites [e.g., K335 (33)] did not have this effect. These data suggest that an anion binding site does, indeed, lie at or very close to position S341.

Spatial dependence of discriminating power. If S341 and T338 lie in the region of highest discrimination between monovalent anions, we would expect that mutations here would have the greatest effect on the ability of CFTR channels to distinguish between monovalent anions. To test this notion, we determined the relative affinity (18) for each anion in WT CFTR and for each of the alanine and glutamic acid substitution mutants as relative affinity [(Px/PCl)/(Gx/GCl)]. Intuitively, relative affinity for ion x could be elevated by mutations that promote anion entry into the channel (reflected as an increase in Px/PCl) and/or promote an increase in anion binding (reflected as a decrease in Gx/GCl). For example, SCN- accesses the WT pore easily and binds tightly, resulting in a high value for relative affinity (13.4 in WT CFTR). In contrast, acetate experiences a barrier to pore entry and then does not bind well, resulting in a low value for relative affinity (0.3 in WT CFTR). For each CFTR variant, the degree of spread between the lowest value for relative affinity (usually acetate) and the highest value for relative affinity (usually SCN-) provides a measure of the discriminating power of the pore. A channel that cannot discriminate between monovalent anions would exhibit a narrow range of relative affinities. We excluded isethionate, glutamate, and gluconate from this analysis because Gx/GCl values for these anions are not clearly indicative of their intrinsic behavior due to the conductance arising from residual Cl-.

Figure 8 is an attempt to represent graphically the spatial dependence of changes in the discriminating power of the channel by two types of substitutions, alanine (Fig. 8A) and glutamic acid (Fig. 8B). The overall discriminating power was approximately the same for WT, K335A, T339A, and T1134A CFTR. However, alanine mutations at T338 and S341 effectively diminished discriminating power. These data show that even the unbiased approach of alanine-scanning mutagenesis can be used to identify positions that contribute to the ability of the channel to distinguish between various ions. Glutamic acid substitution had even greater effects (Fig. 8B). In fact, mutations T338E and S341E led to channels that could barely distinguish between monovalent anions. Positions at which these effects are the greatest identify regions of the pore that confer discriminating power to the WT channel. According to this notion, T338 and S341 make important contributions to this function in the CFTR channel pore and may lie at or adjacent to anion binding sites.

Dependence on the direction of anion movement. Other investigators (41) have described hysteresis in I-V relationships obtained from patches expressing WT CFTR under bi-ionic conditions with I- on one surface and Cl- on the other. In those experiments, PI/PCl was initially >2 but fell to below unity after time. This change in PI/PCl was dependent on the voltage protocol applied to the patch and was interpreted as a shift from an I--permeant state to an I--blocked state. Given these previous results, we compared our results calculated from hyperpolarizing ramps with data calculated from depolarizing ramps. In WT CFTR, both PI/PCl and PClO4/PCl were protocol dependent, although not to such a strong degree as described by others (41) (PI/PCl was 0.52 vs. 0.36 and PClO4/PCl was 0.18 vs. 0.10 for depolarizing vs. hyperpolarizing ramps, respectively). This behavior was retained in K335F and T1134F CFTR but lost in all other mutants. In contrast, mutation T338A induced significant hysteresis for all three of the large anions studied. Px/PCl values for isethionate, glutamate, and gluconate were ~0.13 for depolarizing ramps and ~0.07 for hyperpolarizing ramps. When Cl- exit at negative potentials preceded entry of large anions at positive potentials, the Px/PCl values calculated for those large anions were greater than when the voltage protocol was reversed. The depolarizing ramps begin with a 50-ms step to -80 mV, which induces strong inward current (Cl- exit) and should result in significant loading of Cl- into the pore from the cytoplasmic solution. The hyperpolarizing ramps began with a 50-ms step to +60 mV, which should induce depletion of Cl- from the pore. The observed shift in Px/PCl for large anions suggests that occupancy by Cl- of an anion binding site that is cytoplasmic to the pore constriction reduced the energy barrier height for entry of large anions from the extracellular side.

Effects of mutations on monovalent-to-divalent selectivity. Very little is known about the selectivity between monovalent and divalent anions in CFTR because no polyvalent anions other than ATP4- (23) have been studied in this channel. We have begun to identify the determinants of selectivity according to valence by studying CFTR currents in the presence of thiosulfate (S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>). When the ND96 bath solution was exchanged for one containing 96 mM Na2S2O3 (with all else remaining equal), both inward and outward currents in WT CFTR were greatly reduced (Fig. 1) and the Vrev shifted to the right to 22.52 ± 1.98 mV (n = 16). PS2O3/PCl was very low under these conditions and was highly variable in both WT CFTR and the CFTR variants. On the other hand, GS2O3/GCl was well above zero (Table 6) and could be reliably measured in WT CFTR and the CFTR variants. Hence we focused on the effects of mutations in terms of Gx/GCl alone. S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP> has a diameter that is very close to the predicted minimal pore diameter (Fig. 3) and yet the conductance in S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>-containing solutions fell below the baseline conductance carried by the residual Cl-. Hence GS2O3/GCl values in WT CFTR reflected a significant degree of pore block by this anion. In confirmation, we found that substitution of S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP> had a greater concentration-dependent impact on macroscopic conductance than did a similar substitution with the nonconductive, nonblocking anion gluconate (Fig. 4).

The effects of pore-domain mutations on GS2O3/GCl are given in Table 6. Most of the glutamic acid substitutions significantly affected monovalent-to-divalent selectivity expressed in this way. Mutation K335E led to a slight decrease in GS2O3/GCl, whereas mutations T338E and S341E greatly increased GS2O3/GCl. In contrast, among alanine substitution mutants, only S341A CFTR exhibited a significant effect on GS2O3/GCl. Even mutation T338A, which had the most pronounced effects on selectivity between monovalent anions, did not affect selectivity between Cl- and S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP>. These data suggest that selectivity between monovalent anions was conferred predominantly in the region of T338, whereas selectivity between monovalent and divalent anions arose from structures closer to S341.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several approaches have been used to identify amino acids in CFTR that contribute to anion selectivity. However, most studies have not been performed in a manner allowing comparison of the effects of mutagenesis at multiple positions in the pore. A single mutation almost always has a measurable effect on at least one parameter of selectivity. To facilitate interpretation, experimental designs that allow one to look for patterns avoid the potential pitfall of generating conclusions based on the effects of mutations at a single position. By studying the effects of a limited number of substitutions made at several positions, one can look for patterns in the aggregate results, which may provide information about the structure of the pore in the WT channel. This study provides proof of concept because we report the effects of two classes of mutations at five positions in the CFTR channel. The magnitude of the effects of alanine or glutamic acid substitution should be largest at positions that contribute most strongly to selectivity. These data allow us to identify a region of strong discrimination between monovalent anions and between monovalent and divalent anions, which appears to reside between T338 and S341 in TM6. Selectivity between monovalent anions was most sensitive to mutation at T338 or S341 depending on the anion. Selectivity between Cl- and a divalent anion was most sensitive to mutation at S341. K335 and T1134, near the predicted ends of TM6 and TM12, respectively, were relatively insensitive to mutation. These observations are consistent with a narrowing of the pore from the extracellular end toward a constriction near S341.

Amino acids involved in permeation in CFTR. Several individual amino acids in the TM domains of CFTR have been shown to contribute to permeation properties such as single-channel conductance (21, 24, 33, 36, 37, 41, 42, 47, 49), interaction with pore blockers (20-22, 26, 33, 44, 49, 50), anion or cation selectivity (6, 14), and selectivity between monovalent anions (3, 19, 22-24, 26, 41, 49). These studies have focused on TM6, where several amino acids appear to play important roles in establishing the character of the CFTR pore (for a review, see Ref. 29). It should be pointed out that all studies attributing a functional role to R347 must be reconsidered because substitution of this amino acid with anything other than lysine or histidine grossly disrupts the conformation of the channel (8). K335 in TM6 appears to make only indirect contributions to selectivity between small monovalent anions (3, 26; present study); however, this study shows that K335 does directly contribute to selectivity between Cl- and large anions. When the alanine in K335A CFTR was replaced by the bulkier phenylalanine, the effects on anion selectivity were greater (Tables 2 and 4). Glutamic acid substitution here had limited effects on discrimination between anions (Fig. 8). These observations suggest that the pore may be wide at the cross-sectional level of K335 such that anions do not make intimate contact with the walls at this putative end of the pore. K335 may lie near the innermost edge of the outer vestibule and not within a region of strong discrimination between anions. Alanine and phenylalanine mutations at T1134 in TM12 had effects very similar to those of mutations at K335, suggesting that this amino acid also may contribute only indirectly to anion selectivity. T1134 is the first amino acid in TM12 of CFTR for which selectivity data have been described after mutation. In most cases, the effects of equivalent mutations were somewhat smaller at T1134 than at K335; this is consistent with our hypothesis that T1134 projects into the pore at a level slightly more extracellular than does K335.

T338 and T339 in TM6 have received considerable attention. Based on the results of cysteine-scanning mutagenesis, neither of these amino acids was predicted to line the pore (5). Linsdell et al. (23) showed that the concurrent mutation of both of these sites (TT338,339AA) led to increased channel conductance and increased permeability to several large anions (23), as if this locus controlled the functional pore diameter. However, a subsequent study (24) found that the single mutation T338A led to an even larger single-channel conductance and that mutations here did not strongly affect the functional minimum pore diameter. These authors concluded that T338 did not play a major role in contributing to the narrow pore region. Unfortunately, the single-channel conductance of charge mutants at this position (e.g., T338E compared with T338Q) was not studied to determine whether electrostatic repulsion occurred due to the introduction of charge. This would clarify whether or not T338 is a pore-lining residue. T339A CFTR expressed poorly in Chinese hamster ovary (CHO) cells (24), although it expressed well in oocytes (33; present study). T339V CFTR, which did express in CHO cells, exhibited limited alterations in selectivity compared with mutants at T338 (24), consistent with a non-pore lining role for T339.

McDonough et al. (33) previously showed that T339A CFTR was identical to WT CFTR with respect to blockade by DPC, whereas T338A CFTR exhibited identical affinity for DPC (at -100 mV) but slightly altered voltage dependence. These data are consistent with the notion that both T338 and T339 reside at positions extracellular to the binding site for DPC. In our hands, T339A CFTR exhibited selectivity very similar to WT CFTR. We interpret these results as indicating that T339 is not a pore-lining residue. In contrast, alanine substitutions at T338 had the greatest impact on selectivity between Cl- and all monovalent anions larger than NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 6). Hence, by comparing the effects of mutations at T338 with the effects of identical mutations at other loci in TM6 and TM12, we conclude that T338 does indeed contribute to the region of strongest discrimination between monovalent anions.

Previous experiments by McDonough et al. (33) and Zhang et al. (50) also showed that S341, near the middle of TM6, provides the majority of the energy for binding DPC and NPPB. In the absence of this binding site, DPC appears to be able to permeate further toward the extracellular end of the pore as the voltage dependence of block is increased in S341A CFTR. S341 also appears to be a Cl- binding site because single-channel conductance was greatly reduced in S341A CFTR (33). Alanine substitution at S341 significantly affected selectivity between Cl- and small anions but not large anions. For the smallest anions tested (NO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Br-), selectivity was affected more by mutations at S341 than by mutations at T338. Mutation S341E had a greater effect on the ability of the channel to discriminate between monovalent anions than did any other glutamic acid substitution (Fig. 7). Therefore, S341 appears to make an important contribution to the region of strong discrimination between monovalent anions. S341 also determines the selectivity between monovalent and divalent anions (Table 6). Given the effects of mutations at this position on selectivity between monovalent anions, selectivity between monovalent and divalent anions, single-channel conductance, and open channel block, it appears that S341 plays a very prominent role in determining the permeation properties of CFTR.

The importance of anion binding. One significant difference between our approach and that of some other investigators (e.g., Ref. 23) is that we can ascertain the effects of mutations on Gx/GCl as well as on Px/PCl because we made measurements in the presence of Cl- and substitute anions in the same cell and we can correct for changes in the activation status of the CFTR channels. Most other studies (e.g., Ref. 19) of selectivity in CFTR have only reported the effects of mutations on Vrev values; single-channel conductance (or Gx/GCl) was not reported for most mutants. Anion binding, which affects Gx/GCl, is known to be an important determinant of selectivity in CFTR (9, 26). Consistent with this notion, we found that for small anions, Gx/GCl values were generally more prone to change on mutation at T338 and S341 than were Px/PCl values (note the differences in ordinate scales for Fig. 6, A vs. B). This was true for both alanine and glutamic acid substitutions at T338 and S341. In contrast, for ions larger than SCN-, Px/PCl values were more sensitive to mutations than Gx/GCl values. Superficially, this observation appears to be inconsistent with the work by Mansoura et al. (26). However, those investigators only studied the effects of mutations on Px/PCl and Gx/GCl of ions as large as I- or smaller, and they did not study mutations in TM6 at positions cytoplasmic to K335. Hence, given the aggregate picture, anion binding is clearly important for selectivity between Cl- and small anions but less important for selectivity between Cl- and large anions.

Distributed determinants of selectivity. It has been suggested by others (19) that the CFTR pore includes a discretely localized selectivity filter, which confers on the CFTR channels most, if not all, aspects of anion selectivity. Alternatively, lyotropic permselectivity may be a characteristic imposed on the CFTR pore by virtue of distributed interactions of different anions with several points along the length of the pore (9). If this were the case, one might expect to find regions of the pore that are highly sensitive to mutation and other regions that are less sensitive; our results suggest that this is the case. One might also expect to find that some anions may be most sensitive to mutation at one position, whereas other anions are more sensitive to mutation at different positions. This also appears to be indicated by our alanine substitution data that show that the relative affinities for NO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Br- were affected most by mutations at S341, the relative affinity for SCN- was equally affected by mutations at S341 and T338, the relative affinities for I- and ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> were affected most by mutations at T338, and the relative affinity for acetate was equally sensitive to mutations at S341 and T338. This does not appear to reflect a dependence on anion size because the results differed for ClO<UP><SUB>4</SUB><SUP>−</SUP></UP> and acetate, which are very similar in radius. Further evidence against a discrete selectivity filter is the greater than sixfold increase in relative affinity for I- exhibited by T1134F CFTR. These data suggest that the pore contains features that provide for selectivity between anions at several positions along its length. Furthermore, mutation of a non-pore lining residue could grossly alter pore structure along the full length of the helix, thereby affecting selectivity indirectly (19, 24).

Reconciling the results of alanine and glutamic acid substitutions. The alanine substitution experiments had the greatest impact on anion selectivity at T338 (Fig. 6). In contrast, the effects of glutamic acid substitution were greatest at position S341 (Fig. 8). This discrepancy likely arises from the fact that alanine substitutions represent loss-of-function mutations where the side chain present in the WT protein is replaced by the methyl side chain of alanine, whereas glutamic acid substitutions represent gain-of-function mutations that introduce a point-negative charge. Hence the electrostatic consequence of a glutamic acid substitution at S341 may reach well past this locus, whereas the effects of an alanine substitution may be more localized. Also, it may be true that ions more closely approach a charge placed at S341 than a charge placed at T338 by virtue of the constriction, leading to greater destabilization of anion binding in S341E CFTR. Our results with K335E (outside the region of high discrimination) and S341E (inside the region of high discrimination) show that this interpretation is feasible.

Localizing the narrowest point in the pore. By comparing the effects of a common mutation at multiple positions, as shown in Figs. 6 and 8, we can interpret these data in a structural context that allows us to make predictions about the shape of the pore. It is clear that the largest effects of alanine and glutamic acid substitutions are found at T338 and S341, suggesting that the narrowest region of the pore lies in the vicinity of these two positions. If these amino acids are separated by one turn of the alpha -helix, then selectivity between some ions is very strong over this ~5.4-Å distance. We can localize the narrowest point even further by considering the following scenarios and predictions from their effects on permeation properties.

In scenario 1, the narrowest point lies at T338. Scenario 1 would predict that 1) selectivity between small anions would be more sensitive to mutations at T338 than at S341, 2) the effects on selectivity between Cl- and large anions would be missing for S341A CFTR, and 3) mutations at T338 would greatly affect block by DPC. None of our data support this scenario.

In scenario 2, the narrowest point lies cytoplasmic to S341. This scenario would predict that 1) the effects on selectivity between Cl- and both small and large anions would be much greater for mutations at S341 than for mutations at T338 and 2) there would be minimal effects of mutations at S341 on blockade by DPC from the cytoplasmic side because the drug would not be able to reach this site. Our results suggest that these predictions are not realized.

In scenario 3, the narrowest point lies slightly extracellular to S341. Scenario 3 would predict that 1) selectivity between Cl- and small anions would be more sensitive to mutations at S341 than to mutations at T338, 2) the effects of alanine substitution at S341 on selectivity between Cl- and large anions would be small but still present, 3) selectivity between Cl- and some anions may be affected oppositely by comparable mutations at T338 and S341, and 4) macroscopic currents in Cl- may show rectification in opposite directions after mutations at T338 and S341.

Our data support these predictions as follows. 1) Selectivity between Cl- and NO<UP><SUB>3</SUB><SUP>−</SUP></UP> as well as between Cl- and Br- was affected more in S341A CFTR than in T338A CFTR. 2) Although relative permeabilities for the largest anions (acetate and larger) were affected greatly in T338A CFTR, they were also reduced significantly in S341A CFTR. 3) GClO4/GCl and Gacetate/GCl were oppositely affected by mutations T338A and S341A, as if these amino acids lie on opposite sides of a barrier that determines selectivity between these anions of very similar size. 4) T338E CFTR exhibits pronounced inward rectification in the macroscopic I-V, whereas S341E CFTR exhibits pronounced outward rectification (Fig. 5). These data suggest that T338 and S341 lie on opposite sides of the narrowest region in the CFTR pore. These observations are not consistent with a model recently proposed by Akabas (2), which has the narrowest region of the pore at a much more cytoplasmic position.

Evidence against the dual-pore hypothesis. Yue et al. (46) recently proposed that the amino-terminal half of CFTR forms one Cl- conducting pore and the carboxy-terminal half forms a separate pore with distinct anion selectivity and conductance. The amino-terminal pore was suggested to contribute to the main conductance state, whereas the carboxy-terminal pore contributed only to a subconductance state. If this were the case, mutations at comparable positions in TM6 and TM12 should not have similar effects on selectivity in macroscopic currents. However, our data show that mutations K335A and T1134A had nearly identical effects on selectivity patterns between large and small anions, as if these amino acids occupy nearly homologous positions in TM6 and TM12. This similarity of effects argues strongly that TM6 and TM12 contribute to the same pore, in contradiction of the one-channel, dual-pore model. Other observations against the dual-pore hypothesis include the ability to transfer the DPC binding site from TM6 to TM12, which resulted in affinity and voltage dependence for block by DPC that was very similar to that of WT CFTR (33). This would not be the expected result if the second-membrane-spanning domain only contributed to the subconductance state as the dual-pore model proposes.

It has also been suggested that CFTR channels may be formed from a dimer of CFTR peptides (12, 28, 43, 48). However, the data are not yet clear enough to tell whether a CFTR dimer forms a single channel pore or two channel pores. Further work is required to distinguish between these two possibilities. If a dimer of CFTR peptides forms a single pore, our data would suggest that this pore would be lined by two copies of TM6 and two copies of TM12.

Structural predictions for the pore of CFTR. CFTR, like other anion channels, does not exhibit strong selectivity. It may be generally true that channels with pores lined by alpha -helices are poorly selective (CFTR, gamma -aminobutyric acid type A receptor, and nicotinic ACh receptor channels), whereas channels lined by combinations of alpha -helices and beta -strands, such as the prototypical voltage-gated K+ channel (11), which exhibit 100-fold discrimination between ions that differ in radius by <0.5 Å (16), are much more selective. Because the lyotropic selectivity pattern in CFTR suggests that the barriers to permeation reflect ion-water interactions rather than ion-channel interactions, whereas anion binding appears to play a critical role in anion selectivity in this channel, studies that identify anion binding sites in CFTR may be very informative. We have suggested that S341 contributes to a Cl- binding site (33); T338 may contribute to another. These observations suggest that hydroxylated residues may play very important roles in coordinating anions in the permeation pathway of the CFTR channel. We originally proposed this concept based on studies of ligand-gated cation and anion channels (33). We are now fortunate to be able to interpret our results in CFTR in relation to the recently published crystal structure of halorhodopsin, the light-driven anion pump of bacteria (17). Only five TMs in halorhodopsin are required to contain the Cl- and a protonated Schiff base of retinal, which serves as a cofactor. There is no evidence of contributions to Cl- binding from beta -strands or carbonyl dipoles. The only amino acid in halorhodopsin that directly interacts with Cl- in the binding pocket is a serine. In fact, the interaction between the hydroxyl group in this serine, Cl-, and two waters of hydration contributes 8.9 of the 21.6 kcal/mol of the stabilization energy, far greater than the contribution by any other component. These observations in halorhodopsin add credibility to our proposal that hydroxylated amino acids make important contributions to anion binding and selectivity in the pore of CFTR.

Limitations of this study and future directions. A few limitations of this study are obvious. First, as always, the effects of any one mutation may not be specific but may reflect a structural change experienced at some distance from the site of mutation (1, 4). However, there was no evidence of gross structural changes because the Cl- currents in oocytes retained their dependence on activation by cAMP-dependent pathways, the currents were time independent, and most mutations did not affect all selectivity patterns. Second, because the results of all mutations described in this study were presented as Px/PCl, Gx/GCl, or relative affinity, it is impossible to determine directly whether the mutations affect changes in the interaction of Cl- with the pore. To answer this question, we will have to perform single-channel experiments in WT CFTR and in those variants that exhibited large changes in selectivity. Finally, although we have separately described the effects of mutations on Px/PCl and Gx/GCl, there is some interdependence of these two parameters. The use of the Goldman-Hodgkin-Katz equation to estimate Px/PCl implicitly assumes that the behavior of one ion is independent of the behavior of another ion or of the occupancy of the channel by any ion. This assumption fails for CFTR and for any other channel with multiple ion-binding sites (42) because tight binding of one anion would be expected to increase the mean occupancy of the binding sites in the pore and, thereby, alter the permeability of another anion. However, this approach is intuitively useful and has been widely used for assessing selectivity in a broad range of channels (9). Furthermore, changes in relative affinity should not be impacted by this interdependence if the concentration of substitute anions is held constant, as was the case in our experiments.

Based on our results, we position T338 and S341 at the +3' and 0' positions, respectively, in our model of TM6 (29). To clarify the picture further, other positions between K335 and S341 that were not studied here should be investigated. It will also be important to extend these studies to other positions more cytoplasmic than S341 to identify the cytoplasmic end of the region of strong discrimination. Similarly, this approach should be applied to TM domains other than TM6, as we have begun here, to determine whether amino acids in other TM domains also contribute to the region of strong discrimination.


    ACKNOWLEDGEMENTS

We thank H. Turki for assistance with the preparation of the constructs used in this study and C. Hartzell for comments.


    FOOTNOTES

This work was supported by the American Heart Association (9820032SE) and the National Science Foundation (MCB-077575).

Address for reprint requests and other correspondence: N. A. McCarty, Dept. of Physiology, Emory Univ. School of Medicine, 1648 Pierce Dr., Atlanta, GA 30322-3110 (E-mail: nmcc{at}physio.emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 January 2001; accepted in final form 10 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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