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 |
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 |
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).
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MATERIALS AND METHODS |
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
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 M
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
), isethionate,
perchlorate (ClO
), 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
). 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 ). 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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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 ( hydrGo)]. Relative permeability
(Px/PCl) values for
I and ClO 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 . 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 . 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 -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 ;
, Cl ; , I ;
, Br ; ,
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.
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Analysis.
Reversal potentials (Vrev) for Cl
(V
) and for each test anion were used
to calculate Px/PCl
values according to the Goldman-Hodgkin-Katz equation in the following form
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);
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 |
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
,
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
.
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
, 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
, 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
). 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)
, 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
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
(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
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
(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
, 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
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
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
(Table 5). In contrast, T1134E CFTR
exhibited a leftward shift in V
. S341E
CFTR exhibited a larger shift in V
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
(Table 2). Px/PCl values for all anions except SCN
and ClO
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
). 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
has a diameter that is very close
to the predicted minimal pore diameter (Fig. 3) and yet the conductance
in S2O
-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
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
. 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 |
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
(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
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
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
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
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
-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
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
-helices
are poorly selective (CFTR,
-aminobutyric acid type A receptor, and
nicotinic ACh receptor channels), whereas channels lined by
combinations of
-helices and
-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
-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.
 |
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