 |
INTRODUCTION |
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 contains an
anion-selective pore (1-6). Earlier work has shown that amino acid
residues in the two membrane-spanning domains, MSD1 and MSD2, determine
the properties of this pore and harbor a number of disease-associated
mutations (7-12). Despite the importance of this region to CFTR
function, an understanding of the pore and MSD structure is limited.
Studies of the effect of cystic fibrosis (CF)-associated mutations have
been of value in identifying structurally and functionally important
regions of CFTR. At least four CF-associated mutations have been
identified at position 347 in M6: R347C, R347H, R347L, and R347P,
suggesting that Arg-347 is important for CFTR structure and function
(13-15).2 Early studies by
Sheppard et al. (7) showed that mutation of Arg-347 to
proline significantly decreased single-channel conductance with little
effect on CFTR trafficking to the plasma membrane. Other work by
Tabcharani et al. (8, 16) and Linsdell and Hanrahan (17)
emphasized the importance of Arg-347 for anomalous mole-fraction
behavior, iodide permeability, and voltage-dependent block
by DIDS, in addition to single-channel conductance. Interestingly, mutation of Arg-347 to a histidine (R347H) produced a channel that
displayed pH-dependent conductance and anomalous
mole-fraction behavior (8). These studies suggested that Arg-347 may
line the pore and that a positive charge at position 347 is sufficient for wild-type conductance. Because mutation of Arg-347 eliminated anomalous mole-fraction behavior, Arg-347 itself was proposed to be an
anion binding site in the CFTR pore (8), and the presumed positive
charge introduced upon protonation of His-347 was thought to facilitate
interactions with permeating anions. An alternative interpretation is
that Arg-347 may be important for maintenance of pore architecture
without contributing directly to the permeation pathway. For example,
mutation of this site could lead to a change in MSD conformation and
loss of an anion-binding site(s) elsewhere; protonation of His-347
might then rescue the conformation of the R347H mutant.
As discussed by Perutz (18), charged residues within proteins reside in
locations where they are either solvated or can interact with and be
neutralized by oppositely charged residues. These electrostatic
interactions mediate an important stabilizing effect, providing
increased thermostability and resistance to denaturation. Based on
these considerations, it is possible that Arg-347 may line the pore
where it can interact with either water or permeant anions. However, it
is also possible that Arg-347 may mediate a structural role in the
MSDs; there are a number of negatively charged residues with which
Arg-347 might interact. Both possibilities are consistent with the
present data.
To better understand the role of Arg-347 in CFTR structure and
function, we examined the effect of mutating Arg-347 to cysteine, aspartic acid, glutamic acid, lysine, and leucine on CFTR conductance. We examined the cytosolic pH (pHc)-dependent
behavior of CFTR-R347H and that of the other residue 347 mutants both
with (R347C, R347D, R347E, and R347K) and without (R347L) a
pHc-titratable residue. The conductance of CFTR-R347H is
pHc-dependent. Because the site of protonation may
exist in one of two states, either protonated or deprotonated, we
tested the hypothesis that CFTR-R347H may display two
pHc-dependent conductance states, which it did. If
the protonatable site lines the pore, then the two ionization states of
the protonatable residue might yield two distinct conductance states.
Alternatively, if the protonatable site influences structure, then the
two conductance states might represent two distinct conformational
states of CFTR. Moreover, like CFTR-R347H, all the other residue 347 mutants, except R347K, displayed two pHc-dependent
conductance states over a similar pHc range. The residue at
position 347 did not influence current flow through either conductance
state. These data suggested that residue 347 probably does not line the
pore but likely stabilizes CFTR structure. To pursue this, we studied the effect of mutating residues elsewhere in the MSDs that might interact with Arg-347.
 |
EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis and Transfection--
All mutants were
constructed in the pTM1-CFTR4 plasmid by the method of Kunkel (19). The
mutagenesis was confirmed by restriction digestion of silently
introduced restriction sites and by sequencing around the introduced
mutation site. In vitro transcription and translation of
each mutant was performed to confirm expression of full-length protein.
Wild-type and mutant channels were expressed transiently in HeLa cells
using the vaccinia virus/T7 bacteriophage hybrid expression system as
described previously (20). Cells were routinely studied 12 to 24 h
after infection-transfection.
Patch-Clamp Technique--
Methods used for excised, inside-out
patch clamp recordings were as described previously (21-23). Voltages
were referenced to the extracellular side of the membrane. All studies
were done at room temperature (22-24 °C) to facilitate kinetic
analysis. The membrane potential was clamped at
120 mV unless
otherwise indicated.
CFTR was activated by excising patches into a bath solution
(pHc 7.3; Tricine or TES) containing 1 mM ATP and
75 nM catalytic subunit of cAMP-dependent
protein kinase (Promega Corp., Madison, WI). During the pHc
studies, cAMP-dependent protein kinase was removed, and
bath (cytosolic side) ATP concentration was adjusted (usually to less
than 0.05 mM ATP) to resolve single-channel bursting
activity within multichannel macropatches. For experiments with
excised, inside-out patches, the pipette (extracellular) solution
contained (in mM): 140 N-methyl-D-glucamine (NMDG), 140 aspartic acid,
10 Bis-Tris, 5 CaCl2, 2 MgSO4, pHo 6. The bath (intracellular) solution contained (in mM): 140 NMDG, 10 Bis-Tris, 3 MgCl2, 4 CsOH/1 EGTA, pHc 6.5 (unless indicated) with HCl ([Ca2+]free < 10
8). We selected Bis-Tris as a buffer to avoid the
blocking effect that Good's type buffers such as MOPS have on CFTR
(24). pH was adjusted at room temperature (22-24 °C) using a Model
10 Accumet meter fitted with a gel-filled combination electrode
calibrated with pH 6.0 or 7.0 standardized buffer solutions, where
appropriate (all pH analytical equipment was from Fisher Scientific,
Pittsburgh, PA).
Data Analysis--
Single-channel current amplitudes were
determined from the peaks in all-points histograms. Single-channel
conductances were derived from the slope of the single-channel linear
I-V relationship. Single-channel data were filtered at 1000 Hz using an
8-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA),
digitized at 5000 Hz, and digitally filtered at 500 Hz. The resolution
of the A/D converter was 0.0012 pA. Events lists were generated using a
half-height transition protocol; transitions less than 1 ms were
excluded. For events list generation, a prompt was positioned by eye at
each current level representing the closed state, the little
conductance state (OL), and the big conductance state
(OB). The lifetimes of individual sojourns in
OL or OB were collected, binned (10 bins per
decade), and fitted with a one-component exponential using the maximum
likelihood method. Wild-type CFTR enters a short-lived intraburst
closed state; for simplicity we omitted this state in our analysis of
residue 347 mutants since it is rare in wild-type CFTR in the absence
of MOPS or other blocking buffers (approximately once every 213 ms
within bursts of duration equalling 649 ms; n = 2 at
pHc 6.0,
120 mV, 22-24 °C) on the time-scale of
transitions between the OL and OB state (24).
Single-channel current variance analysis was done as described in the
legend for Fig. 3 and in Ref. 25. Data acquisition and analysis were done using the pClamp software package (Axon Instruments Inc., Foster
City, CA) and Excel 5.0 (Microsoft Corp., Redmond, WA).
 |
RESULTS |
pHc-dependent Conductance of Residue 347 Mutants--
To determine whether R347H exhibits two discrete
conductance states, we studied single channels in excised, inside-out
patches. Fig. 1 shows a representative
single-channel trace from R347H displaying two
pHc-dependent conductance states, OL
and OB. Unexpectedly, all the other variants at residue
347, except R347K, showed two pHc-dependent
conductance states over a similar pH range. Even R347L, which does not
have a titratable side chain, displayed this behavior (Fig. 1). The
conservative substitution of Arg-347 by Lys (R347K) showed only a
single conductance state and no pHc dependence. Similar results
were observed when NMDG-Cl was replaced with NaCl or when Bis-Tris was
replaced with MES (n = 2 each, not shown), suggesting
that this behavior is not due to block by a
pHc-dependent change in one of the pH-titratable
bath reagents, NMDG or Bis-Tris. The pH of the pipette solution did
influence the results (not shown). Also, there was no
state-dependent bias in opening or closing. Channels opened
into or closed from the OL or OB states as
predicted based solely upon pHc.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Single-channel current tracings from excised,
inside-out membrane patches containing the wild-type or indicated
residue 347 mutant channels. All current tracings were collected
with membrane voltage clamped at 120 mV at the indicated
intracellular pHc; the pipette pHo was 6.0 throughout.
All-points histograms were derived from 7-40 s of data using a
binwidth of 9.8 or 19.5 fA and are shown to the right of
current tracings. C refers to closed state.
|
|
The all-points histograms (Fig. 1) illustrate qualitatively that as
pHc increased, each mutant except R347K spent an increased
fraction of time in OL and a correspondingly decreased fraction of time in OB. Points in the histogram from the
zero current state (C), which are largely a function of ATP
concentration (26-28), are included only for purposes of clarity.
Wild-type CFTR and R347K possess only one predominant conductance state
over the pHc range 5.5-7.3 (Fig. 1, data not shown, and Ref. 8). This result suggests that a large positively charged residue at
position 347 (Arg or Lys) in CFTR stabilizes the OB state
relative to the OL state. Visual inspection suggested that
the lifetimes of OL and OB states were also
influenced by the nature of the residue at position 347: R347E and
R347H tended to have longer dwell times in the OL and
OB states, whereas R347L, R347C, and R347D tended to
display shorter dwell times. In general, the smaller the residue, the
more rapid the kinetics. Perhaps steric hindrance imposed by the
residue at position 347 might explain the size dependence of the
kinetics. For R347D, the lifetime in the OB state was so
short that a discrete OB was not apparent on the all-points
histogram; instead, as pHc decreased, a shoulder developed on
the OL state distribution in the all-points histogram (Fig.
1). Since R347L displayed two pHc-dependent
conductance states and since leucine is aliphatic and non-ionizable,
the pHc dependence of residue 347 mutants cannot be attributed
simply to protonation of residue 347.
Single-channel Conductance of Residue 347 Mutants--
To
determine whether the residue at position 347 affects single-channel
conductance and not merely the conductance state of the channel, we
examined the I-V relationship and slope conductance of the mutants with
slower pHc-dependent kinetics, R347H and R347E, as
well as R347K. Filtering obscured the true current amplitudes of the
other pHc-dependent mutants with more rapid
kinetics. Fig. 2 shows that the I-V
relationships and slope conductances for the OL and
OB states were not significantly affected by the nature of
the residue at position 347. The single-channel conductance at
pHc 6.0 of wild-type CFTR, R347K, and the OB state
of R347E and R347H were all very similar (in pS): 7.7 ± 0.4, 8.3 ± 0.6, 7.4 ± 0.4, and 6.9 ± 0.2, respectively
(n = 3 for each). The single-channel conductance of the
OL states of R347E and R347H at pHc 6.0 were also
very similar (in pS): 1.5 ± 0.1 and 1.6 ± 0.1, respectively
(n = 3 and 4 for each). These data suggest that the
amino acid at residue 347 does not affect single-channel current
amplitude, rather the predominant effect is on the lifetime of the
OL and OB conductance states. The lack of
effect of the specific residue at position 347 on the rate of
Cl
flow through the pore in either the OL or
OB conductance state suggests that residue 347 does not
interact directly with permeating anions.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Single-channel I-V relationships for R347E
(OL and OB states), R347H (OL and
OB states), R347K, R347D/D924R, and wild-type CFTR at
pHc 6.0. n = 2-4 at each data
point.
|
|
There are at least two possible explanations for the
pHc-dependent behavior of residue 347 mutants. One
explanation is that mutations at residue 347 reveal the effect of a
protonatable residue in the permeation path that directly interacts
with anions. In this case, the lifetime of the OB and
OL state represent the lifetime of the protonated and
deprotonated ionization states of this unknown site, respectively. A
second explanation is that when Arg-347 is mutated, the
membrane-spanning domains that form the pore fluctuate between two
conformational states, OL and OB; protonation
or deprotonation of some unknown site favors the OB or
OL conformational states, respectively. The first
explanation seems unlikely because the reciprocal lifetime of the
OL state which represents the "on" rate for the proton
is very slow (e.g. it is 6 × 107
M
1 s
1 for R347E). This rate is
~200-fold slower than proton transfer onto an imidazole in free
solution (29, 30) and ~10-fold slower than transfer onto an imidazole
in the pore of ROMK1 (25). The slow rate, however, might be explained
in part by residence of the protonatable site in a sterically and
electrostatically shielded position. In addition, the site of
protonation revealed by a residue 347 mutation would have to perfectly
replace the lost anion-binding site incurred through the mutagenesis;
this seems exceedingly fortuitous. This consideration convinced us to
favor a model involving a conformational change in CFTR.
Dwell-time Analysis of OL and OB
States--
We performed a dwell-time analysis of the lifetimes of the
OL and OB states of R347E and R347H to enable
more quantitative comparisons between them and to better understand
their pHc dependence. Both OL and OB
dwell-time histograms were best fit by a single exponential function at
all pHc values examined. This suggests that the distribution
between OL and OB state does not change
markedly throughout the gating cycle; this can also be observed
visually in Fig. 1. Fig. 3A
shows that the reciprocal lifetime of the OL state
increased relatively linearly with increasing proton concentration.
This indicates that the rate of entry into the OB state
increased with increasing proton concentration. This linear, first
order dependence on proton concentration suggests that protonation of
only a single site limits the rate of movement from the OL
to the OB state. The rate of exit from the OB
state decreased non-linearly with increasing proton concentrations. The
kinetics and the equilibria between OL and OB
were very similar for both mutants (Fig. 3A). The observable
pK (0 mV) for the equilibrium between OL and
OB of R347E and R347H were 6.4 and 6.3, respectively. The
faster kinetics of R347D, R347C, and R347L made dwell-time analysis for
these mutants less reliable. Therefore, to obtain an estimate of the
approximate pK of these mutants, we examined changes in the
variance of the open-state current with changes in pHc (25).
The current variance should go through a maximum when the pHc
equals the observable pK. Each mutant displayed an increase
in current variance with increasing proton concentration
(i.e. decreasing pH). Fig. 3B shows that R347C, R347D, and
R347L did not reach a peak variance over the range of pHc
studied, suggesting that their apparent pK is less than
5.0-5.5. Visual inspection of the tracings suggested that the mutants
were largely in their OL state over the range of
pHc employed such that we did not miss a peak in the variance.
In this analysis, we assumed that the mutants would display minimal
pH-independent movement between OL and OB
states at very acidic pHc so that the variance should pass
through a maximum as pHc decreases. As a control for the
variance analysis, we examined the R347E mutant on which we had also
done dwell-time analysis (Fig. 3A); as expected, Fig.
3B shows that the variance of R347E goes through a maximum
between pHc 6.5 and 5.5.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
pHc dependence of residue 347 mutants. A, dwell-time analysis in the OL
and OB conductance states versus pHc for
R347E and R347H. n equals 3-7 for each. B,
open-channel current variance of the R347C, R347D, R347L, and R347E
mutants versus pHc. Open-channel current variance
was calculated by subtraction of current variance in the
closed state ( c2) from current
variance in the open state ( o2). Each data point
was derived from 2-4 excised, inside-out membrane patches; in general,
10 measurements ( o2 c2) were averaged per patch and were obtained
from individual openings and adjacent closed periods. Error bars are
smaller than the symbols.
|
|
Voltage Dependence of OL and OB--
To
learn more about the nature of the two conductance states of residue
347 mutants, we examined the voltage dependence of the OL
and OB lifetimes. Fig.
4A shows qualitatively that
both conductance states of R347E were voltage-dependent.
Fig. 4B shows quantitatively that at pHc 6.0 the
OL and OB states for R347E and R347H were both
influenced by the transmembrane voltage. The voltage dependence may
derive from alterations in the distribution of protons near the
protonatable site or from charged regions of CFTR moving through the
voltage field. We assumed the Boltzman distribution to quantify the
effect of voltage on the equilibrium between OL and
OB states such that
|
(Eq. 1)
|
in which
= electrical distance from the cytosolic surface,
z = valence, F = Faraday's constant,
E = transmembrane potential, r = gas
constant, T = temperature, pK = the
negative log of the equilibrium constant between OL and
OB conformations. The degree of voltage dependence was
similar for both mutants despite the charge differences at residue 347 and yielded a
z of 0.25 and 0.21 for R347E and R347H,
respectively. The voltage dependence was asymmetrically disposed
between the rate of entry into the OB state and the rate of
exit from OB (Fig. 4B). To quantify these differences we used the following:
|
(Eq. 2)
|
|
(Eq. 3)
|
in which
= symmetry factor which partitions voltage dependence
(31, 32). The rate of exit from the OB state
(
B
1) was more
voltage-dependent than the rate of exit from the
OL state (
L
1) for both mutants
(
= 0.8 versus 1
= 0.2 for R347E and
= 0.7 versus 1
= 0.3 for R347H). This observation
may be explained by the protonatable site moving through the voltage
field; protonation and deprotonation of this site before and after the
conformational change will affect the net charge migrating through the
applied voltage. Since voltage dependence arises from charge movement through a voltage field and since R347E and R347H displayed similar voltage dependences and carry different charges at position 347, residue 347 is not likely moving through a transmembrane potential during interchange between OL and OB
states.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 4.
Voltage dependence of OL and
OB states. A, current records from excised,
inside-out membrane patch containing R347E channel. Traces were
collected at pHc 6.0 and pHo 6.5 at the indicated
transmembrane voltage. B, dwell times (pHc 6.0) in
the OL state (closed symbols) or
OB state (open symbols)
versus voltage for R347E (left,
circles) and R347H (right, squares).
Error bars are smaller than the symbols. n equals
4-6 for each.
|
|
The Phenotype of R347D Is Suppressed by the D924R
Mutation--
The data suggest that Arg-347 and Lys-347 may stabilize
the structure of the pore; in their absence, the channel "flickers" between two conductance states. Arginine and lysine residues through electrostatic interactions with anionic residues are important for the
structure of membrane-spanning domains in other proteins such as the
Lac permease and the inward-rectified K+ channel, IRK1
(33-35). As discussed by Perutz (18), salt bridges within proteins are
an important structural feature that confers thermostability and
resistance to denaturation. We hypothesized that Arg-347 may mediate a
stabilizing influence by contributing to a salt bridge within the MSDs.
There are multiple glutamates and aspartates within transmembrane (M)
regions with which Arg-347 or Lys-347 might interact: Glu-92 (M1),
Glu-873 (M7), Asp-924 (M8), Asp-993 (M9), and Glu-1104 (M11). To
identify the Arg-347 interaction partner, we replaced Arg-347 with an
anionic residue (R347E or R347D) and introduced an arginine residue in
the place of candidate partners in a salt bridge. We studied the
conductance properties of the following double mutants: R347D/D924R,
R347D/D993R, and R347E/E1104R. The R347D/D993R and R347E/E1104R mutants
each had two conductance states with pHc-dependent
behavior (Fig. 5). For R347D/D993R the
increased entry into the OB state was apparent as a
shoulder on the amplitude histogram at pHc 5.5. Accordingly,
for R347D/D993R and R347E/E1104R the current variance in the open state
increased with decreasing pHc (Fig. 5B).
Qualitatively, the lifetimes of the OL and OB
conductance states in the R347D/D993R and R347E/E1104R were similar to
that of the R347D and R347E mutants, respectively. The amplitude of the
OL state was larger for both of these double mutants as
compared with the single mutants (Figs. 1B and
5B). We also observed an infrequent, additional small
conductance state in the R347E/E1104 mutant (see amplitude histogram in
Fig. 5A); this is likely due to the E1104R mutation
itself.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
A, single-channel current tracings from
excised, inside-out membrane patches containing R347E/E1104R,
R347D/D924R, and R347D/D993R. Membrane voltage was 120 mV, and
pHc is indicated; pipette pHo was 6.5 throughout.
All-points histograms were derived from 6-39 s of data using a
binwidth of 9.8 or 19.5 fA and are shown on the right.
B, current variance of R347E/E1104R, R347D/D924R, and
R347D/D993R at the indicated pHc was collected as in Fig. 3.
Each data point was derived from 2-3 patches. Error bars are smaller
than the symbols.
|
|
In contrast to the other double mutants, the R347D/D924R mutant did not
display the pHc-dependent flicker found in the
R347D single mutant (Fig. 5, A and B), and there
was no effect of pH on open-channel variance (Fig. 5B). The
single-channel conductance was similar to that of wild-type CFTR
(6.1 ± 0.1 pS; n = 3; Fig. 2). These data suggest
that the D924R mutation compensates for or rescues the phenotype of the
R347D mutation. This result predicts that the D924R mutation alone
(with Arg at position 347) would generate an unstable channel with at
least two open conductance states. Fig. 6
shows that the D924R single mutant displayed multiple (~3)
conductance states that appeared to be pHc-independent.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
Single-channel currents from the D924R
variant. Currents were obtained at indicated pHc with
membrane voltage maintained at 120 mV. Similar results were obtained
in three patches.
|
|
 |
DISCUSSION |
Function of Arg-347 in CFTR--
Previous work from our and
Hanrahan's laboratories (7, 8, 17) has led to the speculation that
Arg-347 may be an anion-binding site in the CFTR pore. However in
contrast to earlier interpretations, our current data show that residue
347 does not influence permeation properties via a direct interaction
with permeating anions. Instead they suggest that Arg-347 may be more
important for maintenance of pore architecture.
We found that mutation of residue 347 to glutamate, aspartate,
cysteine, histidine, or leucine all produced channels with two distinct
conductance states, OL and OB. pHc, and voltage influenced the movement between these two states over a similar
range for all mutants. Additionally, the single-channel slope
conductances of R347H and R347E were the same in both OB and OL states. The OB state for each had the
same conductance as wild-type CFTR. The average calculated
pKa for a glutamate and a histidine within a protein
are 4.0 and 6.9 (36), respectively. If His-347 or Glu-347 line the
permeation pathway, at pHc 6.0 (bath solution) or pHo
6.5 (pipette solution) His-347 is predicted to be protonated and to
have at least a partial positive charge, and Glu-347 should be fully
deprotonated and have a negative charge. Since the charge or the
structure of the residue at position 347 failed to affect
single-channel conductance, it seems unlikely to be either an
anion-binding site or to be positioned such that it interacts
sterically or electrostatically with permeating anions. Thus, mutation
of Arg-347 may decrease single-channel conductance and anomalous
mole-fraction behavior by disrupting pore architecture and the function
of some other anion-binding site(s).
The OB and OL Conductance States--
All
the mutants except R347K showed two pH-dependent
conductance states. The equilibria between OB and
OL states were similar despite the nature of the mutation
at residue 347 with values of pK ranging from
~5-7.3 Because the values
of pK were roughly similar, the data suggest that the
structure, titratability, and pK of residue 347 do not markedly influence the distribution between OL and
OB states. The linear dependence on proton concentration
for OB entry suggests that a single protonatable site
largely determines entry into this state.
The OB and OL states may represent two
protonation states of the CFTR molecule or two distinct conformational
states that are influenced by protonation. For the reasons discussed
above, we favor the latter possibility, that alternating residence in two discrete conformations is responsible for the two conductance states. How might this occur? We hypothesize that Arg-347 forms a salt
bridge with another negatively charged residue in CFTR. Mutation of
Arg-347 would leave a negatively charged residue unpaired within the
membrane. Charged molecules within a low dielectric constant are
unstable (37). Therefore, the protein might reorient to solvate the
charge, i.e. enter the OL state. Subsequent
protonation of that site would neutralize its charge and allow the
protein to reorient back to its native conformation, i.e.
return to the OB state.
This model suggests that the residue(s) with which Arg-347 interacts
may be the protonatable site. Several observations are consistent with
this hypothesis. First, we found that a positively charged lysine,
which can support a salt bridge, was able to supplant arginine.
However, the other residues tested were not able to substitute for
arginine. Perhaps histidine was not able to replace arginine because
its side chain was not long enough or it may have an anomalously acidic
pKa within the low dielectric constant of the membrane.
Second, and more importantly, we found that a second-site complementary
mutation at position 924 (D924R) largely eliminated the
pHc-dependent flickering phenotype of the R347D
mutation and restored current amplitude to near wild-type values. We
also recognize the possibility that Arg-347 may interact with
additional yet untested residues, e.g. Glu-873 in M7.
Third, a salt bridge between Arg-347 and Asp-924 should be disrupted by
mutation of Asp-924. As predicted, we found that the D924R mutant
displayed erratic flickery, pHc-independent behavior.
Presumably, this mutation also generates an unstable channel. The
pHc independence of D924R is also consistent with the
hypothesis that Asp-924 is the site of protonation in the residue 347 mutants. Interestingly, mutation of a glutamate in the putative salt
bridge in the P-loop of the IRK1 channel leads to a single-channel
flickering phenotype reminiscent of residue 347 mutations (35).
Tertiary Structure of the Membrane-spanning Domains--
At the
simplest level, the data suggest that both MSDs functionally interact
in a manner that influences permeation. The studies of R347D/D924R are
consistent with a salt bridge between Arg-347 and Asp-924 and thus an
interaction between M6 and M8. This further suggests that mutations in
MSD2 may alter the phenotype of mutations in MSD1. Consistent with
this, mutation of D993R and E1104R in MSD2 increased the relative
amplitude of the OL conductance state in the context of
Arg-347 mutations.
The Arg-347 residue is targeted by several CF-associated mutations,
R347C, R347H, R347L, and R347P (13-15).2 Our data suggest
that CF-associated as well as other mutations at residue 347 affect
CFTR similarly. They disrupt pore architecture by disrupting an
interaction between Arg-347 and another residue(s), one possibly being
Asp-924 in M8. These data highlight the importance of residue 347 for
CFTR function and may explain in part why this residue is targeted by
multiple CF-associated mutations.