Block by MOPS reveals a conformation change in the CFTR pore
produced by ATP hydrolysis
Hiroshi
Ishihara and
Michael J.
Welsh
Howard Hughes Medical Institute, Departments of Internal Medicine
and Physiology and Biophysics, University of Iowa College of
Medicine, Iowa City, Iowa 52242
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ABSTRACT |
ATP hydrolysis by the cystic fibrosis transmembrane
conductance regulator (CFTR)
Cl
channel predicts that
energy from hydrolysis might cause asymmetric transitions in the
gating cycle. We found that 3-(N-morpholino)propanesulfonic acid (MOPS) blocked the open channel by binding to a site 50% of the
way through the electrical field. Block by MOPS revealed two distinct
states, O1 and O2, which showed a strong asymmetry during bursts of
activity; the first opening in a burst was in the O1 state and the last
was in the O2 state. Addition of a nonhydrolyzable nucleoside
triphosphate prevented the transition to the O2 state and prolonged the
O1 state. These data indicate that ATP hydrolysis by the
nucleotide-binding domains drives a series of asymmetric transitions in
the gating cycle. They also indicate that ATP hydrolysis changes the
conformation of the pore, thereby altering MOPS binding.
chloride channel; gating; cystic fibrosis transmembrane conductance
regulator; buffer; anion; 3-(N-morpholino)propanesulfonic
acid
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INTRODUCTION |
THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) is an epithelial Cl
channel with complex regulation (7, 22, 29). Amino acid sequence
analysis, biochemical characterization, and functional studies indicate
that CFTR is composed of five domains: two membrane-spanning domains
(MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain
(RD). The MSDs, which are each composed of six transmembrane sequences,
contribute to the formation of the
Cl
conducting pore. The RD
contains a number of consensus phosphorylation sites; phosphorylation
of the RD by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is required for the channel to
open.
The NBDs in CFTR hydrolyze ATP to control channel activity. This
conclusion is based on several findings:
1) hydrolyzable forms of MgATP are
required to open the channel, 2)
several nucleotide species and inorganic phosphate analogs alter
channel gating in specific ways, 3)
site-directed mutations in the NBDs produce specific alterations in
channel gating, and 4) biochemical
studies show that CFTR and an isolated NBD1 function as ATPases by
hydrolyzing ATP (1-3, 6, 9, 14-16, 23, 30). This hydrolysis
is required for the channel to open (1). As first suggested by
Baukrowitz et al. (3), ATP hydrolysis is also required for the channel to close. On the basis of the effect of site-directed mutations in key
residues in NBD1 and NBD2 and on the effect of agents such as
5'-adenylylimidodiphosphate (AMP-PNP),
pyrophosphate, and orthovanadate, it has been proposed
that ATP hydrolysis at NBD1 opens the channel into a burst of activity
and that ATP hydrolysis at NBD2 terminates the burst and closes the
channel (4, 5, 8).
The input of energy from ATP hydrolysis predicts that CFTR will have
discrete conformations that do not exist at thermodynamic equilibrium.
Moreover, the sequence of ATP hydrolysis at the two NBDs predicts that
the channel will progress through an ordered, relatively irreversible
series of distinct conformational states. Previous studies with the
patch-clamp technique have identified two conformations of the protein,
which are resolved as open and closed states. However, with only two
recognizable gating conformations, open and closed, it is not possible
to observe directly asymmetric transitions in the gating cycle. An
indication that additional conformations exist comes from the recent
observation of Gunderson and Kopito (9) that the open state may have
two discrete conductances. We found that the buffer
3-(N-morpholino)propanesulfonic acid (MOPS) produced a
flickery block of the channel that allowed us to discern additional
conformations of the channel linked to ATP hydrolysis.
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MATERIALS AND METHODS |
Cells and CFTR expression systems.
We used two different expression systems to study wild-type CFTR: HeLa
cells transiently expressing CFTR using the vaccinia virus-T7 RNA
polymerase expression system and C127 mouse mammary epithelial cells
stably expressing CFTR. We have previously described both systems (18,
21). Data obtained from both cell types were identical and were
combined for analysis.
Chemicals and solutions.
MOPS was obtained from Fisher Scientific (Fair Lawn, NJ). The catalytic
subunit of PKA was from Promega (Madison, WI). All other reagents were
from Sigma Chemical (St. Louis, MO). For excised, inside-out
patch-clamp experiments, both sides of the membrane were bathed with a
solution containing 140 mM
N-methyl-D-glucamine chloride and 5 mM MgCl2 and
buffered with the indicated concentrations of MOPS or 10 mM tricine (pH
7.3). To activate CFTR, 75 nM of PKA and 1 or 3 mM of
Na2ATP were added to bath
(cytoplasmic) solution; PKA was present for all conditions.
Patch-clamp methods and data analysis.
An Axopatch 1C amplifier (Axon Instruments, Foster City, CA) was used
for voltage clamping and current amplification. A microcomputer and the
pCLAMP software package (version 6.0.1, Axon Instruments) were used for
data acquisition and analysis. Currents were recorded on videotape
following pulse-code modulation with a PCM-2 analog-to-digital videocassette recorder adapter (Medical System, Greenvale, NY) for
later analysis. The methods used for the experimental setup and the
excised, inside-out patch configuration were previously described (4,
6). Voltages are referenced to the extracellular side of the membrane.
Bath temperature was maintained by a temperature-controlled microscope
stage (Brook Industries, Lake Villa, IL).
Replayed data were filtered at a 1-kHz corner frequency with a variable
eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA) and
digitized at 10 kHz. For analysis, data were digitally filtered with
pCLAMP software using a Gaussian filter at either 500 Hz
("lightly" filtered) or 10 Hz ("heavily" filtered). These
records were used to make all-points histograms for determination of
current amplitude and to make events lists for open- and closed-time analyses. Transitions of <1 ms in duration were excluded. In some cases, current amplitudes of the O1 and O2 state were measured manually
in the heavily filtered records. Single-channel open- and closed-time
histograms were plotted from the idealized records with a logarithmic
x-axis with 10 bins/decade. Histograms
were fit with a one or more component exponential function using the maximum likelihood method. Burst analysis was performed from the idealized records as described previously (4), using a discriminator of
20 ms to separate interburst closures from intraburst closures. This
value was determined from the single-channel recording closed-time histogram of wild-type CFTR activated by 1 mM ATP and 75 nM PKA in 10 mM tricine solution at 25°C. Similar values were obtained with
other conditions.
In experiments in which the membrane patch contained more than one
channel, regions of data with no superimposed openings were used for
burst analysis (4). There was no statistical difference between burst
durations derived from patches with only one active channel compared
with patches with more than one active channel nor was there any trend
toward an increase or decrease of lifetimes by inclusion of data from
patches with more than one channel.
Results are expressed as means ± SE of
n observations. Statistical
significance was assessed with a paired or unpaired Student's t-test and a log-likelihood ratio
test, where appropriate. P values of
<0.05 were considered statistically significant.
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RESULTS |
Two open-gating states in CFTR studied in solutions containing MOPS.
Figure 1 shows recordings of several bursts
of activity obtained from an excised, inside-out patch of membrane that
contained a CFTR Cl
channel. Within a burst of activity, we often observed two different gating patterns. For example, in trace
a in Fig. 1, during the first two-thirds of the burst
the channel showed a flickery pattern of gating in which the open state
was frequently interrupted by very short closings. In contrast, during
the last one-third of the burst the open state was less frequently
interrupted. For convenience, we refer to the time that the channel is
in the flickery gating pattern as the O1-gating state and the time that
it shows the less flickery pattern as the O2-gating state. In general, most bursts of activity contained both patterns of gating. However, traces a-e in Fig. 1 show the
gating pattern was variable. Inspection of the tracings also indicates
that rigorous discrimination between the O1 and O2 states was difficult
for several reasons. First, the closures and openings in the O1 state
were very short. The short duration plus the small single-channel
current made accurate measurements difficult. Second, the O2 state also
contained short closures, although they appeared to be less frequent
than in the O1 state (e.g., note traces
a-d). Third, the relative durations of the O1
and O2 states were variable; for example, the flickering O1 state was a
small proportion of the burst shown in trace
b but occupied most of the burst in
trace e. These considerations made it
difficult to be certain whether both states were always present within
a burst; for example, trace e might
have been composed only of the O1 state or it may have ended in the O2
state. Therefore, as a way of qualitatively evaluating the gating
behavior, we examined the effect of filtering the data. At the bottom
of Fig. 1, we show the recording from which traces
a-e were obtained after they were filtered at 10 Hz. In this heavily filtered recording, the very rapid kinetics are not
resolved and the observed current is a mean value, reflecting the
proportion of time that the channel spent in the open and closed
states. Thus the O1 state "appears" to have a lower conductance
than the O2 state. The two gating states we observed likely explain the
two conductance states reported by Gunderson and Kopito (10) in which
they heavily filtered the data from CFTR studied in planar lipid
bilayers.

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Fig. 1.
Examples of cystic fibrosis transmembrane conductance regulator (CFTR)
Cl channel gating in the
presence of MOPS. Top: tracings were
filtered at 500 Hz and were taken at positions indicated by letters
a-e in bottom trace (which was filtered at
10 Hz). Tracings were obtained from excised, inside-out patches.
Holding potential was 80 mV, and temperature was
25°C. Scale bars are shown. Dashed lines indicate closed (C) state;
O1 and O2 states are indicated in bottom
trace. cAMP-dependent protein kinase was present in the
bath solution of this and all subsequent experiments.
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The flickery pattern of gating suggested the possibility of channel
block by something in the solution. Our solutions contained 10 mM MOPS
as a buffer, and previous reports have suggested that related buffers
such as
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) can block other
Cl
channels (11, 32).
Therefore, we substituted tricine for MOPS because it did not block an
outwardly rectifying Cl
channel (11). With 10 mM tricine as the buffer (Fig.
2A), the gating pattern resembled only the O2 state; we saw none of the fast
flickery behavior observed in the O1 state with MOPS as the buffer
(Fig. 1). Instead, gating resembled what we observed at positive
voltages with MOPS (see Fig. 4).

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Fig. 2.
Examples of CFTR Cl channel
gating in the presence of 10 mM tricine at 25°C
(A), 10 mM MOPS at 15°C
(B), and 10 mM MOPS at 35°C
(C). Tracings shown at
top (of
A-C)
were filtered at 10 Hz, and data at
bottom (of
A-C)
were filtered at 500 Hz. * Site of bottom
traces. See legend of Fig. 1 for other details.
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In the past, we had not observed two different gating patterns;
however, we usually studied CFTR at 35-37°C. Therefore, we studied patches under conditions identical to those used in Fig. 1A, except that temperature was
varied. When temperature was reduced to 15°C, both the O1 and O2
states were readily apparent and the duration of the O2 state appeared
to be prolonged (Fig. 2B). In contrast, when temperature was increased to 35°C, we could not resolve a distinct O2 component (Fig.
2C); the records showed only fast
flickery behavior similar to that observed in the O1 state at 25°C.
It seems probable that channels studied in MOPS buffer at 35°C also
have two open states. However, perhaps because the duration of the O2
state decreases as temperature increases, we could not distinguish O2
and O1. Of note, the conditions under which the two gating states were
readily apparent, temperatures less than 35°C and solutions that
contain MOPS, were the same as those employed by Gunderson and Kopito
(10) when they reported two conductance states.
Previous studies performed under different conditions have shown that
open times for CFTR Cl
channels were well fit by a single exponential function (30). Figure
3 shows that, when we used tricine as the
buffer at 25°C, the open times were well fit with a single
exponential function. In contrast, when we used 10 mM MOPS as the
buffer, open-time histograms were fit better by two than by one
exponential function. This suggests the presence of two populations of
open times. Table 1 shows values for short
and long open times within bursts of activity as well as closed times.
The data suggest that there are two closed times within bursts of
activity, but, because the short closed time is near the limits of
resolution for our recordings, we may have underestimated their
occurrence and, consequently, absolute values of open times. The
average open time in tricine was longer than in MOPS, primarily because
of a paucity of short closings. With both MOPS and tricine, a reduction
in temperature prolonged the long open time (Table 1).

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Fig. 3.
Examples of open-time histograms obtained from single-channel
recordings used to produce the tracings in Figs. 1 and 2,
A and
B. Tracings were obtained in 10 mM
tricine at 25°C (A) or 10 mM
MOPS buffer at 25°C (B) or
15°C (C). Data are open
dwell-time analysis of 1 channel. Line shows the fit to a single
exponential in the case of 10 mM tricine and a double exponential for 2 tracings shown with MOPS buffer. With tricine as the buffer
(A), use of 2 exponential functions
did not produce a significantly better fit than a single exponential
function (n = 5). With MOPS as the buffer, in 6 of 6 cases at 25°C
(B) and 4 of 4 cases at 15°C
(C), open-time histograms were fit
better by 2 than by 1 exponential function. Note the different scale of
the x-axis for 10 mM tricine (A).
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MOPS block of CFTR Cl
channels.
To evaluate further the possibility that MOPS blocks the channel, we
examined the effect of voltage on single-channel currents. Figure
4 shows data filtered at 500 Hz
(left) and data filtered at 10 Hz
(right). At a voltage of +80 mV, the
flickering pattern of gating with short closures was not observed.
However, as the voltage became more negative, the short closings and
flickering gating behavior of the O1 state became more and more
prominent. This was readily apparent after the data were heavily
filtered.

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Fig. 4.
Effect of voltage on gating in the presence of 10 mM MOPS at 25°C.
Tracings on left were filtered at 500 Hz, and tracings on right show the
same data filtered at 10 Hz.
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Figure 5A
shows current-voltage
(I-V)
relationships for channels studied in tricine at 35 and 25°C. The
I-V
relationship of lightly filtered (500 Hz) data was linear, and the
slope conductance was greater at 35°C (11.0 ± 0.21 pS,
n = 3) than at 25°C (7.9 ± 0.02 pS, n = 4, P < 0.0001). Figure
5B shows the
I-V
relationship of channels studied in 10 mM MOPS at 25°C. When the
data were lightly filtered at 500 Hz, the
I-V
relationship was relatively linear with slight rectification at the
most negative voltages. After the data were filtered at 10 Hz, there
was a clear distinction between the O1 and O2 states, with the O1 level
showing greater rectification. At positive voltages, the
I-V
relationship was linear with a slope conductance of 7.4 ± 0.1 pS
(n = 4) at 25°C and 10.2 ± 0.5 pS (n = 4, not shown in Fig.
5B) at 37°C. These conductance
values are similar to those obtained from channels studied in tricine.

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Fig. 5.
Current-voltage relationships of channels studied in tricine and MOPS.
A: buffer was 10 mM tricine at
25°C ( ) and 35°C ( ). Data were filtered at 500 Hz;
n = 3-5.
B: channels were studied at 25°C
with 10 mM MOPS buffer. Recordings of O1 and O2 current levels were
filtered at 10 Hz ( and , respectively). , Current-voltage relationship of data filtered at 500 Hz;
n = 2-5 for each. In most cases,
SE bars are hidden by symbols.
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We also examined the effect of increasing concentrations of MOPS (Fig.
6). At 0.5 mM MOPS, there was only a small
amount of flicker in the lightly filtered tracing. However, both O1 and O2 states were apparent. As the concentration of MOPS increased to 25 mM, the fast flickery behavior became more prominent. In addition, when
the data were filtered at 10 Hz, the distinction between O1 and O2
states became more obvious as the concentration of MOPS increased. The
single-channel
I-V
relationships for the heavily filtered data are shown in Fig.
7, A and
B. As the MOPS concentration
([MOPS]) increased, the
I-V
relationships for both O1 (A) and O2
(B) states showed increasing
rectification, with the effects more pronounced for the O1 level.

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Fig. 6.
Effect of concentration of MOPS. Left:
records filtered at 500 Hz at the indicated concentration of MOPS.
Right: records filtered at 10 Hz.
Traces at left were obtained at the
points indicated by asterisks. Temperature was 25°C.
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Fig. 7.
Effect of concentration of MOPS on current-voltage relationships
(A and
B) and dissociation constant
(Kd;
C and
D). Data for the O1 state are in
A and
C, and data for the O2 state are in B and
D. Temperature was 25°C, and
voltage was 80 mV. , 25 mM MOPS. , 10 mM MOPS. , 5 mM
MOPS. Data are means ± SE of 4-5 experiments for each
condition. Data were filtered at 10 Hz. In most cases, error bars are
hidden by symbols. Kd at 0 mV
[Kd(0)] for the O1 state
was 71 mM, and electrical distance sensed by MOPS ( ) was 0.50 ± 0.01. Kd(0) and for
the O2 state depended on the MOPS concentration: with 25 mM MOPS they
were 89 mM and 0.25 ± 0.01, and with 10 mM MOPS they were 261 mM
and 0.54 ± 0.02, respectively.
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The flickery pattern of gating in the O1 state at hyperpolarized
voltages is consistent with classic open channel block. We calculated
the voltage-dependent dissociation constant
(Kd) for MOPS
inhibition using the single-channel current amplitude of heavily
filtered data in the O1 state as follows
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(1)
|
where
Kd(V)
is the voltage-dependent
Kd at voltage
V,
iMOPS is the
single-channel current of the O1 state in the presence of MOPS, and
i0 is the
single-channel current in the absence of MOPS (with tricine as the
buffer), respectively. Figure 7C shows that Kd was
strongly voltage dependent and the predicted
Kd at 0 mV was
71 mM.
Voltage dependence suggests that MOPS binds within the electric field
of the membrane. The electrical distance sensed by MOPS (
) can be
calculated with the relationship (31)
|
(2)
|
where
z is the valence of MOPS, which is
assumed to be
1 (see Fig. 8),
Kd(0) is the Kd at 0 mV, and F,
R, and
T represent Faraday's constant, the
gas constant, and absolute temperature, respectively.
Assuming a single binding site for MOPS, we calculated that
= 0.50 ± 0.01 (n = 5) measured over the
voltage range of
120 to
40 mV.
Comparisons of the tracings in Fig. 1 with those in Fig.
2B and the I-V relationships in Fig.
7B with those in Fig. 5A indicate that MOPS also
affected the channel in the O2 state. Therefore, we calculated the
voltage-dependent Kd for O2. Figure
7D shows that the Kd for the effect
of MOPS on the O2 state was voltage dependent. However, when the
concentration of MOPS changed,
varied. These results indicate that
the effect of MOPS in the O2 state is not consistent with a simple
voltage-dependent block of an open channel as described by Woodhull
(31). Thus MOPS block of CFTR in the O2 state is different from the
open channel block observed in the O1 state.
Channel block by the anionic form of MOPS.
MOPS is a buffer that exists in solution as the zwitterion
(MOPS0) or the anion
(MOPS
), and the ratio
depends on the pH. To determine whether
MOPS0 or
MOPS
produced the rapid
flickery block in the O1 state, we used the equation pH = pKa + log([MOPS
]/[MOPS0]),
where pKa, the
dissociation constant of MOPS, is 7.2 at 25°C. We altered the pH
and the total [MOPS] to vary the concentration of
MOPS
and
MOPS0. Figure
8A shows
that an increase in the concentration of
MOPS
had a greater effect
on gating than did MOPS0. For
example, increasing the
[MOPS
] from 2.8 to 13.9 mM at a constant pH of 7.3 markedly increased the flickery
block (compare the first and second trace in Fig. 8A). Likewise, increasing the
[MOPS
] from 2.8 to 13.8 mM at a nearly constant total [MOPS] had a similar
effect (compare the third and fourth trace in Fig.
8A). The effect of
[MOPS
] may be
more readily apparent from examination of current amplitude of data
obtained at negative voltages and filtered at 10 Hz (Fig. 8B). When
[MOPS
]
increased, the current amplitude in the O1 state decreased, whereas
changes in the concentration of
MOPS0 or in the pH did not
correlate with effects on current amplitude. We obtained similar
results when we examined the effect of
MOPS
on the O2 state. Our
previous unpublished observations have suggested little effect on
gating of pH changes in this range. These data suggest that
MOPS
, rather than
MOPS0, blocked the channel in both
the O1 and the O2 states, results consistent with a voltage-dependent
effect.

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Fig. 8.
Effect of MOPS (anion) and
MOPS0 (zwitterion).
A: example of traces obtained at
different MOPS and
MOPS0 concentrations produced by
variation of total MOPS concentration and pH. Specific conditions are
indicated above each tracing. All tracings were obtained at 25°C
and 80 mV. B: current amplitude of heavily filtered data for O1 (solid bars) and O2 state (open bars)
under same conditions as in A.
Concentrations of MOPS ,
MOPS0, total MOPS, and pH are
indicated. Data are means ± SE of 3-5 experiments for each
condition.
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To learn whether MOPS was able to block the channel from one or both
sides, we substituted tricine as the buffer in either the intracellular
or extracellular solution. Figure 9 shows
that the flickery block was evident when MOPS was in the internal
(bath) solution alone. In contrast, when only the external solution
contained MOPS, flickery block was not observed at either
hyperpolarizing or depolarizing voltages. These results indicate that
MOPS can access its binding site only from the internal surface of the channel.

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Fig. 9.
Effect of addition of MOPS to the intracellular or extracellular
surface of the patch. Examples were obtained at 25°C and 80
or +80 mV as indicated. MOPS was applied to the intracellular (bath) or
extracellular (pipette) surface with the indicated concentrations of
MOPS or tricine. Similar results were obtained in 3 other experiments. Records were filtered at 500 Hz.
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Sequential passage of the channel through O1 and O2 states.
As is evident from an examination of Figs. 1,
2B, 4, 6,
8A, and 9, bursts of activity usually
started with the flickery O1 state and ended with the O2 state. To
evaluate this pattern more objectively, we measured the duration of the
first opening and the last opening within a burst using pCLAMP
software. Table 2 shows that the first
opening was significantly shorter than the last opening. Moreover, the
average durations of the first (7.3 ms) and last (26.1 ms) openings
were very close to the short (6.1 ms) and long (31.7 ms) open times
obtained from the open-time histograms (Table 1). These data indicate
that bursts of activity usually began with the O1-gating state and
ended in the O2-gating state. In contrast, for channels studied in 10 mM tricine at 25°C, the average duration of the first opening was
not significantly different from that of the last opening. We could not
distinguish a difference between the first and last openings for
channels studied in 10 mM MOPS at 35°C, but, as indicated above,
our resolution is limited under those conditions.
AMP-PNP inhibits the transition from the O1 to the O2 state.
The finding that the O1 state usually preceded the O2 state indicates
that the two states are not randomly distributed but rather have a
specific order in their occurrence. This would require energy input
that would probably come from ATP hydrolysis by the NBDs. To evaluate
this possibility further, we examined the effect of AMP-PNP. Previous
studies have shown that, in the presence of ATP and PKA, AMP-PNP
increases the duration of a small fraction of the bursts of activity
(4, 14). The fact that AMP-PNP affects only a fraction of bursts is
consistent with the observation that AMP-PNP competes poorly compared
with ATP in inhibition of 8-azidoadenosine 5'-triphosphate
photolabeling of CFTR (28). Previous work with site-directed mutants
has suggested that hydrolysis at NBD2 terminates a burst of activity.
These results suggest that AMP-PNP binds to NBD2 and, because it cannot
be hydrolyzed, prevents the hydrolysis that ultimately terminates a
burst and closes the channel. When we added 1 mM AMP-PNP to a channel
studied in a 10 mM MOPS solution at 25°C, we observed occasional
long bursts of activity (Fig. 10), as we
and others had previously reported (4, 14). The heavily filtered
tracings in Fig. 10 (top) show that
when the channel was in a long burst of activity it was in the O1
state, whereas bursts of normal duration exhibited both O1 and O2
states. The tracings in Fig. 10
(bottom) show fast flickery behavior
during the long AMP-PNP-induced bursts. These data suggest that the
transition from the O1 to O2 state requires ATP hydrolysis.

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Fig. 10.
Effect of 5'-adenylylimidodiphosphate (1 mM) on single-channel
currents. Solution contained 10 mM MOPS and 1 mM ATP. Experiments were
performed at 25°C and 80 mV.
Top: records filtered at 10 Hz.
Bottom: records were filtered at 500 Hz and were obtained at the position indicated by
a and
b. Similar results were obtained in 2 other experiments.
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DISCUSSION |
MOPS blocks CFTR Cl
channels.
Our data indicate that MOPS blocked CFTR
Cl
channels by two
different mechanisms, manifest as the O1- and the O2-gating states. In
both cases, block was concentration dependent and was due to the
anionic form of MOPS
(MOPS
) rather than the
protonated form (MOPS0). The
data suggest that MOPS was not able to pass through the channel because
it did not block when added to the extracellular surface. These
findings are consistent with the voltage dependence: block was only
observed at negative voltages that would drive MOPS
from the intracellular
solution into the channel. In the O1 state, MOPS produced a flickering
block with frequent short closures characteristic of classic
open-channel block. When the data were heavily filtered, the O1 state
appeared to have a reduced single-channel conductance that reflected
the proportion of time the channel spent in the open and closed states.
In the O2 state, block was not resolved as discrete closings, probably
because the kinetics were too fast to be resolved with our recording
system. However, as in the O1 state, block in O2 was voltage dependent
and appeared as a decreased conductance at negative voltages. Thus
block in the O1 state corresponds to block of "intermediate"
speed and block in the O2 state may correspond to "very fast"
block as described by Hille (13).
The voltage-dependent
Kd for block in
the O1 state suggests that MOPS binds at a site ~50% of the way
across the electrical field. Electrical distance does not necessarily
indicate physical distance. However, in the two cases in which it has
been determined, there has been excellent correlation between
electrical distance and physical distance across the pore formed by the
MSDs. Tabcharani et al. (26) found that
SCN
bound within the CFTR
channel at a site ~20% of the electrical distance through the
membrane from the cytoplasmic side, in good agreement with the
predicted location of Arg-347 with which it interacted (assuming an
-helix). McDonough et al. (20) found that diphenylamine-2-carboxylic
acid bound at a site ~40% of the electrical distance through the
membrane from the cytoplasmic side; this value is in good agreement
with the predicted location of Ser-341 to which it bound. MOPS is
predicted to have dimensions of 7 Å × 5.5 Å × 12 Å, and the pore of CFTR is predicted to have a
diameter of ~5.5 Å (25). These considerations suggest that
MOPS on the internal side can enter a pore that has a wide cytosolic
mouth and progress at least 50% of the distance through the electrical
field to its binding site. While it is bound, MOPS appears to occlude
the pore, preventing the flow of
Cl
. At some point external
to the MOPS binding site, the pore presumably narrows to a diameter of
~5.5 Å. This model of the channel pore and its interaction
with MOPS
in the O1 state
are shown schematically in Fig.
11A.

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|
Fig. 11.
Model of MOPS interaction with CFTR in the O1 (A) and O2
(B) state. Membrane is represented by hatched area, and
domains of CFTR are labeled. MSD, membrane-spanning domain. NBD,
nucleotide-binding domain. Model shows the channel only during a burst
of activity; long closed state between bursts, ATP hydrolysis at NBD1,
and phosphorylation of the regulatory (R) domain are not represented. Model also does not show any interaction with MOPS during O2 state, although an interaction does occur that is different from that during
the O1 state. B: ADP and
Pi are shown dissociating from the
channel for purposes of illustration; we have no data to determine if
they remain bound or if one or the other dissociates. Tracing (middle) represents the gating of
CFTR between the open (O) and closed (C) state.
|
|
The mechanism of MOPS interaction in the O2 state is more difficult to
characterize. The concentration-dependent change in the proportion of
the electrical distance sensed by MOPS indicates that the voltage
dependence was not consistent with a simple open channel block.
Therefore, for simplicity, in the model in Fig. 8 we do not show an
interaction with MOPS during the O2 state.
Previous studies have shown that anionic buffers can block other
Cl
channels. Hanrahan and
Tabcharani (11) showed that an outwardly rectifying
Cl
channel was blocked by
HEPES and MOPS but not by tricine. However, in contrast to the MOPS
block of CFTR, HEPES was able to block the outward rectifier from the
extracellular surface and both the anionic and the zwiterionic forms of
HEPES were effective. Yamamoto and Suzuki (32) also showed that a 35-pS
Cl
channel from cultured
Drosophila neurons was blocked by
HEPES and MOPS. In CFTR, Sheppard et al. (24) and McCarty et al. (19) reported that CFTR showed brief flickery closings at hyperpolarizing voltages, an effect that may have been due to the use of 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.
Our observations also have practical implications for future studies of
CFTR function. For studies designed to evaluate the conductive
properties of the channel, the use of tricine as a buffer may be
preferable to MOPS because the lack of the flickery kinetics would
allow a more accurate determination of single-channel current. In
addition, study of channels at 35°C will increase single-channel
conductance. In contrast, for studies designed to evaluate channel
regulation and gating, the use of MOPS as a buffer and temperatures
<35°C may be useful because these conditions allow the
identification of two different conformational states within bursts of
activity. These findings also reconcile the report of Gunderson and
Kopito (10) with previous studies from our and other laboratories that
did not find evidence for two different "conductance" levels:
those earlier studies did not use conditions that would make the two
different gating states apparent. Finally, previous studies have shown
that single CFTR Cl
channel
currents can occasionally be observed to enter subconductance states
(27, 30). The effect of MOPS we report here is different from those
earlier studies in that the "appearance" of a subconductance is
only evident with heavy filtering and there is a specific pattern of
its occurrence during a burst of activity.
The block we observed with MOPS reminds us of the behavior of CFTR
studied in cell-attached patches. For example, Haws et al. (12) and
McCarty et al. (19) showed that, in cell-attached patches, CFTR had a
pronounced flickery behavior at hyperpolarizing but not depolarizing
voltages. This gating is similar to the voltage-dependent behavior we
observed with MOPS on the cytosolic surface of excised patches of
membrane. Thus it is interesting to speculate that an intracellular
anion might be a physiological blocker of CFTR. By analogy,
intracellular polyamines are proposed to block and thereby regulate
inwardly rectifying K+ channels
(17).
Conformational changes in CFTR during an asymmetric gating cycle.
The results show that CFTR can exist in three distinct protein
conformations that can be distinguished as three different gating
states. 1) The C state is a long
closed state between bursts of activity.
2) The O1 state is an open state
within bursts during which the channel conformation allows MOPS to bind
a site in the pore and intermittently occlude
Cl
flow.
3) The O2 state is an open state
during which MOPS has a different, less well-defined interaction with
the channel, perhaps producing a very fast block.
We found a strong asymmetry or directionality in the movement between
these conformational states; they did not exist at thermodynamic equilibrium. The channel first moves from C to O1. This transition probably represents a series of steps that include binding of ATP to
NBD1 and NBD2, but it appears to be hydrolysis of ATP at NBD1 that
ultimately provides the energy for the entry into O1 and a burst of
activity (4, 5, 8, 14). Then the protein is driven from the O1
conformation to the O2 state (Fig. 11) by ATP hydrolysis at NBD2.
Consistent with this conclusion, AMP-PNP (which is thought to bind at
NBD2) prevented the transition to O2 and prolonged the O1 state. This
is also consistent with observations on the effect of pyrophosphate
(10). Finally, the channel progresses from O2 back to the C state, a
transition that appears to be irreversible because the channel will not
open without ATP. In several respects, this model is similar to the one
proposed by Gunderson and Kopito (10); an important difference is that
we propose that ATP hydrolysis is required for the channel to open,
based on previous work (1, 4, 8, 14, 16). Although the mechanisms
involved in the O2-to-C transition are not known, it is interesting
that it is temperature sensitive.
In Figs. 1, 2, 4, 6, and 8-10 we showed examples of
these gating patterns and their variations. The most common pattern was
a burst of activity that began with the O1 state and ended with the O2
state. This is consistent with our kinetic analysis that showed that,
within a burst of activity, the first opening was shorter than the last
opening, and the durations of the first and last openings were similar
to the short and long open times identified by dwell-time analyses.
However, other patterns were occasionally observed. For example,
channels may have rarely closed from the O1 state
(trace e in Fig. 1), especially in the
presence of AMP-PNP (trace b in Fig.
10). Perhaps in these cases, ATP dissociated from CFTR (presumably
NBD2) without undergoing hydrolysis. Nevertheless, our kinetic analysis
suggests that the O1-to-C transition is uncommon.
Our results link the activity of the NBDs to alterations in a MOPS
binding site. They indicate that input of external energy from ATP
hydrolysis causes a conformational change in the pore. Thus they
suggest that there may be a direct physical link between the NBDs and
the MSDs in CFTR, a conclusion that provides a new insight into how ATP
may gate the channel.
 |
ACKNOWLEDGEMENTS |
We thank Pary Weber, Phil Karp, Theresa Mayhew, Virginia Song, and
Amanda Niehaus for excellent assistance and our laboratory colleagues
for helpful discussions. We especially appreciate the thoughtful
comments and suggestions of Dr. David Sheppard. We thank Drs. John
Marshall and Seng Cheng for the gift of C127 cells. We thank Boyd Knosp
in the Image Analysis Facility for modeling MOPS.
 |
FOOTNOTES |
This work was supported by the National Heart, Lung, and Blood
Institute and the Howard Hughes Medical Institute (HHMI). H. Ishihara
was an associate and M. J. Welsh is an investigator at the HHMI.
Present address of H. Ishihara: Second Dept. Med., Yamanashi Medical
Univ., 1110, Shimogato Tamaho-cho, Nakakoma-gun, Yamanashi-ken 409-38, Japan.
Address for reprint requests: M. J. Welsh, Howard Hughes Medical
Institute, Univ. of Iowa College of Medicine, 500 EMRB, Iowa City, IA
52242.
Received 10 March 1997; accepted in final form 14 June 1997.
 |
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