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
chloride channel; gating; cystic fibrosis transmembrane conductance regulator; buffer; anion; 3-(N-morpholino)propanesulfonic acid
THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR) is an epithelial Cl2 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 nucleotidebinding domains (NBDs), and a regulatory domain
(RD). The MSDs, which are each composed of six
transmembrane sequences, contribute to the formation
of the Cl2 conducting pore. The RD contains a number
of consensus phosphorylation sites; phosphorylation of
the RD by adenosine 38,58-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 58-adenylylimidodiphosphate
(AMP-PNP), pyrophosphate, and orthovanadate, it has
C1278
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.
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-Dglucamine 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
0363-6143/97 $5.00 Copyright r 1997 the American Physiological Society
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 18, 2017
Ishihara, Hiroshi, and Michael J. Welsh. Block by
MOPS reveals a conformation change in the CFTR pore
produced by ATP hydrolysis. Am. J. Physiol. 273 (Cell Physiol.
42): C1278–C1289, 1997.—ATP hydrolysis by the cystic
fibrosis transmembrane conductance regulator (CFTR) Cl2
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.
CONFORMATIONAL CHANGES IN CFTR
appropriate. P values of ,0.05 were considered statistically
significant.
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 Cl2 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
Fig. 1. Examples of cystic fibrosis transmembrane conductance regulator (CFTR) Cl2 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 280 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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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 allpoints 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. Singlechannel 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 6 SE of n observations.
Statistical significance was assessed with a paired or unpaired Student’s t-test and a log-likelihood ratio test, where
C1279
C1280
CONFORMATIONAL CHANGES IN CFTR
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).
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 Cl2 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
Fig. 2. Examples of CFTR Cl2 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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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.
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-N8-2-ethanesulfonic acid (HEPES) can block other Cl2 channels (11,
32). Therefore, we substituted tricine for MOPS because it did not block an outwardly rectifying Cl2
channel (11). With 10 mM tricine as the buffer (Fig. 2A),
the gating pattern resembled only the O2 state; we saw
C1281
CONFORMATIONAL CHANGES IN CFTR
Table 1. Effect of MOPS and temperature on open and closed times within bursts of activity
Open Time Within Bursts, ms
10 mM MOPS
10 mM tricine
Closed Time Within Bursts, ms
Temperature,
°C
n
Short
Long
Short
Long
Mean Burst
Duration, ms
25
15
35
25
6
4
4
5
6.1 6 0.2
5.4 6 0.6
31.7 6 2.6
57.3 6 9.0*
92.8 6 8.4
249.4 6 33.7*
1.2 6 0.2
1.6 6 0.1
6.9 6 3.1
4.5 6 0.7
2.5 6 0.3
11.8 6 2.7
411.0 6 54.3
607.0 6 42.7
205.5 6 35.7
608.5 6 156.8
1.1 6 0.1
Values are means 6 SE of n observations. Long closed times between bursts were not included in the analysis (see MATERIALS AND METHODS).
Open and closed times were measured only in bursts of openings. We were not able to obtain accurate estimations of the short open and short
closed time for channels that were studied at 35°C in 10 mM MOPS because the average short closed time was ,1 ms, below the limit of
resolution of our experimental setup. * Value different from that obtained at higher temperature (P , 0.05).
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Fig. 3. Examples of open-time histograms obtained from singlechannel 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 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).
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).
MOPS block of CFTR Cl2 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 180
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.
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 6
0.21 pS, n 5 3) than at 25°C (7.9 6 0.02 pS, n 5 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 6 0.1 pS (n 5 4) at 25°C
and 10.2 6 0.5 pS (n 5 4, not shown in Fig. 5B) at 37°C.
These conductance values are similar to those obtained
from channels studied in tricine.
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 appar-
C1282
CONFORMATIONAL CHANGES IN CFTR
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.
Kd(V) 5 ([MOPS] · iMOPS)/(i0 2 iMOPS)
(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 (d) can be calculated with the
relationship (31)
Kd(V) 5 Kd(0)exp[(2zdFV)/(RT)]
Fig. 5. Current-voltage relationships of channels studied in tricine
and MOPS. A: buffer was 10 mM tricine at 25°C (k) and 35°C (s).
Data were filtered at 500 Hz; n 5 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 (r and j, respectively). n, Currentvoltage relationship of data filtered at 500 Hz; n 5 2–5 for each. In
most cases, SE bars are hidden by symbols.
(2)
where z is the valence of MOPS, which is assumed to be
21 (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 d 5 0.50 6
0.01 (n 5 5) measured over the voltage range of 2120 to
240 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.
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ent. 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.
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
CONFORMATIONAL CHANGES IN CFTR
C1283
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.
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 )
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
280 mV. k, 25 mM MOPS. s, 10 mM MOPS. n, 5
mM MOPS. Data are means 6 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
(d) was 0.50 6 0.01. Kd(0) and d for the O2 state
depended on the MOPS concentration: with 25
mM MOPS they were 89 mM and 0.25 6 0.01,
and with 10 mM MOPS they were 261 mM and
0.54 6 0.02, respectively.
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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, d varied. These results
indicate that the effect of MOPS in the O2 state is not
C1284
CONFORMATIONAL CHANGES IN CFTR
or the anion (MOPS2 ), and the ratio depends on the pH.
To determine whether MOPS0 or MOPS2 produced the
rapid flickery block in the O1 state, we used the
equation pH 5 pKa 1 log([MOPS2]/[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 MOPS2 and MOPS0. Figure 8A shows
that an increase in the concentration of MOPS2 had a
greater effect on gating than did MOPS0. For example,
increasing the [MOPS2] from 2.8 to 13.9 mM at a
constant pH of 7.3 markedly increased the flickery
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Fig. 8. Effect of MOPS2 (anion) and MOPS0 (zwitterion). A: example of traces obtained at different
MOPS2 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 280 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 MOPS2, MOPS0,
total MOPS, and pH are indicated. Data are means
6 SE of 3–5 experiments for each condition.
block (compare the first and second trace in Fig. 8A).
Likewise, increasing the [MOPS2] 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 [MOPS2] may be more readily apparent from
examination of current amplitude of data obtained at
negative voltages and filtered at 10 Hz (Fig. 8B). When
[MOPS2] 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
CONFORMATIONAL CHANGES IN CFTR
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 58-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
Fig. 9. Effect of addition of MOPS to the intracellular or extracellular surface of the patch. Examples
were obtained at 25°C and 280 or 180 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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we examined the effect of MOPS2 on the O2 state. Our
previous unpublished observations have suggested little
effect on gating of pH changes in this range. These data
suggest that MOPS2, rather than MOPS0, blocked the
channel in both the O1 and the O2 states, results
consistent with a voltage-dependent effect.
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.
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
C1285
C1286
CONFORMATIONAL CHANGES IN CFTR
Table 2. Duration of first and last opening
within bursts of activity
10 mM MOPS
10 mM tricine
Temperature,
°C
n
Duration of First
Opening, ms
Duration of Last
Opening, ms
25
25
6
5
7.3 6 0.5
262.2 6 58.6
26.1 6 1.6*
242.4 6 33.1
Values are means 6 SE of n observations. See MATERIALS AND
for detail of analysis. * Value significantly different from
duration of first opening (P , 0.0001).
METHODS
bursts. These data suggest that the transition from the
O1 to O2 state requires ATP hydrolysis.
DISCUSSION
Fig. 10. Effect of 58-adenylylimidodiphosphate (1 mM)
on single-channel currents. Solution contained 10 mM
MOPS and 1 mM ATP. Experiments were performed at
25°C and 280 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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MOPS blocks CFTR Cl2 channels. Our data indicate
that MOPS blocked CFTR Cl2 channels by two different mechanisms, manifest as the O1- and the O2gating states. In both cases, block was concentration
dependent and was due to the anionic form of MOPS
(MOPS2 ) 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 MOPS2
from the intracellular solution into the channel. In the
O1 state, MOPS produced a flickering block with
frequent short closures characteristic of classic openchannel 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 SCN2 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 a-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 3 5.5 Å 3
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
CONFORMATIONAL CHANGES IN CFTR
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.
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 Cl2
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
K1 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 Cl2 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.
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pore, preventing the flow of Cl2. 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 MOPS2 in the O1 state are
shown schematically in Fig. 11A.
The mechanism of MOPS interaction in the O2 state
is more difficult to characterize. The concentrationdependent 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 Cl2 channels. Hanrahan and Tabcharani
(11) showed that an outwardly rectifying Cl2 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 Cl2 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
C1287
C1288
CONFORMATIONAL CHANGES IN CFTR
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.
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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Consistent with this conclusion, AMP-PNP (which is
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