Functional Geometry of the Permeation Pathway of
Ca2+-activated Cl
Channels Inferred from
Analysis of Voltage-dependent Block*
Zhiqiang
Qu and
H. Criss
Hartzell
From the Department of Cell Biology, Emory University School of
Medicine, Atlanta, Georgia 30322-3030
Received for publication, February 8, 2001, and in revised form, February 28, 2001
 |
ABSTRACT |
We examined the voltage-dependent
block of Ca2+-activated Cl
channels by
anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid
(DPC), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and
niflumic acid (NFA) in excised inside-out and outside-out patches from
Xenopus oocytes. The fraction of the voltage field (
)
experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from
the outside.
was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for
NFA. Because the voltage dependence of the drugs differed, the order of
potency was also voltage-dependent. At +100 mV the order of
potency was NFA > A9C > DIDS > DPC
(Ki (µM) = 10.1, 18.3, 48, and
111, respectively). Because the drugs are hydrophobic, they can cross
the bilayer when applied from the inside and block the channel from the
outside. The equilibrium geometries of the blockers were determined by
molecular modeling and compared with their blocking positions (
).
This analysis suggests that the channel is an elliptical cone with the
largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 × 0.75 nm, because
C(CN)3
, which has these dimensions,
permeates. The external opening is at least 0.60 × 0.94 nm,
because DPC has these dimensions and penetrates the channel
~30%.
 |
INTRODUCTION |
Ca2+-activated Cl
(Cl(Ca))1 channels play
important roles in physiological processes, including epithelial
secretion, repolarization of the cardiac action potential, regulation
of vascular tone, olfactory transduction, and neuronal excitability
(1-4). Xenopus oocytes have long served as a model system
for studying Cl(Ca) channels because these channels are the predominant
channel type natively expressed in this cell and because they are
expressed at extremely high levels (0.5 mA/cm2) (5).
Recently, we have been investigating the mechanisms of anion permeation
(6), gating (7), and regulation (8-11) of Ca2+-activated
Cl
channels in Xenopus oocytes, where these
channels play a key role in fast block to polyspermy (12).
Xenopus oocyte Cl(Ca) channels have many features in common
with Cl(Ca) channels in epithelial cells, cardiac myocytes, and
vascular smooth muscle cells (see references in Ref. 6), and we think
that elucidating the mechanisms of operation of Xenopus
oocyte channels will provide important insights into the function of
these channels in other tissues.
The roles of Cl(Ca) channels in human disease are not yet firmly
established. However, there are reasons to think that they may be
involved in diseases as diverse as cystic fibrosis and cardiac
arrhythmias. For example, there appears to be a reciprocal relationship
between the level of expression of CFTR and Cl(Ca) channels in airway
epithelial cells. Cells from the airway of cystic fibrosis patients can
secrete in response to elevations of intracellular Ca2+
(13, 14), and ICl(Ca) is up-regulated in the
airway of CFTR knockout mice (15). The up-regulation of
ICl(Ca) in the airway of CFTR knockout mice can
apparently compensate for the lack of CFTR and ameliorate the lung
pathology in this mouse model (16). Furthermore, overexpression of CFTR
in cultured airway epithelial cells from cystic fibrosis patients
results in a decrease in Cl(Ca) current (17). In the heart, the
transient outward current (Ito), which plays an
important role in repolarization of the cardiac action potential, is
composed of several components, one of which (Ito2) is mediated by Cl(Ca) channels (18-20).
Changes in Ito2 can alter cardiac rhythmicity by
affecting action potential duration (19, 21). Recently, it has been
shown that dogs that are genetically prone to cardiac sudden death have
an abnormal Ito (22), implying that Cl(Ca)
channels play a role in sudden cardiac death. Cl(Ca) channels also
contribute to the arrhythmogenic transient inward current
(Iti) in some species (22-25). During
Ca2+ overload, Iti can
trigger oscillatory afterpotentials resulting in serious cardiac
arrhythmias (26, 27). Cl
channels clearly can participate
in arrhythmogenesis, because anion substitution or pharmacologic
Cl
channel blockade protects against reperfusion and
ischemia-induced arrhythmias (28-30).
Understanding the nature of the pore of this channel is an important
step in elucidating how ions permeate anion-selective channels in
general and also in developing reagents that can be used to block or
activate these channels. In our previous study (6), we concluded that
the pore of the Cl(Ca) channel must be at least 0.74 nm in one
dimension, because the pseudo-halide anion C(CN)3, which is
0.33 × 0.75 nm in its smallest cross section, is permeant through
the channel. In the present study (6), we have examined the voltage
dependence and sidedness of block of several classical Cl
channel blockers and have related these to the molecular size and
structure of the blocking molecules. These studies provide insights
into the functional geometry of the permeation pathway of this channel.
 |
EXPERIMENTAL PROCEDURES |
Isolation of Xenopus Oocytes--
Stage V-VI oocytes were
harvested from adult Xenopus laevis females
(Xenopus I) as described by Dascal (5). Xenopus
were anesthetized by immersion in tricaine (1.5 g/liter). Ovarian
follicles were removed, cut into small pieces, and digested in normal
Ringer's solution with no added calcium containing about 2 mg/ml
collagenase type IA (Sigma) for 2 h at room temperature. The
oocytes were extensively rinsed with normal Ringer's, placed in L-15
medium (Life Technologies, Inc.), and stored at 18 °C. Oocytes were
used 1-6 days after isolation. For excised patch experiments, oocytes were placed in a hypertonic solution (200 mM potassium
aspartate, 20 mM KCl, 1 mM MgCl2,
10 mM EGTA, 10 mM HEPES, pH 7.2 with KOH) for
1-10 min to facilitate manual removal of the vitelline membrane, and
then they were placed in a standard solution (see "Solutions" below) until use.
Electrophysiological Methods--
Recordings were performed
using the inside-out and outside-out excised patch configurations of
patch clamp technique. Pipettes were made of borosilicate glass (Sutter
Instrument Co.), pulled by a Sutter P-2000 puller, and fire-polished.
Patch pipettes had resistances of 6-10 megohms. Unless noted, they
were filled with a standard solution (see "Solutions"), which was
always the same as the solution in the bath. The bath was grounded via
a 3-M KCl-agarose bridge connected to a Ag-AgCl reference electrode.
Solution changes were performed by positioning the patch at the end of
a battery of sewer pipes having 100-µm internal diameter connected to
the gravity feed solution containers. Patches were usually obtained from the animal hemisphere of oocytes, because Cl(Ca) channels are
concentrated here (9). The patch was typically held at 0 mV, and
current was measured in response to a 200-ms voltage ramp from
100 to
+100 mV.
Patch clamp data were acquired by an Axopatch 200A amplifier controlled
either by Clampex 8.1 via a Digidata 1322A analog-to-digital and
digital-to-analog converter (Axon Instruments) or by Curcap 3.0 and a
Challenger DB voltage stimulator (W. Goolsby, Emory University).
Solutions--
Symmetrical solutions containing ~150
mM Cl
were used. In different experiments,
the cation was either Na+ or NMDG, as indicated in
the figure legends. Na+ was always used in experiments with
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) because
of possible interaction of DIDS and NMDG. The NMDG standard solution
contained 150 mM NMDG-Cl, 10 mM NMDG-HEPES, 4 mM MgCl2, pH 7.3. Zero Ca2+
solution contained 10 mM EGTA. High Ca2+
solution contained 10 mM EGTA titrated with
Ca2+ to give ~50 µM free Ca2+.
The standard Na+-containing solution contained 150 mM NaCl, 10 mM HEPES, pH 7.2. Zero
Ca2+ solution contained 10 mM EGTA, and the
high Ca2+ solution contained 0.1 mM
CaCl2 with no added EGTA. With inside-out patches, to be
certain that the currents recorded were
Ca2+-dependent, we always measured the current
in a zero Ca2+ solution at the start and end of the
experiment. If the Ca2+-independent current was >5% of
the total current in Ca2+, the patch was discarded. For
outside-out patches, it was not practical to change the
Ca2+ bathing the cytosolic face. Therefore, with
outside-out patches, the identity of the current as a Cl
current was verified at the start and end of an experiment by bathing
the external face of the patch in solution in which all Cl
was replaced with
SO
. Patches were not analyzed if the
outward current under these conditions was >5% of the current with
symmetrical Cl
.
Anion Channel Blockers--
Diphenylamine-2-carboxylic acid
(DPC) and niflumic acid (NFA) were from Sigma; anthracene-9-carboxylic
acid (A9C) was from Aldrich; DIDS was from Molecular Probes. DIDS from
Sigma and Molecular Probes differed significantly in their color, and
some preliminary experiments with Sigma DIDS yielded irreproducible
results. DIDS (Molecular Probes) was suspended in water at 0.3 M as a stock before working solutions were made. Other
compounds were dissolved in Me2SO at 0.3 M as stocks to keep the [Me2SO] in working
solutions < 0.1%.
Display and Analysis of Data--
For the calculations and
graphical presentation, we used Origin 6.0 software (Microcal). Curve
fitting was performed using the iterative algorithms in Origin. Results
are presented as means ± S.E., and n refers to the
number of patches in each experiments.
Molecular Modeling--
The equilibrium geometries of the
blockers used were calculated using both MMFF94 molecular mechanics
models and Hartree-Fock molecular orbital calculations with the 3-21G*
basis set (31). Calculations were performed using PC Spartan Pro
software (Wavefunction, Inc., Irvine, CA) run on an Intel
Pentium-III-based PC running Windows 2000. To determine the minimal
cross-sectional dimensions of the molecule, the molecule was rotated
manually to fit it into the smallest possible rectangle. The
center-to-center atomic distances were measured using utilities in PC
Spartan Pro; the molecular dimensions were calculated by adding the van
der Waals radii of the terminal atoms to the measured center-to-center
distances. In the cases of C(CN)3 and A9C, the geometries
were confirmed by comparison to crystal structure data available in the
Cambridge Crystallographic Database.
 |
RESULTS |
The goal of these experiments was to characterize the inhibition
of Ca2+-activated Cl
channels by various
Cl
channel blockers. Excised patches in either inside-out
or outside-out configurations were pulled from stage VI
Xenopus oocytes. The solutions on the inside and outside of
the patch were always the same, except that 0.1 mM
CaCl2 was added to the cytosolic face to activate Cl(Ca)
channels. The effects of the blockers applied to either the cytosolic
face of the membrane in inside-out patches or the extracellular face in
outside-out patches were then determined.
A9C Blocks from the Outside
Outside-out Patches--
Fig.
1A shows
I-V curves of ICl(Ca) in
an outside-out excised patch from Xenopus oocytes recorded
with symmetrical 150 mM Cl
solutions. The
I-V curve in the absence of A9C was approximately linear (Fig. 1A). The current was a Cl
current, because the outward current was completely blocked by replacement of extracellular Cl
by impermeant anions such
as SO
, and replacing Na+
with NMDG had little effect on Erev (data not
shown, but see Ref. 6).

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Fig. 1.
ICl(Ca) block by
extracellular A9C in an outside-out patch. A,
I-V relationships showing outward
ICl(Ca) block by A9C applied to the
extracellular side of an outside-out patch. The patch was
voltage-clamped from the holding potential of 0 mV with a 250-ms
duration voltage ramp from 120 to +120 mV. Both the pipette and the
bath contained NMDG standard solution. The extracellular side of the
patch was perfused with bath solution containing different
concentrations of A9C (0, 3, 10, 30, 100, 300 µM).
B, voltage-dependent block of
ICl(Ca) by A9C. Each curve from +10 to 110 mV in
A was divided by the curve obtained in 0 µM
A9C. The fractional currents were plotted versus membrane
potentials. Note that A9C at every concentration blocks more at the
higher voltages. C, concentration-dependent
block of ICl(Ca). The fractional currents at
various potentials in B were replotted as a function of
[A9C]. The data were fitted to Equation 1. D,
apparent Ki of extracellular A9C as a function of
membrane potential. The apparent Ki at each voltage
was determined from the fits in C in three separate
experiments and averaged. The points were fitted to Equation 2 (the
Woodhull equation (32)).
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|
The outward current was blocked in a concentration- and
voltage-dependent manner by addition of A9C to the
extracellular face of the membrane (Fig. 1A). The inward
current was only slightly affected. In Fig. 1B, the currents
from Fig. 1A were expressed as a fraction of the current in
the absence of A9C (I/IA9C = 0) and plotted as a function of voltage. Relative current amplitude decreased with depolarization, confirming that inhibition of
Cl
current by A9C was voltage-dependent. The
data in Fig. 1B were replotted in Fig. 1C for
each voltage as a function of [A9C]. These data were fitted to an
equation of the form
|
(Eq. 1)
|
where Imax and Imin
are the maximum and minimum current amplitudes, Ki
is the concentration of A9C required to reduce the current amplitude to
(Imax + Imin)/2, and
n is the slope factor. Ki was determined
for each potential and plotted in Fig. 1D. The apparent
Ki decreased ~10-fold per 100-mV depolarization. The Ki at 0 mV was estimated to be 158 µM, and the Ki at +100 mV was 18.3 µM.
From Fig. 1D, one can estimate the fraction of the voltage
field experienced by the blocking particle at its blocking site from
the equation derived by Woodhull (32, 33),
|
(Eq. 2)
|
where log Ki (V) is the
Ki at each voltage, Ki (0 mV) is
the Ki at 0 mV, z is the electronic charge of the blocking particle,
is the fraction of the voltage field sensed by the blocker from the outside of the membrane, R, F, and T have their usual
thermodynamic meanings, and V is voltage. The solid line is the
best fit of the antilog of Equation 2 to the data.
was estimated to
be ~0.6 the distance of the voltage field from the extracellular side.
The inhibition of currents by voltage-dependent blockers is
often time-dependent; as membrane potential is
changed, the current changes as the blocker accumulates or is removed
from the blocking site. Fig. 2 shows
ICl(Ca) currents in response to 100-ms duration voltage pulses from a holding potential of 0 mV to voltages between +100 and
100 mV. In the absence of A9C (Fig. 2A), outward
currents were time-independent. Inward currents at the most negative
potentials exhibited a slow decrease in current; currents decreased
<10% in 100 ms. I-V curves were plotted for the
current at the beginning (squares) and at the end
(circles) of the 100-ms pulses (Fig. 2B). Both
curves were approximately linear. In the presence of 10 µM A9C (Fig. 2C), the outward currents
exhibited pronounced time dependence. The currents decayed with time as
A9C block developed (Fig. 2C), and the
I-V curves measured at the beginning and end of
the voltage pulses diverged at positive potentials. Increasing the
[A9C] increased both the rate and the degree of block of the outward
current (Fig. 2, E-H).

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Fig. 2.
Time dependence of
voltage-dependent block by A9C. The extracellular face
of an outside-out patch was exposed to NMDG standard solution (also in
pipette) with different [A9C]: 0 µM, a and
B; 10 µM, c and D; 30 µM, e and F; 100 µM,
g and H. The patch was voltage-clamped by
stepping to various potentials between 120 and +120 mV for 0.1 s
with 20-mV increments for each step from the holding potential of 0 mV,
followed by a step to +120 mV for 0.1 s (protocol shown above
A). A, C, E, and
G show current traces of A9C block. Note that the higher
[A9C] made the outward current deactivate faster. B,
D, F, and H show
I-V relationships obtained from the corresponding
current traces in A, C, E, and
G, respectively, at 105 ms (closed squares) and
195 ms (closed circles).
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The decay of the current upon stepping from 0 to +100 mV was fitted to
a single exponential. The time constants were
concentration-dependent;
= 42.5 ± 1.2 ms at
10 µM A9C, 16.4 ± 1.1 ms at 30 µM
A9C, and 11.6 ± 0.4 ms at 100 µM A9C
(n = 3 for each). These data are consistent with a
model in which A9C enters the pore of the channel and blocks it in a
voltage-dependent manner.
Inside-out Patches--
To determine whether A9C also blocked from
the inside, we obtained inside-out patches (Fig.
3). Surprisingly, A9C applied from the
cytoplasmic side of the patch also blocked outward current (Fig.
3A). If A9C had blocked the channel from the inside in a similar manner to block from the outside, we would have expected A9C
applied on the cytoplasmic surface to block inward, not outward, current. Woodhull analysis of the voltage-dependent block
showed that
= 0.6 (Fig. 3B). Thus, A9C blocked
outward current at the same site in the channel with the same voltage
dependence whether it was applied from the inside or outside.
The most logical interpretation of these data is that A9C blocks only
from the outside but that it crosses the lipid membrane and gains
access to the outside when applied from the inside. If this is true, we
might expect that the apparent Ki for block by A9C
applied on the inside would be larger than when applied from the
outside, because the actual concentration of A9C on the outside when
applied from the inside would be lower than expected. In support of
this idea, the Ki for A9C applied to the inside at 0 mV was 945 µM and at +100 mV was 103.7 µM,
~5-6-fold greater than the Ki for A9C applied
from the outside at the same voltage. In addition, DPC, which has a
similar structure to A9C, has been shown to cross lipid membranes
readily at neutral pH (see references and "Discussion" in Ref.
34).

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Fig. 3.
ICl(Ca) block by intracellularly
applied A9C. A, I-V relationships
showing block of ICl(Ca) by A9C applied to the
intracellular side of an inside-out patch. The voltage ramp protocol,
solutions, and [A9C] were the same as in Fig. 1. A, note
that only outward currents were blocked. B, apparent
Ki of A9C applied intracellularly versus
membrane potentials. Apparent Ki values were
obtained by Woodhull analysis as in the legend to Fig. 1.
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DPC Blocks from the Outside
A similar analysis was performed for DPC (Fig.
4). In outside-out patches DPC blocked
outward current in a dose-dependent manner with an apparent
Ki at 0 mV of 323 µM and at 100 mV of
111 µM. In the experiment illustrated, there was also a small effect on inward current, but it was difficult to determine whether this was a true inhibitory effect or was related to channel rundown, which occurs in some patches.

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Fig. 4.
ICl(Ca) block by extracellular
DPC. A, I-V relationships showing
ICl(Ca) block by DPC in an outside-out patch.
B, voltage-dependent block of
ICl(Ca) by DPC. C,
concentration-dependent block of
ICl(Ca). D, apparent
Ki of extracellular DPC as a function of membrane
potential. For details, see the legend to Fig. 1.
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|
Woodhull analysis of DPC is presented in Fig. 4, B-D. The
voltage dependence of the relative current
(I/IDPC = 0) in Fig.
4B shows that the block by DPC is clearly
voltage-dependent. However, the voltage dependence is less
than that of A9C (Fig. 1, C and D).
was
estimated to be 0.3. These data show that DPC blocks in a
voltage-dependent manner but that the blocking site is not
as deep in the pore as the A9C blocking site.
The kinetics of voltage-dependent block by DPC were faster
than block by A9C (Fig. 5). Fig. 5,
A and B, shows current traces in response to a
series of voltage pulses from 0 mV to voltages between
100 mV and
+100 mV for 0 and 300 µM DPC, respectively. Fig.
5C shows current traces from the voltage pulses from
100 to +100 mV on a faster time scale for four different DPC
concentrations. As [DPC] was increased, a more pronounced
time-dependent tail current was observed at the onset of
the pulse. The time constant was faster than 1 ms. The
I-V curves showed clearly increasing inward
rectification with increasing DPC concentration (Fig.
5D).

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Fig. 5.
Time dependence of ICl(Ca) block
by DPC. An outside-out patch experiment was performed in the same
way as in the legend to Fig. 2. The patch was exposed to extracellular
DPC at 0 µM DPC (A) and 300 µM
DPC (B). C, superimposed current traces elicited
by voltage steps from 100 to +100 mV on a faster time scale in
different [DPC] as marked. D, I-V
relationships for currents measured at 195 ms in A and
B.
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DPC, like A9C, can cross the membrane when applied from the inside in
inside-out patches and block the channel from the outside (Fig.
6). DPC applied from the inside blocked
outward current with a Ki of 212 µM at
100 mV, which was about twice the Ki for inhibition
from the outside. The estimated
was 0.2, which was similar to the
estimate for DPC applied from the outside. As noted above, DPC has been
shown to partition readily into lipid membranes at neutral pH (34).

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Fig. 6.
ICl(Ca) block by DPC applied
intracellularly to inside-out patch. For experimental conditions
refer to the legend to Fig. 3.
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DIDS Blocks from the Outside
Outside-out--
DIDS applied to the extracellular side decreased
outward Cl
current in a dose-dependent manner
(Fig. 7A). Block was clearly voltage-dependent (Fig. 7B). The apparent
Ki at 0 mV was 562 µM and at +100 mV
was 48 µM.
was estimated to be 0.3 (Fig. 7,
C and D).

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Fig. 7.
Voltage dependence of ICl(Ca)
block by external DIDS in outside-out patch. For all experimental
conditions refer to the legend to Fig. 1; however, the cation in these
experiments was Na+. A,
I-V relationships. B,
voltage-dependent block by DIDS. C,
concentration-dependent block by DIDS. D,
voltage-dependent Ki of DIDS
block.
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Inside-out--
100 µM DIDS applied to the
cytoplasmic side blocked inward current >50% and outward current
~30%, but the block required >1 min to develop (Fig.
8). In contrast, DIDS (and the other
blockers used here) applied to outside-out patches produced a
steady-state block within several seconds. Because this slowness of
block suggested that the mechanisms were significantly different from
block from the outside, we did not analyze these results
quantitatively.

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Fig. 8.
Block of current by intracellular DIDS in
inside-out patch. The patch was clamped with a voltage ramp from
100 to +100 mV. The currents at 90 and +90 mV are plotted
versus time. The patch was exposed to 100 µM
DIDS in Na+ standard solution or zero Ca2+
solution during the periods indicated.
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NFA Block Is Not Significantly Voltage-dependent
Outside-out--
NFA has been widely used to block
Ca2+-activated Cl
channels in
Xenopus oocytes (35). In outside-out patches, NFA applied to
the extracellular face blocked both inward and outward current (Fig.
9A). The apparent
Ki was 12.9 µM at 0 mV and 10.1 µM at +100 mV (Fig. 9D). Although plots of
I/INFA = 0 versus
Vm (Fig. 9, B and C) showed
some curvature at the highest NFA concentrations, the voltage
dependence was rather small. Woodhull analysis (Fig. 9, C
and D) showed that the apparent Ki did
not change significantly between 20 mV (11.6 µM) and 100 mV (10.1 µM). The best linear fit to the data in Fig. 9D yielded a small slope that translated into an estimate
for
of 0.1.

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Fig. 9.
Block of ICl(Ca) by external NFA
in outside-out patch. For all experimental conditions refer to the
legend to Fig. 1. A, I-V
relationships. B, voltage-dependent block by
NFA. C, concentration-dependent block by NFA.
D, voltage-dependent Ki of
NFA block.
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Inside-out--
NFA applied to the cytoplasmic face of inside-out
patches also blocked both inward and outward current (Fig.
10A) with a
Ki of 53.7 µM at +100 mV (Fig.
10B). This Ki is about 4 times greater
than Ki for NFA applied from the outside. We do
not know whether block of NFA from the inside involves NFA crossing the
membrane and blocking from the outside. However, because NFA is very
closely related to DPC, we presume that it crosses the membrane
readily. There was no voltage dependence to the block (Fig.
10C).

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Fig. 10.
ICl(Ca) block by NFA applied
intracellularly to inside-out patch. For experimental conditions
refer to the legend to Fig. 3.
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 |
DISCUSSION |
Summary and Conclusions--
The data on the block of
Ca2+-activated Cl
channels in
Xenopus oocytes by various pharmacological agents is
summarized in Table I. In outside-out
patches, block of Cl
current with externally applied
blockers exhibited an order of potency of NFA > A9C > DIDS > DPC. Block by NFA was not voltage-dependent, but the blocks by A9C, DIDS, and DPC were
voltage-dependent. NFA blocked both inward and outward
current, whereas the other blockers blocked only outward current
significantly. Block by all four blockers was always reversible under
these conditions.
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Table I
Voltage-dependent block of Cl(Ca) currents
The numbers in the parentheses show n (the number of patches
in each experiment); Ki is in µM. is the fraction of the voltage field experienced by the blocker.
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In inside-out patches, block of Cl
currents with blockers
applied on the cytosolic side exhibited the order of potency NFA > A9C > DPC > DIDS. However, block by A9C and DPC from the
inside appeared to be due to the blocker crossing the membrane and
blocking from the outside. There are two reasons we conclude that A9C
and DPC do not block from the inside. First, these blockers block only
outward current, regardless of whether they are applied from the inside
or the outside. Second, the Ki for block from the
inside is larger than from the outside. DIDS block from the inside was
unusual in that it was very slow. This slow block was not analyzed in
detail. Because the block by NFA was only weakly voltage-dependent and because NFA blocked both inward and
outward currents, it was not possible to determine unambiguously the
sidedness of NFA block.
These data provide important insights into the functional structure of
the pore of the Ca2+-activated Cl
channel.
Voltage dependence of block is commonly interpreted to reflect binding
of the blocker to a site in the anion permeation pathway (33). If this
is the case, the calculated fraction of the voltage field (
, from
the outside of the channel) experienced by the blocking particle
provides an estimate of the position of the blocking site in the
channel. We have calculated that
for NFA, DPC, DIDS, and A9C are
<0.1, 0.3, 0.3, and 0.6, respectively. This suggests that A9C
penetrates further into the pore than DPC and DIDS, which penetrate
about the same distance. NFA apparently blocks at a site close to the
outside (and possibly the inside) of the channel.
This interpretation depends on the assumption that the drugs block the
channel by binding to a site in the permeation pathway. However,
because all of the drugs used in this paper are quite hydrophobic, this
raises the possibility that these drugs could access a site external to
the pore from the lipid phase of the membrane. In the case of A9C and
DPC, we think that the data clearly argue against such a mechanism. A9C
and DPC block only outward current regardless of whether they are
applied to the external or internal side of the membrane. In both
cases, the block exhibits the same voltage dependence regardless of the
side of the membrane where the drug is applied. These data argue
strongly that A9C and DPC must access the channel from the external
side and that the block occurs within the permeation pathway. In the
case of DIDS and NFA, other interpretations are possible. DIDS block is voltage-dependent, but the sidedness of its effect is
ambiguous; although block from the outside is rapid and
voltage-dependent, block from the inside is slow and has
much weaker voltage dependence. This suggests that DIDS blocks the
channel by binding to a site in the permeation pathway when applied
from the outside but is capable of blocking the channel by other
mechanisms from the inside. Because DIDS is hydrophobic, one might
expect that DIDS applied from the outside could reach the "inside"
site. However, the access of DIDS to this inside site is quite slow and
may not be evident when we rapidly apply DIDS from the outside. NFA
blocks current in both directions, and the block is very weakly
voltage-dependent, regardless of the side where it is
applied. Thus, the possibility exists that NFA blocks the channel in an
allosteric manner.
Functional Model of the Ca2+-activated Cl
Channel Pore--
To gain additional insight into the mechanisms of
block, we created molecular models of the blocking molecules. The
equilibrium geometry of each of the molecules was calculated using
molecular orbital calculations (see "Experimental Procedures").
Fig. 11 shows the structures of
C(CN)3 (the largest anion that we have shown to be permeant
through this channel (6)) and the blockers used in this paper. The
left column of Fig. 11 shows the structures of these
molecules viewed from the side. The right column of Fig. 11
shows the molecules oriented so that their cross-sectional area is
minimized (viewing the molecule from the "end") in a space-filling model with the molecular dimensions shown.

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|
Fig. 11.
Molecular models of Cl
channel blockers and permeant anions. The equilibrium geometries
of the molecules shown were calculated using molecular orbital
calculations in PC Spartan Pro. Molecules in the left column
are shown in ball-and-stick models viewed from the largest
cross-sectional area. In the right column, the molecules
were rotated to show them in their smallest cross-sectional area as
space-filling models with the molecular dimensions shown in Å.
Black, carbon; white, hydrogen; red,
oxygen; cyan, sulfur; green, fluoride;
blue-gray, nitrogen.
|
|
The minimal dimensions of the channel pore can be estimated by
comparing the minimal dimensions of the molecules with the estimated
distance they penetrate into the channel (
). We propose that these
molecules enter the channel in their end-on dimension, which is the
smallest cross-sectional area. We would argue that if these drugs
entered the channel in a different orientation, the pore diameter would
be so large that ionic selectivity would be nearly impossible to
achieve. In addition, these molecules with rather diverse overall
structure exhibit a certain similarity when viewed end-on; their
vertical dimensions are rather similar (7.5-9.4 Å). Thus, we propose
that in one dimension the channel is at least 7.5 Å for the entire
length of the channel. The width of the molecule correlates with
,
the depth the blocking molecule penetrates into the channel before it
blocks. Because we know that C(CN)3 permeates completely
through the channel, we conclude that the narrowest part of the pore
must be at least as large as C(CN)3, i.e.
3.3 × 7.5 Å. The outer mouth of the channel is at least as large
as DPC (6.0 × 9.4 Å) and may be as large as NFA (7.7 × 9.4 Å) if NFA blocks within the permeation pathway. However, as noted
above, it should be recognized that NFA may not block by entering the
permeation pathway but rather allosterically at some other site. A9C
enters the channel (voltage field) to a distance about 60% from the
outside; so the channel dimensions must be ~4.6 × 9.4 Å at the
A9C blocking site. DPC and DIDS only enter about 30% of the way into
the channel from the outside; so the dimensions at the DPC and DIDS
blocking sites must be ~6.0 × 9.4 Å. The observation that A9C
does not block the channel from the inside suggests that the cytosolic
opening of the channel is less than 4.6 × 9.4 Å. Thus, the shape
of the Ca2+-activated Cl
channel permeation
pathway approximates an elliptic cone with the largest opening facing
the extracellular space. The precision of these estimates is limited by
several factors. First, we cannot be certain that the estimate of
is precise, because the width and shape of the voltage field are
unknown. Second, the estimates of
probably refer to the fraction of
the voltage field experienced by the charge center of the molecule.
However, for simplicity we have assumed that this is the same as the
geometrical center of the molecule. This assumption seems reasonably
well justified because molecular modeling of the charge distribution in
these drugs suggests that the charge is either distributed in the
molecule or concentrated near the center.
The Pore of CFTR Is Oriented in the Opposite Polarity to Cl(Ca)
Channels--
DPC blocks CFTR from the inside at a site about 60%
from the outside surface (36). DIDS does not block from the
extracellular side (34) but does block from the intracellular side in a
voltage-dependent manner (37). These data suggest that the
pore of CFTR is an inverted version of the Cl(Ca) channel; it appears
to have its largest opening on the cytoplasmic side and the
smallest opening extracellularly.
 |
ACKNOWLEDGEMENTS |
We thank Nael McCarty for helpful discussion
and comments and Alyson Ellingson for technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM 60448.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 404-727-0444;
Fax: 404-727-6256; E-mail: criss@cellbio.emory.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M101264200
 |
ABBREVIATIONS |
The abbreviations used are:
Cl(Ca), Ca2+-activated Cl
;
CFTR, cystic fibrosis
transmembrane conductance regulator;
I, current;
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid;
DPC, diphenylamine-2-carboxylic acid;
NFA, niflumic acid;
A9C, anthracene-9-carboxylic acid;
NMDG, N-methyl-D-glucamine.
 |
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