Functional expression of a truncated
Ca2+-activated
Cl
channel and activation
by phorbol ester
Hong-Long
Ji1,
Michael D.
Duvall2,
Holly K.
Patton1,
Cynthia Lyn
Satterfield1,
Catherine M.
Fuller1, and
Dale J.
Benos1
Departments of 1 Physiology and
Biophysics and
2 Anesthesiology, University
of Alabama at Birmingham, Birmingham, Alabama, 35294
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ABSTRACT |
We have isolated a niflumic acid-insensitive,
Ca2+-activated
Cl
channel (CaCC) from
bovine trachea that migrates at 38 kDa (p38) on reducing sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. However, a cloned CaCC
isolated from a tracheal cDNA expression library by screening with an
antibody raised against p38 has a primary cDNA transcript of 2712 base
pairs that codes for a 100-kDa protein and is not susceptible to
dithiothreitol reduction. To test the hypothesis that the functional
channel may be a much smaller posttranslationally processed form of the
100-kDa protein, we generated a mutant construct (CaCCX, 42.5-kDa
protein) truncated at the NH2 and
COOH termini. The whole cell currents of wild-type (wt) CaCC and CaCCX
expressed in Xenopus oocytes were
10-fold higher than those of water-injected oocytes and were further
increased by ionomycin or A-23187 and inhibited by
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and
dithiothreitol. Whole cell currents in wtCaCC- and CaCCX-expressing oocytes could also be activated by phorbol 12-myristate 13-acetate and
could be inhibited by chelerythrine chloride, suggesting that the
cloned CaCC is regulated by protein kinase C. These results suggest
that a smaller form of the full-length CaCC can form a functional
channel.
anions; voltage clamp; ion channel; RNA expression; bovine trachea; Xenopus oocyte; calcium-activated
chloride channel
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INTRODUCTION |
CHLORIDE CHANNELS PLAY AN important role in volume
regulation and electrolyte and water balance in a variety of epithelial cells. Several Cl
channels
have been identified on either the apical or basolateral membrane of
epithelial cells, such as the adenosine 3',5'-cyclic monophosphate-sensitive cystic fibrosis transmembrane conductance regulator (CFTR; Refs. 11, 13) and the ClC family of
Cl
channels (20).
Cl
transport can also be
activated by Ca2+, which in some
cases is attributed to activation of other
Ca2+-sensitive processes, such as
opening of Ca2+-activated
K+ channels, thereby changing the
driving force for Cl
secretion (10). Bona fide Ca2+-
and Ca2+/calmodulin-dependent
protein kinase II (CaMKII)-sensitive
Cl
channels have been
identified in a variety of epithelia, including colonic cells (5, 24,
39), exocrine gland cells (1, 15), biliary and renal epithelia (22,
30), and airway epithelia (4, 34).
Several lines of evidence have suggested that the
Ca2+-activated
Cl
channel (CaCC) pathway
may be a promising alternate therapeutic target in cystic fibrosis (CF)
to compensate for defective
Cl
transport via CFTR.
Studies in freshly excised and cultured CF airway epithelia and in CF
sweat glands have shown that the
Ca2+-sensitive
Cl
conductance functions
normally, whereas CFTR channel activity is compromised or absent (26,
33, 34). Furthermore, the expression of a CaCC seems to be an important
determinant of the severity of organ-level disease in the CF knockout
mouse model (6, 21, 38). Ca2+
ionophores have also been shown to activate a
Cl
conductance in CF airway
epithelial cells (17), and a
Ca2+-sensitive
Cl
conductance is
upregulated in the nasal mucosa of CF mice (16, 32).
We have previously reported the isolation of a
Cl
channel protein from
bovine tracheal epithelia (27, 28). When incorporated into planar lipid
bilayers, the protein formed a small (25-30 pS) linear (under
symmetrical conditions),
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
(DIDS)-sensitive, anion-selective channel that was also sensitive to
reduction by dithiothreitol (DTT) (29) and could be activated by
Ca2+ and phosphorylation by CaMKII
(14). The native protein, which migrates with a relative molecular mass
of 140 kDa on nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE), migrated at 38 kDa when separated under
reducing (50 mM DTT) conditions. This shift in relative molecular mass
was paralleled by a loss of all channel activity in the presence of DTT
(29). We have recently reported the cloning of a
Ca2+-sensitive
Cl
channel from a bovine
tracheal cDNA expression library (8). The cloned channel was inhibited
by DIDS and DTT, was insensitive to niflumic acid, had a linear
current-voltage
(I-V)
plot under symmetrical conditions, and could be activated by
Ca2+ and CaMKII (8, 19). The
sequence of the cloned channel also contained several consensus sites
for phosphorylation by CaMKII, protein kinase C (PKC), and for N-linked
glycosylation.
However, a major difference between the cloned and native CaCCs was
their relative molecular masses. The primary transcript of the cloned
CaCC codes for a protein that migrates at 100 kDa under both reduced
and nonreducing conditions (in the absence of glycosylation),
significantly larger than the smallest polypeptide component of the
native channel, which migrates at 38 kDa under reducing conditions.
This may reflect posttranslational processing of the primary translated
product or that the native and cloned CaCC polypeptides are unrelated
although functionally similar. To test the hypothesis that a much
smaller form of the primary translated product could form a viable
channel, we have generated a mutant CaCC (CaCCX) truncated at both the
NH2 and COOH termini. The
predicted molecular mass of the protein encoded by CaCCX is 42.5 kDa.
Therefore, the purpose of the present study was to compare the
electrophysiological and pharmacological properties of the wt
(wild-type) CaCC and CaCCX constructs functionally expressed in
Xenopus oocytes.
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MATERIALS AND METHODS |
Construction and in vitro translation of a truncated CaCC.
We designed two primers to generate the truncated CaCC construct,
CaCCX, in a polymerase chain reaction (PCR). Both 5' and 3'
primers included a Bgl II site at the
5' end. The 5' primer (5'-GAAGATCTTCACCATGGATGTAATCATG-3') annealed to the wtCaCC
cRNA sequence between bases 819 and 831; translation would be predicted to initiate at Met-277. The 3' antisense primer
(5'-GAAGATCTTCTTATGAATAGATGCCATC-3') annealed to the
CaCC coding region between bases 1984 and 1998, converting
Arg-667 to a premature stop codon. The predicted length of
the PCR product was 1179 base pairs, coding for a polypeptide of 390 amino acids. This PCR-generated fragment preserved the four predicted
transmembrane regions of the wtCaCC and consensus sites for N-linked
glycosylation (6 sites), CaMKII phosphorylation (3 sites), PKC
phosphorylation (4 sites), and tyrosine kinase phosphorylation (1 site). Using the wtCaCC as the template in a PCR
reaction with the above primers, we generated a product of the correct
size under the following conditions: 94°C for 3 min × 1 cycle; 94°C for 1 min, 58°C for 2 min, 72°C for 3 min × 30 cycles; and 72°C for 15 min × 1 cycle, using
Pfu thermostable polymerase
(Stratagene). The product was digested with
Bgl II, phosphorylated, ligated into a
modified pGEM 11 vector (a gift of Dr. D. A. Melton, Harvard
University, Cambridge, MA), and transformed into XL1-Blue. Positives
were selected on the basis of restriction mapping
(Bgl II,
BamH I,
Xba I, and
Xho I).
To prepare methyl guanosine
[m7G(5')ppp(5')G]-capped
cRNA for injection into Xenopus
oocytes, the respective vectors containing either the wtCaCC or CaCCX
insert were linearized with Not I and the insert was in vitro transcribed with T7 polymerase (Ambion). The
integrity of the cRNA was verified by denaturing gel electrophoresis through 1% agarose-formaldehyde gels. In vitro translation was carried
out in the presence of
L-[35S]methionine
(NEN) using micrococcal nuclease-treated rabbit reticulocyte lysate
(Ambion) in the absence of canine pancreatic microsomes. Translated
products were separated by 8% SDS-PAGE under nonreducing conditions.
Oocyte expression.
Toads were obtained from Xenopus I (Ann Arbor, MI) and were kept in
circulating dechlorinated tap water at 18°C. Toads
were fed two times weekly with beef liver. The ovarian tissue was
removed from toads under hypothermal anesthesia through a small
incision in the lower abdomen as previously described (12). Oocytes at maturation stages V and VI were carefully defolliculated by hand in
Ca2+-free OR-2 solution [in
mM: 85.2 NaCl, 2.5 KCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 1.0 Na2HPO4,
pH 7.5] and maintained in OR-2 medium
(Ca2+-free OR-2, 1.0 CaCl2, pH 7.5). Both OR-2 media
contained 0.5% streptomycin (Sigma). Oocytes were incubated in OR-2
for several hours or overnight before cRNA injection. A Nanoject
(Drummond Science, Broomall, PA) was used for injection of cRNA or
ribonuclease-free water (control). The optimized concentrations
(5-500 ng) of cRNA in 50 nl were injected into oocytes in
Ca2+-free OR-2 solution (310 mosM)
and then transferred to normal OR-2 medium; 5% heat-inactivated horse
serum (Sigma) was added to OR-2 culture medium to improve the
expression efficiency and cell viability (25).
Electrophysiological measurements.
After 48-h incubation in OR-2 at 18°C, whole cell currents were
recorded from the oocytes following equilibration at room temperature
(at least 10 min at 20-25°C) and analyzed using pCLAMP version
5.5 software (Axon Instruments, Foster City, CA). Voltage-clamp potentials were evoked using a TEA-200 voltage clamp (Dagan,
Minneapolis, MN) controlled by a personal computer connected via a TL-1
interface (Axon Instruments). The injected oocyte was placed in a small chamber (1 ml) and perfused with ND-96 medium (in mM: 96 NaCl, 1 MgCl2, 2 KCl, 5.0 BaCl2, 5.0 CaCl2, and 5 HEPES, pH 7.4) at a
flow rate of 1.5-2 ml/min for at least 5 min before recording. Microelectrodes filled with 3 M KCl had a resistance of 0.5-3.0 M
. The bath was clamped by two silver-plated wires through 3% agar
bridges in 3 M KCl. The oocytes were clamped at a holding potential of
60 mV. Data filtered at 0.5-1 kHz were digitized and stored
on disk for off-line analysis. Test voltages were stepped from the
holding potential to
100 through +80 mV in 20-mV increments for
1 s. The currents at
80 and +80 mV were monitored at 30-s intervals. An average of samples from 500 to 700 ms of each episode was
used to plot
I-V
curves and to calculate the effect of inhibitors and regulatory agents.
Linear components of capacitance and leak currents were not subtracted.
Solutions and chemicals.
Stock solutions of 1 M DTT in H2O,
10 mM phorbol 12-myristate 13-acetate (PMA; Calbiochem, La Jolla, CA)
in dimethyl sulfoxide (DMSO), and 200 mM DIDS (Sigma) in ethanol or
freshly prepared in H2O, 1 mM
A-23187, or 1 mM ionomycin (both Calbiochem) in DMSO were stored at
0°C in light-protected vials. The working concentrations were
freshly prepared in the perfusing media for each experiment. Niflumic
acid (Sigma; 100 mM stock in DMSO) was diluted to the final
concentration in the perfusing medium immediately before use. The PKC
inhibitor, chelerythrine chloride (10 mM stock in DMSO; Research
Biochemicals International, Natick, MA), was used by pretreating the
cells for 20 min at a final concentration of 10 µM in ND-96 medium
before addition of the PKC activator, PMA. All other reagents not
listed above were obtained either from Sigma (St. Louis, MO) or from
Fisher.
Statistics.
Results are expressed as means ± SE, where
n is the number of oocytes. Student's
t-test was performed to calculate the
significant differences of the recordings before and after each drug
application. Curve fitting of the concentration-response relationship
of PMA was fit with the following equation
where
Imax stands for
the current at the highest concentration of PMA,
Imin is the
current in the absence of PMA, [PMA] is the concentration
applied, EC50 represents the
required concentration of PMA to activate the half-maximal conductance,
and n is the slope of the fitting
curve. Due to the variability of oocyte current expression, the current
amplitudes of CaCC and CaCCX were normalized to the
Imax of the
outward currents (at +80 mV, for
Cl
influx) or to the
Imax of the
inward currents (at
100 mV, for Cl
efflux).
 |
RESULTS |
In vitro translation of CaCCX.
The full-length open reading frames of the CaCC and CaCCX predict that
these polypeptides should migrate (in the absence of cotranslational
glycosylation) at ~100 and 42.5 kDa, respectively. As shown in Fig.
1, in vitro translation of the
two cDNAs resulted in polypeptides that migrated at 102 and 45 kDa,
consistent with their predicted molecular masses under nonreducing
SDS-PAGE.

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Fig. 1.
In vitro translation of wild-type (wt; full length)
Ca2+-activated
Cl channel (CaCC) and
truncated CaCC mutant construct (CaCCX). Appropriate cDNAs were
transcribed and translated in the presence of
[35S]methionine as
described in MATERIALS AND METHODS.
Translated wtCaCC migrated with a relative molecular mass of 102, whereas the truncated CaCCX polypeptide migrated with a relative
molecular mass of 45, consistent with the predicted values of 100 and
43 kDa, respectively.
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Niflumic acid blocks the endogenous CaCC.
Oocytes of the South African clawed toad Xenopus
laevis have been widely used for
Cl
channel expression
studies. There are several endogenous
Cl
channels in the oocytes,
including a CaCC and a
Ca2+-inactivated
Cl
channel. The former is
activated by increased intracellular
Ca2+ (23), and the latter is
activated by decreasing extracellular Ca2+ (35). However, both channels
can be inhibited by niflumic acid and flufenamic acid (37). In the
presence of 2 µM ionomycin (Fig.
2A), the
current of H2O-injected oocytes at
+80 mV was 653 ± 40 nA compared with a basal current of 420 ± 16 nA (n = 4; control). After the
addition of 100 µM niflumic acid, the current returned to the control
level (481 ± 26 nA). The same pharmacological effects of
Ca2+ ionophores and niflumic acid
were observed at a membrane potential of
80 mV (Fig.
2B). Figure
2C shows similar effects on the
endogenous CaCC activated by a second
Ca2+ ionophore, A-23187 (2 µM).
We have previously demonstrated that niflumic acid did not affect the
cloned CaCC expressed in oocytes (8). Thus all of the experiments on
CaCC and CaCCX were done with 100 µM niflumic acid in the perfusing
solutions to eliminate any endogenous CaCC from consideration.

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Fig. 2.
Effects of Ca2+ ionophores and
niflumic acid (NFA) on the endogenous CaCC in the membrane of
Xenopus oocytes.
A: after treatment of
H2O-injected oocytes with 2 µM
ionomycin (IONO; 5 mM extracellular
CaCl2), oocyte whole cell
currents increased above that seen in control (CTL) and are inhibited
by 100 µM niflumic acid (n = 4). Currents recorded at a membrane potential of 80 mV
also reveal activation by ionomycin and blockade by niflumic acid
(B).
C: with 2 µM A-23187, a second
Ca2+ ionophore, the endogenous
CaCC is activated and is also sensitive to 100 µM niflumic acid. To
compare the endogenous CaCC with the expressed CaCC,
y-axis scales are same as those
in Fig. 5. Inset: the same plot at
expanded scale. Results in A and
B are ±SE.
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Expression of wtCaCC and CaCCX in
Xenopus oocytes.
The whole cell currents of oocytes expressing either the wtCaCC or
CaCCX were measured after at least a 48-h incubation following cRNA
injection. Only those oocytes injected with CaCC or CaCCX that had a
basal conductance of at least 1 µA at +80 mV were identified as
functionally expressing cells and selected for further experiments. The
background currents of cRNA-injected oocytes were much higher than
those of H2O-injected cells during
perfusion with ND-96 medium. Figure 3 shows
sample records for wtCaCC (left) and
CaCCX (right) currents. The whole
cell currents in Xenopus oocytes
injected with wtCaCC and CaCCX cRNA at +80 mV were 1,972 ± 282 (n = 7) and 1,783 ± 297 nA
(n = 20), respectively, markedly
higher (P < 0.001) than those of
H2O-injected oocytes (124 ± 34 nA, n = 7). The current amplitudes at
80 mV for oocytes injected with H2O, wtCaCC cRNA, or CaCCX cRNA
averaged
40 ± 2,
399 ± 30, and
466 ± 38 nA, respectively. These results are also similar to those previously
reported for H2O-injected and
wtCaCC cRNA-injected oocytes (8).

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Fig. 3.
Representative pharmacological responses recorded from
Xenopus oocytes expressing wtCaCC or
CaCCX. Left: traces recorded from 1 oocyte injected with 25 ng wtCaCC cRNA.
Right: records from 1 oocyte injected
with 25 ng CaCCX cRNA. Cells were held at 60 mV with a 20-mV
step test potentials to +80 mV from 100 mV (see
MATERIALS AND METHODS). Perfusing
medium was ND-96 plus 100 µM niflumic acid. Ionomycin-sensitive and
dithiothreitol (DTT)-sensitive currents are the differences before and
after application of 2 µM ionomycin or 2 mM DTT.
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Extracellular Cl
sensitivity.
For oocytes injected with wtCaCC cRNA or CaCCX cRNA, large currents
were recorded at the test membrane potential of
100 mV. To
ensure that these currents were due to a
Cl
conductance, sodium
gluconate was used to replace sodium chloride isosmotically in the
superfusing solutions. Three
Cl
concentrations, 114, 50, and 6 mM, were used to assess the
Cl
sensitivity of the
current (Fig. 4). The current for the
wtCaCC at
100 mV in the presence of 114 mM
Cl
(n = 6) was
1,924 ± 108 nA
and
2,819 ± 263 nA at 50 mM
Cl
and
4,240 ± 433 nA at 6 mM Cl
.
Readdition of Cl
led to the
restoration of the normal
Cl
current level (data not
shown). Therefore, the increased inward currents recorded for oocytes
injected with cRNA are specific Cl
-dependent conductance
pathways. Subsequently, the same procedure was applied to oocytes
injected with CaCCX cRNA and perfused with ND-96 containing variable
extracellular Cl
concentrations. As shown in Fig. 5, the
expressed inward currents were increased when the normal ND-96 was
replaced with the low-Cl
medium. The current amplitude at
100 mV in 114 mM
Cl
(n = 7) was
2,259 ± 71 nA
and increased to
2,838 ± 108 nA in 50 mM
Cl
. The largest current
averaged
3,441 ± 118 nA and was recorded after 6 mM Cl
ND-96 was perfused
for more than 5 min (Fig. 5). These results indicate that CaCCX
cRNA-injected oocytes exhibited a similar dependence on external
Cl
as did wtCaCC.

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Fig. 4.
External Cl dependence of
wtCaCC. A: summary of currents
recorded at 100 mV (n = 6).
B: current-voltage
(I-V)
relationships for 1 typical cell in the presence of different
extracellular Cl
concentrations
([Cl ]o).
Reversal potential shifted from 31.2 to 12.5 and +2.4 mV,
respectively, when the superfusing medium was switched to 50 mM
[Cl ]o
and 6 mM
[Cl ]o
from 114 mM
[Cl ]o,
suggesting that these currents represent a
Cl channel. Current
increased significantly when the oocytes were perfused in 6 mM
Cl .
* P < 0.05 for 6 mM vs. 114 mM
Cl . Data in
A are ±SE.
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Fig. 5.
External Cl dependence of
CaCCX. A: averaged currents at
100 mV (n = 7) plotted as a
function of the
[Cl ]o
(* P < 0.05, 6 mM
Cl vs. 114 mM
Cl ).
B:
I-V
relationships of a typical recording. Reversal potentials in 114, 50, and 6 mM Cl were
29.8, 10.1, and +4.3 mV, respectively. Data in
A are ±SE.
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Ca2+
ionophore activation of CaCCX and CaCC.
The hallmark of the epithelial
Cl
channel from bovine
trachea is its Ca2+ sensitivity
(14, 19). As expected, newly expressed
Cl
channels were activated
by increasing intracellular Ca2+
via the application of the Ca2+
ionophores A-23187 or ionomycin. Figure 6
shows that the wtCaCC- and CaCCX-mediated
Cl
currents were elevated
by 2 µM A-23187 or ionomycin. Activated currents in the presence of 2 µM ionomycin or A-23187 were recorded for oocytes expressing CaCCX
(4,396 ± 551 nA, P < 0.01 vs.
control, n = 7) or wtCaCC (5,832 ± 425 nA, P < 0.01 vs. control,
n = 5) with 5 mM
CaCl2 in the perfusing ND-96
medium. The
I-V
curve of wtCaCC- and CaCCX-induced currents were plotted in the
presence and absence of the Ca2+
ionophores, the anion channel inhibitor, and the reducing agent as
shown in Fig. 6. The plots yielded an outwardly rectified current at
depolarizing potentials. The reversal potential of the control oocytes
expressing wtCaCC and CaCCX was approximately
30 mV.

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Fig. 6.
I-V
relationship of wtCaCC and CaCCX. Expression of wtCaCC or CaCCX induces
an outwardly rectified conductance with a reversal potential of
approximately 30 mV. A:
activation effect of 2 µM A-23187 on the expressed wtCaCC currents in
20-mV steps from 100 to +80 mV. Blocking effect of 200 µM DIDS
appears slightly greater than that of 2 mM DTT.
B:
I-V
curve of wtCaCC in the presence of 2 µM ionomycin, 2 mM DTT, and 200 µM DIDS. C:
I-V
curve of CaCCX and the current change in the presence of 2 µM
ionomycin, 200 µM DIDS, and 2 mM DTT. Data are ±SE.
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DTT and DIDS inhibit wtCaCC and CaCCX.
Studies on the native tracheal CaCC (29) and the cloned CaCC (8)
revealed that both epithelial
Cl
channels
are sensitive to the anion channel inhibitor DIDS and the
reducing agent DTT. To verify the sensitivity of the expressing CaCC
and CaCCX in Xenopus oocytes, 200 µM
DIDS and 2 mM DTT were applied separately to the ionophore-activated
oocytes (Figs. 2 and 6 and Table 1). DTT (2 mM) or DIDS (200 µM) significantly inhibited ionomycin-stimulated
currents and further reduced the basal amplitudes. DIDS (200 µM)
inhibited the currents to a greater extent (CaCCX: 565 ± 213 nA,
n = 3; wtCaCC: 769 ± 241 nA,
n = 3) than did 2 mM DTT (CaCCX: 1,222 ± 289 nA, n = 9; wtCaCC: 1,636 ± 254 nA, n = 10), indicating that
both wtCaCC and CaCCX were sensitive to DIDS
(P < 0.001; decrease of 87.1 ± 7.3% for CaCCX and 86.8 ± 3.1% for wtCaCC) and DTT
(P < 0.001; decrease of 72.2 ± 5.8% for CaCCX and 71.9 ± 2.9% for wtCaCC;
P < 0.001). These inhibitors had similar effects when A-23187 was used to increase intracellular Ca2+. These data are
consistent with wtCaCC or CaCCX cRNA expressing a DTT- and
DIDS-sensitive Cl
conductance in oocytes. The inhibitory effect of DIDS could be reversed
by washout unlike that of DTT. Addition of the same concentration of
vehicle (0.1% ethanol) had no apparent effect on oocyte currents. In
contrast, the endogenous CaCC was not sensitive to the reducing agent
DTT (data not shown).
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Table 1.
Summary of whole cell currents from wild-type CaCC or CaCCX
expressing oocytes under different conditions
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Chord conductances.
The chord conductances at +80 mV were estimated to evaluate the
functional expression of CaCC cRNA- or CaCCX cRNA-injected Xenopus oocytes and the
pharmacological responses of the channels. A concentration of 2 µM
ionomycin induced conductances of wtCaCC (70.9 ± 7.4 µS,
n = 5) and CaCCX (50.0 ± 8.3 µS,
n = 8), which were double those
observed in the absence of ionophore (31 ± 4 µS for wtCaCC,
n = 9; and 26.1 ± 3.6 µS for
CaCCX, n = 11). The outward
conductances were blocked by application of 2 mM DTT (to 16.7 ± 1.8 µS for wtCaCC, n = 10; and 15.4 ± 5.6 µS for CaCCX, n = 7) and
200 µM DIDS (to 12.7 ± 3.0 µS for wtCaCC,
n = 3; and 8.0 ± 2.4 µS for
CaCCX, n = 3), suggesting that the
outward conductances of wt and truncated
Cl
channel
(Cl
influx) were more
strongly activated by increasing intracellular Ca2+ and more sensitive to DIDS
(results not shown).
PMA activates CaCC- and CaCCX-associated currents.
Activation of PKC by phorbol esters such as PMA has been demonstrated
to induce Cl
secretion
across salt-secreting epithelia (2). Because several PKC consensus
sequences were identified in the CaCC, we tested the hypothesis that
PKC may be a regulatory factor of this cloned epithelial
Cl
channel. Addition of 100 nM PMA to H2O-injected oocytes
(Fig. 7) resulted in no detectable increase
in current at either +80 or
100 mV
(n = 4). The stimulatory effect of PMA
on CaCC- and CaCCX-expressing oocytes is also shown in Fig. 7 and
summarized in Table 1. The average of CaCC- and CaCCX-associated
currents at +80 mV in the presence of PMA alone increased significantly (P < 0.05) to 8,391 ± 761 nA from 2,888 ± 204 nA
(n = 3) and to 8,001 ± 674 nA from
3,226 ± 314 nA (n = 5),
respectively. PMA also induced a larger current at a hyperpolarizing
membrane potential (
80 mV). For the CaCCX, PMA caused a
threefold increase (P < 0.05) to
9,217 ± 691 from
2,913 ± 524 nA
(n = 5). As for the wtCaCC
(n = 3), a fourfold activation by PMA
was observed from
1,580 ± 230 to
6,593 ± 759 nA
(P < 0.05). The activating effect of
PMA was found across the whole range of step-clamping voltages (from
100 to +80 mV), and no significant difference was found between
the responses of wtCaCC and CaCCX to PMA treatment.

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Fig. 7.
Effect of phorbol 12-myristate 13-acetate (PMA) on the endogenous CaCC,
the wtCaCC, or CaCCX. Injected oocytes were exposed to 100 nM PMA,
which stimulated both the wtCaCC and CaCCX currents but slightly
inhibited the endogenous CaCC. Left:
bar plot of the averaged currents at 80 mV.
Right: averaged currents at +80 mV.
* P < 0.05. Data are
±SE.
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DIDS and DTT inhibit the PMA-activated currents of wtCaCC and CaCCX.
To further determine if the currents elicited from CaCC or CaCCX
cRNA-injected oocytes with 100 µM PMA were mediated by
Cl
channels, 2 mM DTT and
200 µM DIDS were applied to the PMA-pretreated oocytes. Both DTT and
DIDS significantly inhibited PMA-induced currents of the wtCaCC and
CaCCX (Table 1). In the presence of 2 mM DIDS, the current at +80 mV of
wtCaCC decreased to 2,184 ± 145 nA from 7,092 ± 168 nA
(PMA-pretreated control, n = 4), whereas 2 mM DTT decreased the magnitude of the wtCaCC-associated currents from 6,929 ± 246 to 2,184 ± 145 nA
(n = 3). For CaCCX-expressing oocytes,
the currents decreased to 1,583 ± 213 from 5,427 ± 1,632 nA
(n = 3) following the addition of 2 mM
DTT and to 1,343 ± 93 from 5,352 ± 374 nA
(n = 3) after the blockade of 200 µM
DIDS (P < 0.05).
Dose-response relationships of PMA on wtCaCC and CaCCX.
To test whether the pharmacological potency of PMA on the expressed
CaCC and CaCCX was affected by truncation, the concentration response
of PMA up to 1 µM was determined. The half-maximal concentration (EC50) for wtCaCC was 9.2 ± 3.1 nM at +80 mV and 10.1 ± 2.1 nM at
100 mV. PMA (100 nM)
stimulated ~90.3% of the maximal PMA-sensitive current at
100
mV. The EC50 for PMA activation of
the CaCCX was 21.0 ± 9.8 nM at +80 mV and 19.2 ± 4.8 nM at
100 mV. In the presence of 100 nM PMA, 78.8% of the maximal
PMA-induced current was activated. However, the difference between the
EC50 of CaCC and CaCCX was not
statistically significant (P > 0.05). The dose-response curves for PMA stimulation of
Cl
current for the wtCaCC
and CaCCX are shown in Fig. 8.

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|
Fig. 8.
Dose-response curve of PMA on the wtCaCC and CaCCX. Protein kinase C
(PKC) activator PMA evoked wtCaCC ( ) or CaCCX ( ) currents in a
concentration-dependent fashion from 0.1 to 1,000 nM PMA recorded at
+80 mV. Effect of half-maximal concentrations of PMA on the wtCaCC and
CaCCX are similar. Currents are normalized to the maximal inward
currents. See the text for half-maximal effective concentrations. Data
are ±SE.
|
|
Chelerythrine chloride prevents the activation of PMA on wtCaCC and
CaCCX.
To ascertain whether the stimulatory effect of PMA on the CaCC- or
CaCCX-associated currents was due to activation of PKC, we pretreated
the oocytes with 10 µM chelerythrine chloride, a specific PKC
inhibitor, for 20 min before electrophysiological recording. The
currents of CaCC- and CaCCX-expressing oocytes at +80 mV were 1,341 ± 239 nA (n = 3) and 1,649 ± 142 nA (n = 4), respectively, when
treated with chelerythrine chloride (Fig. 9). After a 10-min incubation of 100 nM PMA
in the presence of chelerythrine chloride, the currents were 1,594 ± 301 and 1,759 ± 162 nA for CaCC- or CaCCX-expressing oocytes,
respectively. There were no statistically significant differences
between currents recorded before and after addition of PMA in the
presence of the inhibitor. We also tested the inhibitory effect of
chelerythrine chloride on the currents evoked by PMA. Currents of
wtCaCC and CaCCX in the PMA-pretreated oocytes diminished gradually
following the addition of 10 µM chelerythrine chloride (data not
shown). Addition of 10 mM chelerythrine chloride to CaCC- and
CaCCX-expressing oocytes before exposure to 100 nM PMA was without
effect on the basal current (data not shown).

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|
Fig. 9.
Chelerythrine chloride prevents the activation of PMA on expressed
wtCaCC and CaCCX in oocytes. wtCaCC- or CaCCX-injected oocytes were
exposed to chelerythrine chloride (10 µM) for 20 min before the
addition of agonist (100 nM PMA). Averaged currents at +80 mV are shown
for both wtCaCC and CaCCX. Data are ±SE.
|
|
 |
DISCUSSION |
In the present study, we have examined the roles that the
NH2 and COOH termini of a cloned
CaCC play in essential channel functions such as sensitivity to
Ca2+ and to physiological
regulators and inhibitors and in the maintenance of biophysical
characteristics such as ion selectivity. These studies were initiated
because our earlier observations showed that a native tracheal CaCC
migrates at 38 kDa on reducing SDS-PAGE but as a 140-kDa complex under
nonreducing conditions (8). We therefore designed a construct based on
the cloned CaCC cDNA sequence that might conceivably mimic a
polypeptide that had been processed to form a smaller 38-kDa subunit.
Because membrane proteins are frequently glycosylated in the
endoplasmic reticulum and Golgi and the primary amino acid sequence of
the cloned CaCC protein could be glycosylated in vitro, the truncated
construct was designed to contain the four transmembrane
domains and six glycosylation sites, consistent with a
membrane-spanning protein. Because we have previously shown that the
cloned and native CaCCs are activated by CaMKII-dependent
phosphorylation (14, 19), this construct also preserved 3 of the 10 consensus CaMKII sites. In addition to determining if a smaller form of
the CaCC could make a viable channel, we also examined whether PKC
could be a physiological regulator of this conductance, given that the
primary cDNA sequence encodes 13 consensus sites for PKC
phosphorylation.
We chose Xenopus oocytes as the
expressing cell system because oocytes promiscuously translate and
process injected RNA and because niflumic acid inhibits the endogenous
oocyte CaCC but not the epithelial CaCC (8). Our results demonstrated
that the endogenous CaCC is evoked by an increase in intracellular Ca2+ triggered through incubation
with the Ca2+ ionophore and is
inhibited by niflumic acid as previously reported (37). The possibility
that the recorded currents in CaCC- or CaCCX-expressing oocytes were
contaminated by the endogenous CaCC could therefore be excluded by
inclusion of 100 µM niflumic acid in the bath (Fig. 3), enough to
inhibit all of the endogenous CaCC-associated current (37). It was easy
to distinguish the H2O injected
from the CaCC or CaCCX cRNA-injected oocytes from the respective
current amplitudes and chord conductances. In contrast, there were no
significant differences between wtCaCC- and CaCCX-expressing oocytes as
determined from the current amplitudes, the chord conductances, the
pharmacological profile, or the
Ca2+ ionophore-sensitive
components. The inhibitory actions of DTT and DIDS and the activating
effect of ionomycin are consistent with our previous observations
obtained from oocyte membrane vesicles containing wtCaCC reconstituted
into planar lipid bilayers (19), two-electrode voltage-clamp recordings
from wtCaCC-expressing oocytes, and whole cell currents from
transfected COS-7 cells (8). Because we observed identical
pharmacological properties of the currents in both wtCaCC- and
CaCCX-expressing oocytes, the truncated construct forms a functional
channel.
Our previous results on the native CaCC isolated from the trachea and
the cloned CaCC suggested that these epithelial
Cl
channels are regulated
by at least two signal transduction systems: Ca2+ and inositol phosphate
messengers. Intracellular Ca2+ and
CaMKII activate the native and cloned CaCCs, and
D-myo-inositol 3,4,5,6-tetrakisphosphate downregulates the cloned CaCC incorporated into planar lipid bilayers (14, 19). In epithelial cells, PKC and
CaMKII are activated by increasing intracellular
Ca2+ and are involved in the
regulation of Cl
secretion
(36). In contrast, the endogenous CaCC of
Xenopus oocytes is inhibited by
PKC-activating phorbol esters, as has been demonstrated by Dascal and
colleagues (3). To test the hypothesis that activation of the PKC
pathway could stimulate the expressed CaCC, the potent PKC activator
PMA was used to pretreat oocytes expressing either the wtCaCC or CaCCX.
The upregulatory influence of PMA (Figs. 7 and 8) supports the
hypothesis that PKC can regulate this expressed epithelial CaCC and its
truncated construct. Moreover, the PKC-specific inhibitor,
chelerythrine chloride, prevents the PMA-induced increase in current in
wtCaCC- or CaCCX-expressing oocytes, consistent with PKC-dependent
activation of current in these cells.
The significance of our observations that a severely truncated protein
can form a functional channel lies in the role that posttranslational
processing may play in determining channel structure. Several membrane
receptors and glycoproteins are translated as large
precursor molecules that are subject to posttranslational cleavage to a functional product. These include the human insulin proreceptor (31), cadherin precursors (18), and the hepatocyte growth
factor receptor (7). Proteolytic cleavage would be a novel processing
mechanism for an ion channel. However, our present results suggest that
processing of the wtCaCC polypeptide may account for the disparity in
molecular mass of the native and cloned CaCC, as a severely truncated
form of the protein makes a channel that is indistinguishable from the
wild-type channel when expressed in
Xenopus oocytes. Furthermore, the four
PKC sites that are preserved in CaCCX respond to PMA as does the wtCaCC construct, although 13 predicted PKC sites are available in the full-length protein. These results suggest that the regions of the
channel critical to regulation are preserved in the truncated CaCC,
although we cannot discount the possibility that subtle changes in
kinetics or submicromolar Ca2+
affinities have been altered by this truncation and would therefore reveal differences between full-length and truncated proteins. In
addition to sites for kinase phosphorylation and glycosylation, analysis of the cDNA sequence of the tracheal CaCC revealed the presence of 13 possible monobasic cleavage sites (9). Thus a large
portion of the wtCaCC could be cleaved without deleterious effects on
channel function. Whether the truncated construct truly represents the
smallest 38-kDa subunit of the native tracheal CaCC chloride channel or
whether the cloned and native channels represent two unrelated
polypeptides remains to be determined.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Yongchang Chang (Dept. of Neurobiology, Univ. Alabama at
Birmingham) and Anne Lynn Bradford for technical assistance and Khaled
F. Basiouny for oocyte preparation.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-42017 and DK-53090 and funds from the
Cystic Fibrosis Foundation.
Address for reprint requests: C. M. Fuller, Dept. of Physiology and
Biophysics, Univ. of Alabama at Birmingham, BHSB 735, 1918 Univ.
Boulevard, Birmingham, AL 35294-0005.
Received 21 August 1997; accepted in final form 3 November 1997.
 |
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