Differences in regulation of
Ca2+-activated
Cl
channels in colonic
and parotid secretory cells
Jorge
Arreola1,2,
James E.
Melvin1, and
Ted
Begenisich2
Departments of
1 Dental Research
and of 2 Pharmacology and
Physiology, University of Rochester Medical Center,
Rochester, New York 14642
 |
ABSTRACT |
We investigated
the regulation of Ca2+-activated
Cl
channels in cells from
the human colonic cell line T84 and acinar cells from rat parotid
glands. The participation of multifunctional Ca2+- and calmodulin-dependent
protein kinase (CaM kinase) II in the activation of these channels was
studied using selective inhibitors of calmodulin and CaM kinase II.
Ca2+-dependent
Cl
currents were recorded
using the whole cell patch-clamp technique. Direct inhibition of CaM
kinase II by 40 µM peptide 281-302 or by 10 µM KN-62, another
CaM kinase inhibitor, did not block the Cl
current in parotid
acinar cells, whereas in T84 cells KN-62 markedly inhibited the
Ca2+-dependent
Cl
current. We also used
the calmodulin-binding domain peptide 290-309 (0.5 µM), which
competitively inhibits the activation of CaM kinase II. This peptide
reduced the Cl
current in
T84 cells by ~70% but was without effect on the channels in parotid
acinar cells. We conclude that the
Ca2+-dependent
Cl
channels in T84 cells
are activated by CaM kinase II but that the channels in parotid acinar
cells must be regulated by a fundamentally different
Ca2+-dependent mechanism that does
not utilize CaM kinase II or any calmodulin-dependent process.
exocrine acinar cells; human colon carcinoma cells; fluid and
electrolyte secretion; calmodulin; calmodulin and calmodulin kinase
inhibitors
 |
INTRODUCTION |
FLUID SECRETION ACROSS epithelial cells involves the
activation of several apical
Cl
channels, including
those dependent on intracellular
Ca2+ (1). The properties of
Ca2+-activated currents from
different cell types are similar, conceivably suggesting the existence
of a family of Ca2+-dependent
Cl
channels. One of the
best-studied secretory cell model systems is the human colonic cell
line designated T84. We have investigated Ca2+-activated
Cl
channels in rat parotid
acinar cells (3-5) and found that these channels share many
properties with those in the T84 cell line (7, 8, 25). The similarities
of these Ca2+-activated
Cl
channels include
1) activation that is both voltage
and time dependent, 2) currents that
exhibit an outwardly rectifying current-voltage relation, and
3) channel activation that is
inhibited by intracellular acidification.
The Ca2+-dependent channels
present in the T84 cell line are regulated by a multifunctional
Ca2+- and calmodulin-dependent
protein kinase (CaM kinase) II mechanism. This conclusion results from
several studies of the sensitivity of these channels to activated CaM
kinase II as well as CaM kinase II inhibitors (7, 8, 25, 26).
Ca2+-activated channels in several
other epithelial cells appear to be regulated by a similar mechanism
(9, 12, 18, 24). Because the mechanism of activation of the channels in
rat parotid acinar cells has not previously been investigated, we
examined the possibility that these apparently similar channels are
also activated by a CaM kinase II mechanism. We compared the actions of
several CaM kinase II inhibitors on the
Ca2+-activated
Cl
channels in both T84 and
in rat parotid acinar cells. All these agents markedly reduced
Ca2+-activated
Cl
currents in T84 cells
but, under identical conditions, were without effect on the channels in
parotid cells. These results indicate that it is unlikely that CaM
kinase II is involved in the activation of the
Cl
channels in parotid
acinar cells.
 |
METHODS |
Cell culture.
T84 cells (CCL-248, American Type Culture Collection,
Rockville, MD) were grown at 37°C in a humidified chamber gassed
with 95% air and 5% CO2. Cells
cultured in 50% Ham's F-12 medium-50% Dulbecco's modified Eagle's
medium containing 5% fetal bovine serum were dispersed by
incubation for 20 min with Ca2+-
and Mg2+-free phosphate-buffered
solution. Dispersed cells were plated on glass coverslips (Rochester
Scientific, Rochester, NY) and incubated for an additional 48 h before
use.
Single cell dissociation.
Single acinar cells were dissociated from parotid
glands of male (150-250 g) Wistar strain rats (Charles River,
Kingston, NY) as previously described (2). Glands were minced in
Ca2+-free minimum essential medium
(MEM; GIBCO BRL, Gaithersburg, MD) containing 1% bovine serum albumin
(BSA; fraction V, Sigma Chemical, St. Louis, MO). Tissue was treated
for 20 min at 37°C with a 0.02% trypsin solution
(Ca2+-free MEM + 1 mM EDTA + 2 mM
glutamine + 1% BSA). After the reaction had been quenched with 2 mg/ml
soybean trypsin inhibitor (Sigma), the tissue was further dispersed by
two sequential treatments with collagenase (100 U/ml, type CLSPA,
Worthington Biochemical, Freehold, NJ) in
Ca2+-free MEM + 2 mM glutamine + 1% BSA. The dispersed tissue was then centrifuged and washed with
basal medium Eagle (GIBCO BRL) supplemented with 2 mM glutamine, and
the resuspended cells were plated onto
poly-L-lysine-coated coverslips.
Whole cell patch clamp.
The whole cell patch-clamp technique (13) was used to
record Cl
currents from
single parotid acinar and T84 cells (3). The actions of CaM kinase II
inhibitors were assayed at least 8 min after achieving whole-cell
recording mode to allow for equilibration of the pipette solution with
the cell interior. Cells were bathed in a solution containing (in mM)
135 tetraethylammonium (TEA) chloride, 0.5 CaCl2, 20 N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
(TES), and 60 D-mannitol, pH
7.3. In most experiments,
Cl
currents were activated
by application of the Ca2+
ionophore ionomycin (4 µM). The
Ca2+-sensitive current was
obtained by subtracting the nonstimulated basal current from the
current in the presence of ionomycin. These cells were dialyzed with
(in mM) 135 TEACl, 5 TEAF, 1 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA)-TEA, and 20 TES, pH 7.3. Possible differences in
ionomycin-induced intracellular
Ca2+ levels were eliminated by
directly activating the channels with 250 nM free
Ca2+ in a buffered pipette
solution containing (in mM) 36.2 EGTA-TEA, 26.2 CaCl2, 2.5 TEAF, and 50 TES, pH
7.3. The membrane potential was changed by delivering square pulses of
500 or 2,500 ms every 5 or 10 s, respectively, from a holding potential
of
50 mV. Current-voltage relations were determined from the
currents at the end of the test pulse. Under the conditions used in
this study, these currents are close to steady-state
levels. An Ag-AgCl pellet was used to ground the bath through a 1 M
CsCl agar bridge. Membrane potentials were corrected for liquid
junction potentials. Data were collected using custom-designed software
and hardware.
The calmodulin antagonist peptide 290-309 was obtained from
Calbiochem (San Diego, CA) and the CaM kinase II inhibitor peptide 281-302 was obtained from Research Biochemicals (Natick, MA). These peptides were dissolved in the appropriate pipette solution at 10 times the relevant half-inhibitory concentration. CaM kinase II
inhibition by KN-62 was accomplished by either the addition of 10 µM
KN-62 to the pipette solution or by pretreatment of cells with 10 µM
KN-62 for at least 30 min in the dark. For the pretreated cells, KN-62
was also present in the external solution during the recording period.
Reverse transcription-polymerase chain reaction amplification.
Total RNA was isolated from rat parotid glands using
TRIzol reagent (GIBCO BRL), and cDNA was synthesized according to
previously described methods (5). The cDNA was amplified using
polymerase chain reaction (PCR) primer sets that recognized the
calmodulin-binding region and the association domain of rat CaM kinase
II genes (23). The 50-µl PCR reaction mixture contained 2 µl of the
cDNA-containing solution, 0.2 µM upper (U) and lower (L)
primers, 3 µl of 25 mM MgCl2, 4 µl of 2.5 mM dNTPs, 5 µl of 10× PCR buffer [500 mM KCl and 100 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, pH
8.3] and 0.2 µl (1 U) of AmpliTaq DNA polymerase (Perkin-Elmer,
Norwalk, CT). PCR amplification was carried out for 30 cycles in a DNA thermal cycler (MJ Research, Watertown, MA), and each cycle was set to
94°C for 30 s, 69°C for 30 s, and 72°C for 1 min. A 2-µl aliquot of the first-round PCR reaction mixture was reamplified using
the same PCR conditions. PCR amplification products were separated on
1% Tris base-EDTA-boric acid-agarose gels and further analyzed by DNA sequencing.
Based on published sequences (23), four rat CaM kinase II-specific
oligonucleotide primers for the ubiquitous
- and
-isoforms were
synthesized as follows:
U,
5'-CGCCACCTGCACCCGCTTCACCGAC-3';
U,
5'-CGGAAATTGAAGGGTGCCATCTTGAC-3';
L,
5'-GGATGACCCCGCAGGCCCAGATATCCACAG-3';
L,
5'-TGTAAGCCTCGAAGTCCCCATTGTTGATAG-3'.
Results using the
U and
L primers are shown in Fig.
1. An ~220-base pair product
was amplified. The image was digitized with an IS-1000 imaging system
(Alpha Innotech, San Leandro, CA). Sequence analysis confirmed that
parotid glands contain CaM kinase II-
transcripts.
No PCR products were detected using the
U and
L primers (data not
shown).

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Fig. 1.
Reverse transcription-polymerase chain reaction (RT-PCR) of CaM kinase
II- transcripts in rat parotid gland. Amplified products from
parotid gland (P), molecular weight markers (M; 1-kilobase ladder,
GIBCO BRL), and a negative control ( ) with no template added are
shown. Sequence analysis of ~220-base pair RT-PCR product verified
that CaM kinase II- transcripts are present in rat parotid gland.
|
|
 |
RESULTS |
Ca2+-activated
Cl
currents.
Figure 2 illustrates the general similarity of
Ca2+-activated
Cl
currents in parotid
acinar (Fig. 2, A and
C) and T84 cells (Fig. 2,
B and
D). Figure 2,
A and
B, shows currents recorded in the absence of the Ca2+ ionophore
ionomycin (unstimulated). Illustrated are currents recorded during voltage-clamp steps to
80 and +80 from a holding potential of
50 mV. The currents from both cell types are quite small (<0.2 nA) in the absence of ionomycin and increase to high levels (1-2 nA) in the presence of ionomycin. The magnitude and time course of the currents in the two types of cells are generally similar and consistent with the high levels of intracellular
Ca2+ induced by this concentration
of ionomycin (4). Although we did not do an in-depth pharmacological
analysis of the Ca2+-activated
Cl
currents in rat parotid
acinar cells, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) produced a voltage-dependent block similar to that previously reported for T84 cells (3). In five acinar cells treated
with 500 µM DIDS, the currents were inhibited by 49 ± 4 at +60 mV
and 17 ± 9% at
60 mV, whereas in T84 cells the currents were inhibited by 52 ± 12 at +50 mV and 20 ± 14% at
50
mV with 200 µM DIDS (3).
Effects of CaM kinase inhibition on
Ca2+-activated
Cl
currents.
Although Fig. 2 shows
certain similarities between the
Ca2+-activated
Cl
channels in the two cell
types, Fig. 3 shows that they
differed in their response to the presence in the patch pipette of the CaM kinase inhibitor KN-62. KN-62 selectively inhibits CaM kinases by
competing with calmodulin for the calmodulin-binding site on the kinase
with an inhibition constant
(Ki) of ~1
µM for CaM kinase II (14). Even at 10 times this concentration, KN-62
does not inhibit protein kinase A or protein kinase C (14). As in Fig. 2, currents to potentials of
80 and +80 mV are shown in Fig. 3. Figure 2, A and
C, shows that ionomycin was still able
to substantially activate current in parotid cells in the presence of
KN-62. In contrast, this compound inhibited the ability of ionomycin to activate the channels in T84 cells (Figure 2,
B and
D).

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Fig. 2.
Ca2+-activated
Cl currents from a parotid
acinar cell and from a T84 cell. Raw currents from a parotid acinar
cell (A and
C) and from a T84 cell
(B and
D) before
(A and
B) and during
(C and
D) application of 4 µM ionomycin.
Displayed currents were obtained during voltage-clamp steps to +80 and
80 mV from a 50 mV holding potential. Dashed lines, 0 current.
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Fig. 3.
Actions of KN-62 on Ca2+-activated
Cl currents from a parotid
acinar cell and from a T84 cell. Raw currents from a parotid acinar
cell (A and
C) and from a T84 cell
(B and
D) dialyzed with 10 µM KN-62
before (A and
B) and during
(C and
D) application of 4 µM ionomycin.
Displayed currents were obtained during voltage-clamp steps to +80 and
80 mV from a 50 mV holding potential. Dashed lines, 0 current.
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Results from several experiments like those shown in Figs. 2 and 3 are
summarized in Fig. 4. Figure
4A shows that the ionomycin-stimulated current-voltage relations obtained in the absence
(n = 5) and presence
(n = 4) of KN-62-dialyzed parotid
cells are not significantly different
(P = 0.44). In contrast, KN-62
(n = 5) inhibited ~80-90% of
the ionomycin-activated current in T84 cells over the entire voltage
range (Fig. 4B).

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Fig. 4.
Effects of intracellular dialysis with the multifunctional
Ca2+- and calmodulin-dependent
protein kinase (CaM kinase) II inhibitor KN-62 on current-voltage
relations for currents recorded from parotid acinar cells
(A) and from T84 cells
(B). Ionomycin-induced (4 µM)
Cl currents were recorded
from control cells ( ; n = 5 for
parotid cells and n = 5 for T84 cells)
and from cells dialyzed with 10 µM KN-62 ( ;
n = 4 for parotid cells and
n = 5 for T84 cells). Currents for
parotid cells in presence of KN-62 were not significantly different
from controls (P > 0.05).
Em, membrane
potential.
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The differential effects of KN-62 in parotid and T84 cells was not
dependent on the use of ionomycin to activate the channels. Figure
5 shows current-voltage
relations from parotid (A) and T84
cells (B) obtained at a fixed (250 nM) level of intracellular Ca2+.
There was no difference between the magnitude of
Ca2+-dependent
Cl
currents in parotid
cells obtained under control conditions
(n = 12) and currents after
pretreatment for >30 min with the membrane-permeable KN-62
(n = 6). Under identical conditions,
T84 cell currents in the presence of KN-62
(n = 7) were ~50% smaller than in
control cells (n = 6), less than the
80-90% inhibition seen in Fig. 4, which is likely a result of
dilution of intracellular KN-62 by the pipette solution.

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Fig. 5.
Effects of pretreatment with the CaM kinase II inhibitor KN-62 on
current-voltage relations recorded from parotid acinar cells
(A) and from T84 cells
(B). Current-voltage relations were
constructed for currents activated by 250 nM free
Ca2+ from untreated parotid acinar
and T84 cells ( ; n = 12 for parotid
cells and n = 6 for T84 cells) or
cells pretreated for at least 30 min with 10 µM KN-62 ( ;
n = 6 for parotid cells and
n = 7 for T84 cells).
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CaM kinase II activation of
Ca2+-dependent
Cl
channels in parotid
acinar and T84 cells was evaluated using direct inhibitors of this
particular CaM kinase isoform. One such inhibitor, peptide 281-302, mimics the autoinhibitory domain of CaM kinase II, thus competitively and selectively inhibiting the catalytic site with a
Ki of ~4 µM (22).
It has been shown that 20 µM peptide 281-302 inhibits nearly
75% of Ca2+-dependent
Cl
current in T84 cells
(7). Figure 6 shows the current-voltage relations obtained from parotid acinar cells dialyzed with
(n = 4) and without
(n = 7) 40 µM peptide 281-302.
Plotted are Ca2+-dependent
Cl
currents activated by
increasing
[Ca2+]i
with 4 µM ionomycin. Peptide 281-302 did not significantly affect the Ca2+-dependent
Cl
current in parotid
acinar cells.

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Fig. 6.
Actions of the CaM kinase II inhibitor peptide 281-302 on
activation of Ca2+-dependent
Cl current in rat parotid
acinar cells. Current-voltage relations were constructed for
ionomycin-activated Cl
currents recorded from cells dialyzed without ( ;
n = 7) and with ( ;
n = 4) 40 µM peptide 281-302.
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Effects of a specific calmodulin inhibitor on
Ca2+-activated
Cl
currents.
Unlike peptide 281-302, which binds at the
catalytic site on CaM kinase II, the calmodulin-binding domain (CBD)
peptide 290-309 binds to calmodulin (with a
Ki of 52 nM) and so
inhibits CaM kinase II (6, 15, 20) as well as other
calmodulin-dependent processes. Figure 7
summarizes the current-voltage relations obtained from parotid cells
(A) and from T84 cells
(B) dialyzed with and without 0.5 µM CBD peptide. The magnitude of ionomycin-induced
Cl
currents in T84 cells
was inhibited ~70% by 0.5 µM peptide
(n = 5). In contrast, the currents in
rat parotid cells were not changed by the CBD peptide.

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Fig. 7.
Effects of the calmodulin antagonist peptide 290-309
[calmodulin-binding domain (CBD) peptide] on activation of
Ca2+-dependent
Cl current in parotid
acinar cells (A) and T84 cells
(B). Current-voltage relations were
constructed for ionomycin-activated
Cl currents recorded from
cells dialyzed without ( ; n = 4 for
parotid cells and n = 5 for T84 cells)
and with ( ; n = 4 for parotid cells
and n = 5 for T84 cells) 0.5 µM
peptide 290-309.
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|
 |
DISCUSSION |
Our results demonstrated an inhibition of
Ca2+-dependent
Cl
channel current in T84
cells by KN-62 and the CBD peptide 290-309 and are thus consistent
with the requirement for CaM kinase II activation of the channels in
these cells (7, 8, 25, 26). Conversely, under identical conditions, we
found that selective inhibitors of calmodulin and CaM kinase II were
without effect on the
Ca2+-activated
Cl
channel in rat parotid
acinar cells. Thus CaM kinase II activity is not required for
activation of Ca2+-dependent
Cl
channels in these
exocrine cells. This indicates that the
Ca2+-activated
Cl
channels in parotid
acinar cells are rather different from the channels in T84 cells (7, 8,
25, 26) and in the other cell types (9, 12, 18, 19, 21, 24) in which
CaM kinase II involvement has been established.
Because peptide 290-309 mimics the calmodulin-binding site on CaM
kinase II, it acts by binding to calmodulin and so inhibits all
calmodulin-dependent processes. Consequently, our results indicate that
Cl
channel activation in
parotid acinar cells is not only independent of CaM kinase II but of
calmodulin-dependent activity as well.
The Ca2+-sensitive
Cl
channels in cell-free
patches from human airway epithelia (11), guinea pig hepatocytes (16),
and submandibular gland cells (17) are activated by application of
Ca2+ to the intracellular surface,
suggesting that Ca2+ may directly
activate these channels. Our results showing the failure of CaM kinase
antagonists to inhibit channel activation in parotid acinar cells are
consistent with a direct activation of the
Cl
channel by
Ca2+. We have shown previously
that Ca2+ activation of the
channels in rat parotid acinar cells is voltage dependent (4). This
result indicates that the
Ca2+-binding site is located
within the electrical field of the plasma membrane. One interpretation
of these results is that intracellular Ca2+ directly bind to
Cl
channels to open the
pore.
In conclusion, there appear to be two distinct
Ca2+-dependent
Cl
channels in secretory
epithelia: channels in exocrine gland cells directly activated by
Ca2+ and channels like those found
in other secretory cells, including airway and colonic epithelia
(7-9, 12, 18, 24-26), whose activation requires CaM kinase
II. A calmodulin-dependent
Cl
channel has recently
been cloned from bovine tracheal epithelium (10). Functional expression
of this channel suggests that it shares many of the properties of the
Ca2+-dependent
Cl
channels present in T84
cells and so may represent the prototypical CaM kinase II-dependent
Cl
pathway. Further studies
will be necessary to establish the molecular identity of
Ca2+-activated
Cl
channels that are not
dependent on calmodulin-sensitive processes.
 |
ACKNOWLEDGEMENTS |
We thank Drs. P. Hinkle, A. Persechini, A. Rich, and T. Shuttleworth for critical reading of the manuscript and Dr. Keerang Park for assistance with the reverse transcription-PCR experiments.
 |
FOOTNOTES |
This work was supported in part by National Institute of Dental
Research Grant DE-09692.
Current address of J. Arreola: Instituto de Fisica, Universidad de San
Luis Potosi, San Luis Potosi 78290, Mexico.
Address for reprint requests: J. E. Melvin, Dept. of Dental Research,
University of Rochester Medical Center, 601 Elmwood Ave., Box 611, Rochester, NY 14642.
Received 15 January 1997; accepted in final form 20 September
1997.
 |
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