From the Inositide Signaling and
Membrane
Signaling Groups, Laboratory of Signal Transduction, NIEHS, National
Institutes of Health, Research Triangle Park, North Carolina 27709 and
the ¶ Department of Molecular and Cellular Physiology, College of
Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576
Received for publication, February 6, 2001, and in revised form, February 26, 2001
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ABSTRACT |
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We have studied the regulation of
Ca2+-dependent chloride
(ClCa) channels in a human pancreatoma epithelial cell line
(CFPAC-1), which does not express functional cAMP-dependent
cystic fibrosis transmembrane conductance regulator chloride
channels. In cell-free patches from these cells, physiological
Ca2+ concentrations activated a single class of
1-picosiemens Cl The plasma membrane of most cell types contains
Cl Ins(3,4,5,6)P4 is a cellular signal that accumulates
following PLC activation (6, 7). When polarized epithelial monolayers were treated with a cell-permeant analogue of
Ins(3,4,5,6)P4, Ca2+-dependent
Cl With regard to the nature of the human ClCa
channels that are regulated by Ins(3,4,5,6)P4, these have
not previously been characterized at either the gene or protein level.
The current study focuses on ClCa channels in the CFPAC-1
human pancreatoma ductal cell line (13). Different species of
ClCa channels can be distinguished by biophysical,
pharmacological, and molecular criteria (1). The bCLCA1 channel
mentioned above has a unitary conductance of 25-30 pS (14), and it has
the diagnostic pharmacological property of being insensitive to
niflumic acid (12, 15). Putative 13-pS human homologues of bCLCA1 have
been identified (16, 17), but it is not known if
Ins(3,4,5,6)P4 influences the conductance of these
channels. In the current study using CFPAC-1 cells, we describe an
alternate type of human ClCa channel that has a unitary conductance of 1 pS. This particular channel is activated by both Ca2+ and by CaMKII and is niflumic acid-sensitive. We
further demonstrate that, in contrast to bCLCA1, only the
CaMKII-dependent activation process is inhibited by
Ins(3,4,5,6)P4 in CFPAC-1 cells; direct channel activation
by Ca2+ is not inhibited by Ins(3,4,5,6)P4.
These data provide a new context for understanding the physiological
relevance of Ins(3,4,5,6)P4 in the longer term regulation of Ca2+-dependent Cl Cell Culture--
CFPAC-1 cells were obtained from the American
Type Cell Culture (Manassas, VA). Cells were grown in Iscove's
modified Dulbecco's medium (Hyclone, Logan, UT) supplemented with 10%
fetal bovine serum (Hyclone) in 5% CO2. Cells were
harvested with 0.25% trypsin (Hyclone) and seeded on glass coverslips
(Deutsche Spielglas, Carolina Biological, Burlington, NC) for patch
clamp experiments. All experiments were performed on cells 1-2 days
after seeding, with cell passages between 23 and 30.
Purification of CaMKII--
CaMKII was purified as follows.
Crushed frozen rabbit brain (Pelfreez, Rogers, AK) was homogenized, the
100,000 × g supernatant was applied to
calmodulin-Sepharose in the presence of calcium, and enzyme was eluted
in buffer containing EGTA, as previously described (18, 19). Protein
was analyzed by electrophoresis on a 12% SDS-polyacrylamide gel
(NOVEX) transferred to nitrocellulose, probed with monoclonal
antibodies against anti-CaMKII
Immediately prior to its use, CaMKII was made autonomous of
Ca2+ as previously described (20). In some experiments,
CaMKII was denatured by incubation at 95 °C for 15 min.
Electrophysiology--
All electrophysiological experiments were
performed using an AxoPatch200B amplifier (Axon Instruments, Foster
City, CA). Voltage clamp protocols and data acquisition were controlled
by pClamp8 software (Axon Instruments). Analog data were filtered at 1 kHz and then digitized at 10 kHz with a DIGIDATA 1321A digitizer (Axon Instruments). All experiments were performed at room temperature.
Whole-cell Recording--
Whole-cell Cl
The whole-cell Cl Single Channel Recording--
The pipette solution for
cell-attached and excised inside-out patch clamp recording contained
145 mM NMDG-Cl, 2 mM MgCl2, 1 mM CaCl2, 100 nM charybdotoxin, and
10 mM Hepes pH 7.4. Seals in excess of 70 G
For inside-out recording, patches were first excised into
"base-line" bath solution containing 145 mM NMDG-Cl, 1 mM MgCl2, 0.68 mM
CaCl2, 2 mM BAPTA, and 10 mM Hepes,
pH 7.2 ([Ca2+] ~ 0.1 µM). Where
indicated, the [Ca2+] in the bath solution was then
switched to 0.5 µM. When the effect of CaMKII was to be
studied, the same base-line solution was used, except for the
addition of 1 mM MgATP.
The CaMKII that was used in these experiments was a truncated (1)
type
Results were presented as means ± S.E. of n
observations. Statistical significance was determined using unpaired
Student's t test. p values < 0.05 were
considered statistically significant.
Other Materials--
Calphostin C was purchased from BIOMOL
Research Laboratories. Ins(3,4,5,6)P4 was purchased from
CellSignals (Lexington, KY). Niflumic acid, UTP, and ATP were obtained
from Sigma. Okadaic acid and
2,5-di-(tert-butyl)-1,4-hydroquinone were purchased from Calbiochem. Finally, calmodulin was obtained from New England Biolabs.
Diphenyl-amine-2-carboxylic acid was purchased from Fluka, and
p-tetra-sulfonato-tetra-methoxy-calix[4]arene
was a kind gift from Dr. R. Bridges (University of Pittsburgh)
Purinergic Activation of a 1-pS Cl
Within 150 s following UTP addition, channel activity declined to
the base-line level (NPo ~ 0.05, Fig. 1,
B and C). Similar transient channel activation
was also observed after mobilizing intracellular Ca2+
stores with 50 µM
2,5-di-(tert-butyl)-1,4-hydroquinone (data not shown). The
channels that were recorded after the addition of either UTP (Fig.
1D) or BHQ (data not shown) showed a linear I/V
relationship with an estimated single channel conductance of ~1 pS.
No other channels were ever activated by UTP.
Ca2+ Stimulates the 1-pS Cl
Nevertheless, in 40 of 120 single-cell patches, prior to the
onset of ORCC and with [Ca2+] buffered to 0.1 µM (Fig. 2B), single channel openings were initially infrequent (NPo < 0.1) at all voltages. Elevation
of Ca2+ from 0.1 to 0.5 µM activated
ClCa channels with unitary currents of ~0.1 pA at +70 mV
(Fig. 2B). In 10 patches, the onset of ORCC was sufficiently
delayed to enable us to determine that the ClCa channels
had a conductance of ~1 pS (Fig. 2E). No alternative ClCa channels with a different unitary conductance were
ever activated by raising [Ca2+]. The value of
Po/N was higher at more depolarizing voltages (in a representative experiment with the same patch, Po/N = 0.47 at +90 mV;
Po/N = 0.08 at
In some experiments, instead of elevating [Ca2+], a
partially purified and constitutively active CaMKII was added with ATP
to excised inside-out patches. Provided the onset of ORCC was
sufficiently delayed (see above), a CaMKII-activated Cl Ca2+-mediated Activation of Whole-cell Cl
When intracellular [Ca2+] was elevated from 0.1 to 0.5 µM, the whole-cell Cl
The above experiments indicate that a sustained
ClCa-mediated current can be maintained by an elevated
level of intracellular [Ca2+]. Next, we addressed the
individual contributions to this macroscopic current of direct channel
activation by Ca2+, as compared with channel activation by
CaMKII. To determine the participation of endogenous CaMKII, we used
AIP to selectively inhibit CaMKII. The data from these experiments
(Fig. 4) compare steady-state outward
whole-cell current amplitudes measured at +40 mV and inward whole-cell
current at
We also studied the effects of a synthetic peptide, corresponding to
residues 290-309 of CaMKII. As well as this peptide directly preventing calmodulin from activating CaMKII, it binds and antagonizes other cellular actions of endogenous calmodulin (25). However, the
degree of inhibition of Cl The Effect of Ins(3,4,5,6)P4 upon
Ca2+-activated and CaMKII-activated Whole-cell
Cl
To further test the conclusion that Ins(3,4,5,6)P4
prevents CaMKII from activating whole-cell Cl The major impact of our study comes from the new insight it
provides into the biological significance of Ins(3,4,5,6)P4
as a regulator of a human 1-pS ClCa channel that mediates
both Ca2+-gated and CaMKII-dependent whole-cell
Cl-selective channels. The same channels
were also stimulated by a purified type II
calmodulin-dependent protein kinase (CaMKII), and in
cell-attached patches by purinergic agonists. In whole-cell recordings,
both Ca2+- and CaMKII-dependent mechanisms
contributed to chloride channel stimulation by Ca2+, but
the CaMKII-dependent pathway was selectively inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4). This
inhibitory effect of Ins(3,4,5,6)P4 on ClCa
channel stimulation by CaMKII was reduced by raising
[Ca2+] and prevented by inhibition of protein phosphatase
activity with 100 nM okadaic acid. These data provide a new
context for understanding the physiological relevance of
Ins(3,4,5,6)P4 in the longer term regulation of
Ca2+-dependent Cl
fluxes in
epithelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conducting channels that are activated by increases in
intracellular [Ca2+] (1, 2). These calcium-activated
Cl
(ClCa) channels are believed to have
several important physiological functions, including regulation of
membrane excitability (1, 3) and the maintenance of salt and fluid
balance (2). Pharmacological up-regulation of ClCa channels
in secretory epithelia represents a potential therapeutic strategy for
cystic fibrosis, in which there is defective expression of the
cAMP-activated Cl
channels encoded by the cystic
fibrosis transmembrane conductance regulator gene (4). Thus, there is
increasing interest in understanding the mechanisms through which
Ins(3,4,5,6)P41
specifically inhibits ClCa channels (see Ref. 5 for a review).
secretion was inhibited (6, 8). Direct perfusion of
Ins(3,4,5,6)P4 into single cells has also been shown to
inhibit ClCa channels in whole-cell patch clamp experiments
(9-11). As for the mechanism of action of Ins(3,4,5,6)P4,
experiments with recombinant bovine tracheal ClCa channels
(bCLCA1), incorporated into lipid bilayers, indicate
Ins(3,4,5,6)P4 to be a direct channel blocker (12). The
latter study also indicated that Ins(3,4,5,6)P4 inhibited activation of bCLCA1 by either Ca2+ or CaMKII (12).
fluxes in
human epithelial cells. Our new results also add substantially to our
insight into the complexities inherent in the integration of various
signaling inputs into overall Cl
channel regulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or anti-CaMKII
(Life
Technologies, Inc.), and visualized with 4-chloro-1-naphthol. It is the
usual practice to add 10% (v/v) glycerol to stabilize the purified
CaMKII (18). Unfortunately, these concentrations of glycerol, when they
are introduced into the cell during electrophysiological analyses (see
below), elicit considerable activation of swelling-induced Cl
currents (data not shown). Thus, glycerol was not
added to our preparations of CaMKII, with the result that they
progressively lost activity upon storage at 0-4 °C. Indeed, after 2 weeks of storage, CaMKII activity had declined below a usable level.
currents
across the plasma membrane were isolated pharmacologically by replacing
monovalent cations with N-methyl-D-glucamine, which does not permeate even nonselective cation channels (21). For
conventional whole-cell recording through ruptured membrane patches,
the bath solution contained 145 mM NMDG-Cl, 2 mM MgCl2, 1 mM CaCl2,
15 mM glucose, and 10 mM Hepes, pH 7.4. The
medium added to the pipette contained 40 mM NMDG-Cl, 105 mM NMDG-glutamate, 1 mM MgCl2, 1 mM MgATP, 2 mM BAPTA, 10 mM Hepes,
pH 7.2, and an appropriate concentration of CaCl2
(estimated using MaxChelator software, Stanford University) to give a
[Ca2+]free between 0.1 and 1 µM. The osmolarities of the bath and pipette solutions
were measured with a Micro Osmometer (model 5004; Precision Systems
Inc., Natick, MA), and then, by the addition of 10-20 mM
maltose to the pipette solution, its osmolarity was adjusted to about 5 mosM less than the bath solution; this prevents the activation of volume-activated Cl
currents. The pipette
tip was prefilled, either with or without Ins(3,4,5,6)P4
but always in the absence of any peptides, which improves seal
frequency. When required, CaMKII autoinhibitory peptide (AIP) or
CaMKII-(290-309) (both supplied by BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), were added into the pipette
solution at their final concentration. It was at this point that, in
experiments that required autonomous CaMKII (see above), the latter was
added to the pipette. In this case, recordings did not begin for an
additional 2 min, in order to permit the CaMKII to diffuse toward the
pipette tip. The membrane under the pipette was then ruptured in order
to begin the experiment (seals >5 G
), and the pipette
solution was dialyzed into the cell. Recordings were commenced 30 s after the membrane was ruptured to permit sufficient time for the
pipette solution to equilibrate throughout the intracellular milieu.
Where indicated, calphostin C, okadaic acid, or niflumic acid were
added to the bath solution for at least 5 min before performing
whole-cell recordings.
currents were monitored with a protocol
that began by voltage clamping the cell at
30 mV for 30 ms. Then the
voltage was switched to
100 mV for 200 ms, stepped back to
30 mV
for 30 ms, stepped to 100 mV for 200 ms, and finally stepped back to
30 mV. This sequence of voltage steps was repeated every 5 s for
5-10 min. I/V relationship of Ca2+- or
CaMKII-activated whole-cell Cl
current was obtained with
the following protocol. Membrane potential was held at
30 mV and then
stepped to
110 mV for 50 ms to remove any
voltage-dependent inactivation. Potentials were then
stepped to a 300-ms test pulse ranging from
100 mV to +100 mV (in
increments of 20 mV) before stepping back to
30 mV. All reported
current was measured at 5 ms before the end of test pulse and
normalized by cell capacitance. I/V curves from whole-cell
recording were fitted with a second order polynomial equation. The
value for current density was used to estimate channel density, based
on the current recorded through single channels and the assumption that
membrane capacitance was 1 microfarad/cm2.
were
first obtained in bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM Hepes, pH 7.4. For cell-attached single channel
measurement, the bath solution was then switched to a solution
containing 145 mM KCl, 2 mM MgCl2,
1 mM CaCl2, 10 mM glucose, 10 mM Hepes pH 7.4, in order to depolarize the cell to near 0 mV.
isoform purchased from New England Biolabs (Beverly, MA). This
CaMKII was made autonomous according to the manufacturer's instructions. The ability of CaMKII to phosphorylate autocamtide II, a
synthetic and specific substrate, was evaluated with a commercial kit
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Under these assay conditions (with [Ca2+] buffered to
0.25, 0.5, or 1 µM), Ins(3,4,5,6)P4 at 10 µM had no effect on CaMKII activity (data not shown). All
channel activities were recorded continuously in Gap-Free mode for
30 s at various holding potentials. Data were filtered at 10 Hz
and analyzed for the unitary current and open probability with the
"Single" program (ASF Software, Nova Scotia, Canada). To improve
the signal/noise ratio in all of the single channel recordings,
pipettes were coated with three layers of sylgard before fire polishing
to 10-15 megaohms.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Channel in CFPAC-1
Cells--
The unitary conductance is a biophysical fingerprint that
can catalogue different members of a family of ion channels. All of the
currently known ClCa channels have unitary conductances in
the 1 - 30-pS range (1). Our first goal was to use this biophysical
criterion to identify the nature of receptor-activated ClCa
channels in CFPAC-1 cells. Single channel recordings were obtained from
cell-attached patches on intact CFPAC-1 cells (Fig. 1A). To isolate
Cl
channels, N-methyl-D-glucamine
was substituted for all monovalent cations in the pipette solution, and
K+ channels were blocked with charybdotoxin (see
"Experimental Procedures"). Prior to receptor activation, there was
little channel activity (NPo ~ 0.08; Fig.
1B and t = 0 s trace
in Fig. 1C). When 100 µM UTP was added to the
bath, we observed channels with a unitary current of approximately 0.1 pA (NPo ~ 0.8; Fig. 1B and t = 120 s trace in Fig. 1C). These extremely small
currents were distinguished easily from the background fluctuations in current amplitude by filtering the digitized records at 10 Hz. On
average, the unitary current events had durations greater than 0.1 s, so discrete openings and closing could be resolved.
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Fig. 1.
UTP-activated single Cl channel
recordings. A, a schematic describing the cell-attached
patch recordings at Vm = 100 mV. UTP (100 µM) was
perfused onto the cell for 120 s, and the open probability of the
channel was recorded for an additional 60 s (B and
C). The closed channel state is indicated by the
arrows marked with a C. D describes
the I/V relationships of the UTP-activated Cl
channels. Each data point was averaged from 1-6 patches and fitted
with linear regression. The estimated unitary conductance was 1.1 pS
(R > 0.99).
Channel by
Two Mechanisms--
In order to determine the gating mechanisms for
the 1-pS ClCa channel in CFPAC-1 cells (Fig. 1), we
analyzed patches that were excised from the cells in the inside-out
configuration (cytoplasmic side of the membrane facing the bath; Fig.
2A). A total of 120 patches
were obtained. These experiments were performed in the absence of ATP,
so there could not be any channel phosphorylation. In all but ~5% of
patches, when [Ca2+] was buffered to 0.5 µM, it was possible to observe small conductance ClCa channels. However, in most experiments these channels
could not be analyzed, because they were swamped by a 50-pS outwardly rectifying Cl
channel (ORCC; Fig. 2D). The
latter was generally activated in as little as a few seconds after
excision of the patch, although occasionally the latency period was
several minutes. Membrane depolarization reduced the lag time that
preceded the onset of ORCC. This high conductance ORCC became active
even when [Ca2+] was buffered to 0.1 µM
(Fig. 2D), and elevating [Ca2+] did not
further stimulate ORCC. These results are in agreement with previous
work showing that ORCC is not Ca2+-activated (22). Although
the use of 50 nM
p-tetra-sulfonato-tetra-methoxy-calix[4]arene (23) and 100 µM diphenylamine-2-carboxylic acid (24) are
reported to inhibit ORCC in some cell preparations, these drugs did not prevent the appearance of ORCC in our experiments (data not shown).
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Fig. 2.
Ca2+- and CaMKII-activated single
Cl channel recordings. A, a schematic
showing the inside-out, single-channel patch protocol. B
shows the single Cl
channel recorded at Vm = 70 mV, with [Ca2+] fixed at either 0.1 or 0.5 µM. Unitary current was estimated as 0.07 pA at
Vm = 70 mV. Data are representative of 40 single-channel
recordings obtained prior to the onset of a 50-pS ORCC (D;
see "Results"). C shows the effect of 500 units
of CaMKII upon single Cl
channel recording in an
inside-out patch at Vm = 70 mV with solution containing 0.1 µM Ca2+ and 1 mM ATP. Unitary
current was estimated as 0.08 pA. Data are representative of five
single-channel recordings that did not show the ORCC (for details, see
"Results"); a sixth cell was unresponsive to CaMKII.
E shows the I/V relationships of the
Cl
currents activated by either Ca2+-
(circles) or CaMKII (squares). Each data point
was averaged from 1-10 patches, and data were fitted with linear
regression. The estimated unitary conductance for both the
Ca2+- and CaMKII-activated Cl
channels was
1.2 pS (R > 0.99).
110 mV). A similar
phenomenon has previously been observed with a 13-16-pS ClCa channel in HT-29 cells (26). The
Ca2+-activated channel was also unaffected when endogenous
calmodulin was antagonized by a synthetic peptide (25) corresponding to residues 290-309 of CaMKII (data not shown). Thus, we conclude that
0.5 µM [Ca2+] directly activated these 1-pS
Cl
channels.
current was then observed (unitary current ~0.1 pA at +70 mV; Fig.
2C). The value of Po/N was
higher at more depolarizing voltages (in a representative experiment
with the same patch, Po/N = 0.62 at
+100 mV; Po/N = 0.33 at +50 mV). The
addition of ATP alone had no effect on channel activity (Fig. 2C). The current/voltage relationship for this channel
yielded a unitary conductance of 1 pS (Fig. 2E). We never
observed any alternative CaMKII-activated ClCa channels
with a different unitary conductance.
Current in CFPAC-1 Cells--
The experiments described above
establish the existence of a single class of 1-pS ClCa
channels in CFPAC-1 that are activated by both Ca2+ and
CaMKII. We next investigated to what extent either of these two
regulatory processes contribute to the control of whole cell Cl
current. BAPTA was employed to buffer intracellular
[Ca2+]. The Cl
currents elicited by voltage
steps of 300-ms duration from the holding potential of
30 mV
(ECl) were measured under voltage clamp through
conventional ruptured membrane patches and normalized to cell
capacitance (Fig. 3A). When
the interior of the cell was perfused with pipette solution containing
0.1 µM [Ca2+], voltage steps to either +100
or
100 mV did not elicit any current larger than ~ 4 pA/picofarads (Fig. 3B). Thus, at resting levels of
intracellular [Ca2+], there is very little
voltage-dependent or background Cl
current in
these cells, which corresponds to the low single channel activity of
1-pS ClCa channels under resting conditions (Fig. 2).
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Fig. 3.
Characteristics of a
Ca2+-dependent whole-cell current in CFPAC-1
cells. A, a schematic showing that CFPAC-1 cells were
placed under voltage clamp through conventional ruptured membrane
patches. B shows the development of the whole-cell current
at +100 mV (squares) and 100 mV (circles) when
the pipette solution contained either 0.1 µM
Ca2+ (gray shading) or 0.5 µM
Ca2+ (black shading). Once steady-state current
was obtained (indicated by the arrowhead in B),
the voltage protocol indicated in C was applied, and the
resultant currents were as described in D (representative of
three experiments). For some experiments niflumic acid (NFA)
was added to the bath, at various concentrations (F,
n = 3-7 for each data point, Vm = +100 mV) or
at a maximally effective dose of 100 µM (E and
G). G describes the I/V relationships
of these Cl
currents, recorded after 5 min; data
represent means and S.E. from 4-12 cells.
current was elevated
to a much larger steady-state value of 146 ± 8 pA/picofarads at
100 mV (n = 12), within 2-3 min of rupturing the
membrane under the patch pipette (Fig. 3B). The channel
density was estimated to be ~14 channels/µm2 at 100 mV
(see "Experimental Procedures"). The responses were highly
reproducible; the time course that is shown (Fig. 3B)
represents the average of all 12 cells recorded with intracellular
[Ca2+] buffered to 0.5 µM. Under
steady-state conditions (indicated by the arrowhead in Fig.
3B), a series of voltage steps was applied, each of 300-ms
duration (Fig. 3C); the resultant current traces showed
almost no time dependence and displayed weak outward rectification (Fig. 3D). The current was completely blocked by the
Cl
channel blocker, niflumic acid (IC50 = 3.5 µM; Fig. 3, E and F). The current
reversed at
30 mV (Fig. 3G), close to the Cl
equilibrium potential (ECl) of
32 mV.
100 mV. These two voltages are equidistant from the
ECl at
30 mV, and the two resultant currents,
while opposite in polarity, are nevertheless elicited by electrical
driving forces that are equal in magnitude. Thus, the rectification of
the current can also be estimated from these experiments (Fig. 4).
Under symmetrical electrochemical driving force, 0.5 µM
[Ca2+] activated a whole-cell current (Fig. 4,
Control) with slight outward rectification. The inhibition
of CaMKII by 5 µM AIP elicited a 57% reduction of the
outward whole-cell current at +40 mV and 84% inhibition of the inward
current at
100 mV (Fig. 4). Thus, at 0.5 µM
intracellular [Ca2+], 60-80% of the macroscopic current
was due to channel activation by CaMKII, with the remainder due to
direct channel activation by Ca2+ itself.
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Fig. 4.
Ca2+- and CaMKII-activated
whole-cell Cl current. The figure shows
outward whole-cell currents recorded at +40 mV (white bars)
and inward currents recorded at
100 mV (black bars) with
the pipette solution buffered to 0.5 µM
[Ca2+]. Where indicated, we added either 150 nM calphostin C, 5 µM AIP, 5 µM
CaMKII-(290-309), or 5 µM AIP plus 5 µM
CaMKII-(290-309). Data are presented as means and S.E. from 7-12
cells.
current by CaMKII-(290-309)
was no different from that elicited by AIP (Fig. 4). Moreover, the
effects of CaMKII-(290-309) and AIP were not additive (Fig. 4). The
latter result verifies that both inhibitors were employed at maximally
effective concentrations. Data in Fig. 4 also show that the role of
calmodulin in regulating channel activity is solely due to its
stimulation of CaMKII. A selective inhibitor of protein kinase C (PKC),
calphostin C, was a useful control that had no significant effect upon
whole-cell Cl
current (Fig. 4).
Current: The Influence of
[Ca2+]--
Previous work has shown that whole-cell
receptor-activated Cl
current in CFPAC-1 cells is
inhibited by Ins(3,4,5,6)P4 (11). We next studied how this
action of Ins(3,4,5,6)P4 related to the separate abilities
of Ca2+ and CaMKII to activate ClCa channels.
We examined the effects of Ins(3,4,5,6)P4
upon outward Cl
current
measured at +40 mV and inward Cl
current measured at
100 mV (Fig. 5, Table
I). Three interesting new findings were
made. (i) While increases in [Ca2+] activated
whole-cell current, its inhibition by AIP, as a percentage of total
current, was not substantially affected by changes in [Ca2+] (Fig. 5, Table I). This result indicates that
endogenous CaMKII makes a substantial contribution to whole-cell
current at all tested concentrations of Ca2+. (ii) The
whole-cell current that was observed in the presence of AIP was not
significantly different from the current obtained with AIP and
Ins(3,4,5,6)P4 added together (Fig. 5, Table I). In other
words, the AIP-insensitive current (i.e. the proportion of
the current that was activated by Ca2+ directly) was not
inhibited by Ins(3,4,5,6)P4. These data are consistent with
the inability of Ins(3,4,5,6)P4 to block
Ca2+-activated ClCa channels during
single-channel analysis of excised patches from CFPAC-1 cells (data not
shown). This was an unexpected observation, because
Ins(3,4,5,6)P4 blocks direct activation of bCLCA1 by
Ca2+ (12). Thus, the data in Fig. 5 and Table I indicate
that Ins(3,4,5,6)P4 must be specifically targeting the
process by which CaMKII activates ClCa channels. (iii) We
identified an unexpected interaction between Ca2+ and
Ins(3,4,5,6)P4. For example, at 0.25 µM
[Ca2+], Ins(3,4,5,6)P4 reduced whole-cell
current by 60-70%, but at 1 µM [Ca2+],
only 17% of whole-cell current was inhibited by
Ins(3,4,5,6)P4 (Fig. 5, Table I). Despite this, in the
absence of Ins(3,4,5,6)P4, the proportion of whole-cell
current that was activated by CaMKII was similar at both concentrations
of Ca2+ (see point (i)). Thus, we conclude that increases
in [Ca2+] attenuate the degree to which
Ins(3,4,5,6)P4 inhibited the macroscopic CaMKII-activated
current.
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Fig. 5.
Ins(3,4,5,6)P4-mediated
inhibition of the Ca2+-dependent
Cl current. Shown are the effects of AIP and
Ins(3,4,5,6)P4 upon outward whole-cell currents recorded at
+40 mV and inward currents recorded at
100 mV, at either 0.25, 0.5, or 1 µM Ca2+. Where indicated, we added
either 10 µM Ins(3,4,5,6)P4, 5 µM AIP, or 5 µM AIP plus 10 µM Ins(3,4,5,6)P4. Data are presented as
means and S.E. from 5-17 cells.
Current inhibition
current in
CFPAC-1 cells, we next performed experiments with exogenous CaMKII. A
mixture of the
and
isoforms was used (Fig. 6A). The CaMKII was made
autonomous of Ca2+ by autophosphorylation prior to it being
introduced into the cell through the patch pipette. Exogenous CaMKII
caused a 10-fold increase in whole-cell current (Fig. 6B)
compared with heat-inactivated CaMKII (Fig. 6C), which had
no effect on the whole-cell current. When 10 µM
Ins(3,4,5,6)P4 was included in the pipette solution, exogenous CaMKII was prevented from activating a whole-cell
Cl
current (Fig. 6, D and F),
although Ins(3,4,5,6)P4 has no effect on inherent CaMKII
activity (see "Experimental Procedures"). The dynamic nature of
this process was illustrated by 100 nM okadaic acid
partially reversing the Ins(3,4,5,6)P4-mediated inhibition of CaMKII-activated current (Fig. 6, E and
F).
View larger version (20K):
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Fig. 6.
Effect of Ins(3,4,5,6)P4 and
okadaic acid upon CaMKII-activated whole-cell Cl
current. A, Western blots of CaMKII purified from
frozen rabbit brain; lane 1 shows the purified
CaMKII, stained with Coomassie Blue. Lanes 2 and
3 were stained with antibodies raised against
and
isoforms of CaMKII, respectively (see "Experimental Procedures").
B-E, representative whole-cell current traces for the
voltage protocol indicated in Fig. 3C. The pipette solution
was set to 0.1 µM Ca2+ and supplemented with
either 50-200 ng of active CaMKII (B) or 50-200 ng of
heat-inactivated CaMKII (C) or 50-200 ng of CaMKII plus 10 µM Ins(3,4,5,6)P4 (D) or 50-200
ng of CaMKII plus 10 µM Ins(3,4,5,6)P4 plus
100 nM okadaic acid (E). F, the I/V
relationship for the currents described in B-E
(n = 3-7). All I/V data were collected 10 min after whole-cell recordings commenced.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
current. Our results show that
Ins(3,4,5,6)P4 inhibited this ClCa channel by
specifically targeting the CaMKII-dependent activation process. The alternate pathway for direct activation of this
ClCa channel by Ca2+ was unaffected by
Ins(3,4,5,6)P4. Moreover, as [Ca2+]
approached the 1 µM level, it reduced the efficacy of
Ins(3,4,5,6)P4 as an inhibitor of channel activation by
CaMKII. These various elements of channel regulation are
summarized in Fig. 7. From this model, we
predict that immediately following receptor activation, during the
transient, acute phase of Ca2+ mobilization,
Ins(3,4,5,6)P4 will have little impact upon direct ClCa channel activation by Ca2+ itself.
Subsequently, after the mean [Ca2+] declines below its
peak level, CaMKII remains constitutively active through an
autophosphorylation process, the strength of which depends upon the
intensity of the original Ca2+ stimulus (27). It is this
longer term regulation of ClCa channel activity by CaMKII
that will be countered by Ins(3,4,5,6)P4. The physiological
role of Ins(3,4,5,6)P4 therefore emerges as being primarily
involved in attenuating the longer term activation of ClCa
channel activity by CaMKII. This conclusion places in an important
perspective the observation that Ins(3,4,5,6)P4 is itself a
long lived intracellular signal (6, 8).
View larger version (29K):
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Fig. 7.
Model for regulation of ClCa
channels during PLC activation. The schematic depicts how
ClCa channel activity is regulated following PLC
activation, which elevates cytosolic [Ca2+] and increases
levels of Ins(3,4,5,6)P4 (IP4). + and , pathways
of activation and inhibition, respectively.
Ca2+-activated whole-cell Cl current has been
observed in earlier studies in many cell types (1). However, using
inhibitory peptides (AIP and CaMKII-(290-309)) under conditions where
intracellular [Ca2+] was clamped to controlled
steady-state values, we were able to resolve the individual
contributions that Ca2+ and CaMKII each make toward this
Cl
current (Fig. 4). It is worth pointing out that
AIP-sensitive (CaMKII-dependent) inward current measured at
100 mV is more prominent than the outward current measured at +40 mV
at all levels of [Ca2+] (Fig. 5, Table I). This suggests
that Cl
efflux (represented as inward current)
during physiological stimulation is mostly sustained by a
CaMKII-dependent process. Direct Ca2+
activation makes a larger contribution to the outward
Cl
current measured at +40 mV (Fig. 5, Table I). This is
in agreement with a previous study with parotid acinar cells showing
that the affinity for Ca2+ binding to ClCa
channels increased at depolarizing voltages (28).
Single channel analyses of 120 excised patches only identified two
types of Cl channels in CFPAC-1 cells. One of these was a
50-pS depolarization-activated ORCC (Fig. 2). Earlier work concluded
that this type of ORCC was not active in intact cells (29).
Subsequently, it was found that ORCC could be activated by plasma
membrane cystic fibrosis transmembrane conductance regulator
(22), but this is not present in CFPAC-1 cells (13). ORCC is not
activated by Ca2+ either in intact cells (22) or in excised
patches (Fig. 2). Since ORCC does not contribute to whole-cell
Ca2+-activated Cl
current in CFPAC-1 cells,
the only candidate for mediating this current is the 1-pS
ClCa channel that we have characterized. It is significant
that the 1-pS ClCa channel in CFPAC-1 cells was not only
gated by direct Ca2+ binding but was also activated by
Ca2+ indirectly, via a CaMKII-dependent
mechanism (Fig. 2). The open probability of the 1-pS channel was
considerably higher at more positive, depolarizing voltages (see
"Results"), which can account for the outward rectification of the
whole-cell current in agreement with earlier work with HT-29 cells
(26). This may be due to the influence of the applied voltage upon the
affinity of the ClCa channels for Ca2+
(28).
Our 1-pS ClCa channel can be appended to a subgroup of ClCa channels that have previously been shown to share a unitary conductance of 1-4 pS (30-32). Previously, only the ability of Ca2+ to activate this class of "low conductance" channels was known for sure. Other ClCa channels that have hitherto been shown to be activated by CaMKII all display substantially higher unitary conductances in the 14-40-pS range (15, 33-35).
Earlier molecular studies have distinguished two members of the human
ClCa channel family, hCLCA1 and hCLCA2 (14, 16, 17, 36),
but these all have larger unitary conductances (13-30-pS) than the
1-pS ClCa channel that we have studied. We also used pharmacological criteria to distinguish the 1-pS channel from hCLCA1
and hCLCA2; the latter are blocked by adding 2 mM
dithiothreitol outside the cell (16, 17), whereas the ClCa
channel in CFPAC-1 cells was not significantly affected by
dithiothreitol.2 Thus, the
1-pS endogenous ClCa channel from CFPAC-1 cells represents a different member of the human Cl channel family. As for
the bovine tracheal ClCa channel, bCLCA1 (14), its
Ca2+- and CaMKII-dependent activation were both
inhibited by Ins(3,4,5,6)P4. This is clearly different from
the interaction of Ins(3,4,5,6)P4 with the 1-pS
ClCa channel, where the inositol phosphate only inhibits
CaMKII-activated and not Ca2+-activated Cl
current (Fig. 7). This could reflect species-dependent
differences in Ins(3,4,5,6)P4 function. In any case,
neither our own studies nor those of other laboratories have identified
a human channel that corresponds to the bovine channel in terms of its
unitary conductance (25-30 pS), and its insensitivity to niflumic acid.
One of the major challenges for research in cellular biology is to
understand how multiple positive and negative inputs can be integrated
by protein-based circuitry acting as computational elements (37) that
permit the cell to appropriately respond and adapt to external stimuli.
This has been the achievement of the experiments we describe in this
report, which have provided insight into the regulation of the
Cl conductance of a specific type of ion channel that
resides in the plasma membrane (Fig. 7). These studies also provide new
possibilities for pharmacological up-regulation of ClCa
channels in the treatment of cystic fibrosis. We must now identify the
molecular nature of the various constituents of this signaling network
and ascertain the precise molecular mechanisms that are involved.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Mark Carew, Andrew French, Marek Duszyk, Jerry Yakel, and Christian Erxleben for their comments on the manuscript during its preparation.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant DK 46433 and a grant from the Caroline Spahn Halfter Trust Genetic Research Fund.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.: 919-541-2630; Fax: 919-541-0559; E-mail: ho1@niehs.nih.gov.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M101128200
2 The whole-cell current activated by 0.5 µM Ca2+ was 115 ± 9 pA/picofarads (n = 22) in the absence of dithiothreitol and 83 ± 15 pA/picofarads (n = 6) when 2 mM dithiothreitol was added to the extracellular medium (p > 0.05).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Ins(3, 4,5,6)P4, inositol 3,4,5,6-tetrakisphosphate;
CaMKII, type II calmodulin-dependent protein kinase;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
AIP, autoinhibitory peptide;
ORCC, outwardly rectifying
Cl channel;
pS, picosiemens;
NMDG, N-methyl-D-glucamine.
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