Regulation of a Human Chloride Channel

A PARADIGM FOR INTEGRATING INPUT FROM CALCIUM, TYPE II CALMODULIN-DEPENDENT PROTEIN KINASE, AND INOSITOL 3,4,5,6-TETRAKISPHOSPHATE*

Melisa W. Y. HoDagger §, Marcia A. Kaetzel, David L. Armstrong||, and Stephen B. ShearsDagger

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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--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

The plasma membrane of most cell types contains Cl--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).

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- 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).

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- 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  or anti-CaMKII beta  (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.

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- 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 GOmega ), 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.

The whole-cell Cl- 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.

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 GOmega 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.

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 alpha  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 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)

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purinergic Activation of a 1-pS Cl- 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).

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- 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).

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 -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.

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- 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.

Ca2+-mediated Activation of Whole-cell Cl- 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.

When intracellular [Ca2+] was elevated from 0.1 to 0.5 µM, the whole-cell Cl- 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.

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 -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.

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- 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).

The Effect of Ins(3,4,5,6)P4 upon Ca2+-activated and CaMKII-activated Whole-cell Cl- 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.

                              
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Table I
Current inhibition

To further test the conclusion that Ins(3,4,5,6)P4 prevents CaMKII from activating whole-cell Cl- current in CFPAC-1 cells, we next performed experiments with exogenous CaMKII. A mixture of the alpha  and beta  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).


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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 alpha  and beta  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

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- 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).


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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.

    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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