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(Circulation Research. 1995;76:317-324.)
© 1995 American Heart Association, Inc.

Articles

Unitary Chloride Channels Activated by Protein Kinase C in Guinea Pig Ventricular Myocytes

Mei Lin Collier, Joseph R. Hume

From the Department of Physiology, University of Nevada School of Medicine, Reno.

Correspondence to Dr. Mei Lin Collier, Department of Physiology, University of Nevada School of Medicine, Anderson Medical Science Building, Reno, NV 89557-0046.

	Abstract

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Abstract Recent evidence suggests that protein kinase A (PKA)-activated Cl^- channels in heart are encoded by an isoform of the epithelial cystic fibrosis transmembrane conductance regulator gene (CFTR). Macroscopic current measurements indicate that a similar time-independent Cl^- conductance can be activated through a protein kinase C (PKC)-dependent pathway in guinea pig and feline ventricle. However, it is presently not clear whether PKC is activating the same population of channels as PKA or a separate class of Cl^- channels, even though the regulatory (R) domain of CFTR is known to contain consensus phosphorylation sites for both PKA and PKC. In the present study we directly compare the properties of single Cl^- channels activated by PKC and PKA in cell-attached patches of guinea pig ventricular myocytes. Pipette and bath solutions contained N-methyl-D-glucamine and Cs⁺ or tetraethylammonium as substitutes for Na⁺ and K⁺, respectively, and Cl^- concentration in the patch pipette was either 150 mmol/L or 40 mmol/L. Bath application of phorbol 12,13-dibutyrate or phorbol 12-myristate 13-acetate (PDBu or PMA; 50 nmol/L), activators of PKC, resulted in the appearance of unitary Cl^- channels with a mean conductance of 9.31±0.94 pS (n=8) and 8.8 pS (n=2), respectively, and reversal potentials were close to predicted E_Cl. Addition of staurosporine (500 nmol/L) reduced open probability (P_o) of channels activated by PDBu. Bath application of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 µmol/L) resulted in the activation of Cl^- channels with a conductance (mean 8.76±0.67 pS, n=3) similar to those activated by PDBu. Open probability (P_o) of both PKC- and PKA-activated Cl^- channels exhibited voltage independence and both were insensitive to block by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In patches containing channels preactivated by PDBu, subsequent bath application of IBMX (500 µmol/L) resulted in an increase in P_o without any evidence for the activation of a separate population of channels with a different unitary conductance. These results suggest either that PKC can activate cardiac CFTR Cl^- channels and that once activated by PKC, the channels can be further modulated by PKA or that PKC and PKA activate separate populations of Cl^- channels with similar conductance and kinetic properties.

Key Words: Cl^- channels • protein kinase C • guinea pig ventricle • CFTR • cardiac electrophysiology

	Introduction

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Many properties of the cardiac protein kinase A (PKA)-regulated Cl^- channel, which plays an important role in the regulation of action potential duration and resting membrane potential, closely resemble those of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel. Molecular studies have indicated that cardiac CFTR is an alternatively spliced isoform of epithelial CFTR.¹ Both cardiac and epithelial cAMP-regulated Cl^- currents are time and voltage independent, exhibit Cl^- gradient–dependent rectification properties, and are blocked by carboxylic acid derivatives. The single-channel conductance is between 7 and 13 pS, and phosphorylation of channel proteins by PKA requires hydrolyzable nucleotide triphosphates.^{2 3 4} In human epithelial CFTR, PKA-regulated Cl^- channels are selectively permeable to Br^->Cl^->I^->F^-. In cardiac muscle, PKA-regulated Cl^- channels have been reported to have a permeability sequence of Cl^-I^{-4 5} ; however, a sequence of I^->Cl^- has also been reported.^{6 7}

Recent studies have reported activation of a Cl^- current in guinea pig^{6 8} and feline ventricle⁹ by a protein kinase C (PKC)-dependent pathway. Based upon the properties of macroscopic currents activated by PKC, these studies disagree as to whether PKC and PKA may activate the same or separate populations of Cl^- channels. From studies in feline ventricle it was concluded that both PKA and PKC activated the same Cl^- channels, because once the channels were maximally activated by PKA, PKC did not activate additional current.⁹ In contrast, it was reported that in guinea pig ventricle PKC activated Cl^- channels with properties similar but not identical to PKA-activated Cl^- channels. Although effects of PKA on PKC-activated Cl^- currents were not strongly additive, it was suggested that this was due to a desensitization effect of PKC on the adenylate cyclase system. The relative permeability of the PKC-activated Cl^- currents for I^- was slightly greater than that for Cl^-,⁶ in contrast to the anion permeability of PKA-activated Cl^- channels, in which I^- permeability has generally been reported to be less than or equal to Cl^-.^{5 10} However, such comparisons are complicated by the finding that I^- permeability relative to Cl^- strongly depends upon the direction of anion transport in epithelial CFTR Cl^- channels.¹¹ In cardiac myocytes it was found that I^- readily enters open CFTR channels but leaves them only slowly.⁷ The identification of the unitary Cl^- channels activated by PKC in heart might be helpful in resolving whether or not this kinase activates the same population of Cl^- channels as PKA.

In epithelial cells, there is electrophysiological and biochemical evidence for activation of CFTR Cl^- channels by both PKA and PKC.¹² Molecular studies have also shown that the regulatory (R) domain of epithelial CFTR contains consensus phosphorylation sites for both PKA and PKC.¹³ Recently, the amino acid sequence of the R domain of the rabbit cardiac form of CFTR was determined. The cardiac R domain was found to contain all of the consensus phosphorylation sites for both PKA and PKC and showed 89% homology to epithelial CFTR in this region.¹⁴ Given these molecular similarities, we used the cell-attached configuration of the patch-clamp technique to compare the properties of single Cl^- channels activated by PKA and PKC in cell-attached patches of guinea pig ventricular myocytes.

	Materials and Methods

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Single guinea pig ventricular myocytes were isolated using a modification of the enzymatic dispersion technique described previously.¹⁵ Briefly, the heart was rapidly removed from a euthanized guinea pig (200-300 g) and the coronary arteries were cleared of blood by retrograde perfusion of the aorta with a syringe filled with physiological saline solution (PSS) containing (mmol/L): 126 NaCl, 4.4 KCl, 5 MgCl₂, 1.5 CaCl₂, 22 dextrose, 12 HEPES, 20 taurine, 5 creatine, 5 Na-pyruvate, and 1 NaH₂PO₄, at 37^°C. Once the heart was free of blood it was hung and perfused with PSS for a further 5 minutes. This solution was then exchanged with Ca²⁺-free PSS for 3 minutes prior to the addition of enzymes. Collagenase (Worthington type 2, 0.6 mg/mL) and protease (Sigma type XXIV, 0.02 mg/mL) were added to PSS containing 0.1 mmol/L CaCl₂ and the heart was gently perfused for 10 minutes. The enzymes were washed from the heart during a final perfusion with PSS containing 0.1 mmol/L CaCl₂. The left ventricle was removed, minced, and resuspended in PSS containing 1.5 mmol/L CaCl₂. All cells studied were rod-shaped and exhibited clear striations.

In whole-cell studies where the cytosol is dialyzed with pipette solution, PKC-activated Cl^- currents are small and difficult to detect.⁸ To prevent washout of PKC and to study channels activated in the presence of endogenous levels of PKC and PKA, we kept intracellular pathways intact by using the cell-attached patch-clamp configuration to record single Cl^- channels.¹⁶ Disadvantages in using this technique include uncertainties related to the actual patch membrane potential and uncertainties related to the actual value of intracellular Cl^-. To minimize the former, cells were bathed in an external K⁺-free solution in order to eliminate potential interference from cationic channels and to reduce resting membrane potential. The actual cell membrane potential using this external solution was -24.8±1.14 mV (n=50), which was used to correct all patch potentials reported.

Borosilicate glass electrodes (1.5 mm OD) with resistances of about 500 k when filled with pipette solution were connected to a patch amplifier (Dagan 3900A, Dagan Corporation). These large patch pipettes as used to increase the likelihood of measuring single Cl^- channels, which are presumably less dense than other ion channels in the cardiac sarcolemma.^{2 4 17} A 3 mol/L KCl in agar salt bridge between the bath and a Ag-AgCl reference electrode was used to minimize changes in liquid junction potential, and junction potentials were nulled before formation of a seal. Once a gigaohm seal was established, the patch membrane was clamped to various voltages to verify the absence of channel activity prior to the addition of an agonist. The cell was then superfused with test solution and onset of channel activity was monitored at a test potential of -65 mV. Channel activity was recorded at a gain of 500 mV/pA, filtered with a four-pole low-pass Bessel filter at 100 Hz, stored on videotape, and later digitized (1 kHz) and analyzed on an IBM PC/AT-compatible computer. PCLAMP 5.5.1. software was used for single-channel analysis. Single-channel events at various voltages were manually reviewed, and the open probability of a number of channels (NP_o) at any one potential was obtained from the following equation:

where N is the number of channels in a patch, P_o is the open probability of a single channel, t_s is total time spent at each current level corresponding to s=1, 2, . . . n, and T is total time of recording.

All command voltages and current data displayed were inverted and adjusted according to the relative membrane potential measured under these experimental conditions. A current-voltage (I-V) relationship was plotted for each experiment and conductance of the channel was measured from linear regressions fitted to the most linear portion of the graph. Under conditions of asymmetrical Cl^-, linear regressions were fitted to current data at positive voltages. Outwardly rectifying properties have been attributed to blocking effects of intracellular ions at negative potentials.⁵ All grouped data are expressed as mean±SEM.

Bath solution in all experiments contained 150 mmol/L Cl^- and pipette solution used contained either 150 mmol/L or 40 mmol/L Cl^-. To prevent current flow through cation channels, solutions had Na⁺ and K⁺ ions substituted with N-methyl-D-glucamine (NMG) and Cs⁺ or tetraethylammonium (TEA), respectively, and were nominally Ca²⁺ free. The solution used to give a final Cl^- concentration of 150 mmol/L contained (mmol/L): 140 NMG-Cl, 5 CsCl, 2.3 MgCl₂, 10 dextrose, and 10 HEPES, pH 7.4 (HCl). To obtain a pipette solution containing 40 mmol/L Cl^-, 140 NMG-Cl was replaced with 110 NMG-aspartate and 30 TEA-Cl. Phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA), activators of PKC, and staurosporine, an inhibitor of PKC, were prepared as 1 mmol/L and 2 mmol/L stock solutions in dimethyl sulfoxide (DMSO), respectively. PDBu and PMA were used at a concentration shown to stimulate PKC in isolated myocytes¹⁸ (50 nmol/L), and a high concentration of staurosporine (500 nmol/L) was used to increase the likelihood for maximal inhibition. Final concentration of DMSO did not exceed 0.01%. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the disulfonic stilbene Cl^- transport blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) were added directly to the bath solution on the day of experimentation. As an added precaution against degradation, solutions containing DIDS were made just prior to the experiment and kept in the dark. All compounds were purchased from Sigma Chemical. All experiments were performed at room temperature (22 to 24°C).

	Results

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Under conditions where current flow through K⁺ and Na⁺ channels was prevented and the only permeant ion was Cl^-, 18 of 46 (39%) cell-attached patches exhibited unitary Cl^- currents in the presence of bath-applied PDBu, PMA, or IBMX; no channels were observed in any of the patches in the absence of agonists under these experimental conditions. The percentage of patches that contained Cl^- channels was greater than in previous studies using smaller conventional patch pipettes^{2 17} (5%) but was less than that observed in "giant-patch" studies⁴ (80%).

Addition of PDBu (50 nmol/L) activated Cl^- channels in 10 of 36 (27%) patches after a delay of about 5 minutes. Typically, 1 to 3 channels were present in a patch. Fig 1 shows representative channel currents with 150 mmol/L Cl^- in bath and pipette solutions and composite I-V relation of currents recorded in the presence of 150 mmol/L or 40 mmol/L Cl^- in the pipette. When the pipette contained 150 mmol/L Cl^-, channels reversed at a mean potential of -22.5±3.55 mV (n=8); in the presence of 40 mmol/L Cl^- in the pipette, channels reversed close to 0 mV (n=2). The mean reversal potentials under these conditions are close to that predicted for a Cl^--selective electrode, assuming intracellular Cl^- of 40 to 60 mmol/L. The currents were slightly outwardly rectifying with pipette Cl^- concentrations of 150 mmol/L, and mean single-channel conductance for outward currents was 9.31±0.94 pS. With 40 mmol/L Cl^- in the pipette, linear regression through the mean data points gave a conductance of 5.5 pS. In two patches, 50 nmol/L PMA, another phorbol ester, activated similar Cl^- channels, with a mean unitary conductance of 8.8 pS (pipette Cl^- concentration was 150 mmol/L).

View larger version (17K): [in this window] [in a new window]   Figure 1. Voltage dependence of single-channel currents activated by PDBu in cell-attached patches. A, Schematic diagram showing Cl- conditions (150 mmol/L in the pipette) used to activate PKC-dependent Cl- channels and below, representative channel currents recorded at various estimated transmembrane potentials under these conditions. The dotted line indicates the closed level. Two channels were active in this patch. Upward current deflections at positive potentials indicate the flow of Cl- from pipette to cell interior. Filter frequency was 100 Hz; sample frequency 1 kHz. B, Mean I-V relation of current recorded in 150 mmol/L (; n=8) and in 40 mmol/L (; n=2) external Cl- (pipette) solution. Standard error bars are indicated at each transmembrane potential. With 150 mmol/L external Cl-, the mean single-channel conductance ()=9.31±0.94 pS and channels were outwardly rectifying. With 40 mmol/L external Cl-, =5.5 pS and standard deviations were smaller than the symbols.

Kinetic analysis of PDBu-activated Cl^- channels was done in a patch containing only one channel and held at a transmembrane potential of -65 mV. Although channel openings were often flickery at negative potentials, it has been reported that mean open time, burst time, and open probability show no variation with voltage.² In this patch, the open-time histogram could be fitted to a single exponential with a time constant (_open) of 353 milliseconds. At least two exponentials were needed to fit the closed-time histogram. These channels could be described with faster (_closed1) and slower (_closed2) closed-time constants of 429 and 3058 milliseconds, respectively. Channels also exhibited bursting activity with a mean burst duration of approximately 1 second. NP_o appeared to be voltage independent over the range +15 to +60 mV; mean NP_o=0.48±0.08 (n=4).

To test whether these Cl^- channels were specifically activated by PKC, the PKC inhibitor staurosporine (500 nmol/L) was added to the bath after activating the channels with PDBu. Within 10 minutes, NP_o (in the presence of PDBu) was reduced from 0.29 to 0.089 (Fig 2). After prolonged washout the effect of staurosporine was partially reversed (NP_o=0.16; data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 2. The effect of staurosporine on channels activated by PDBu. Tracings show representative channel currents at an estimated transmembrane potential of +15 mV activated by PDBu (A) and in the presence of staurosporine for 10 minutes (B). The dotted line indicates the closed level. Two channels were active in this patch. Right, Corresponding amplitude histograms from a 3-minute segment of data fitted with a gaussian distribution. The peak at zero amplitude represents the time spent in the closed level; time spent in each open level is represented by two smaller peaks. The mean current amplitude at each open level is the same (0.4 pA). In the presence of staurosporine, the number of peaks and the number of events for the open peak are reduced. In this patch, NPo was reduced from 0.29 to 0.089, with PDBu and PDBu+staurosporine, respectively.

In three patches in which no channels were activated when exposed to PDBu for 10 to 15 minutes, subsequent addition of IBMX (500 µmol/L), however, activated Cl^- channels, with a mean conductance of 8.06±0.59 pS. In one patch, channels appeared to activate and quickly inactivate in the presence of PDBu, and addition of IBMX to this cell reactivated Cl^- channels. In these experiments, it was unclear whether channel activation was due to phosphorylation by PKA or rather due to prolonged delay of activation by PKC.

In separate experiments designed to test whether Cl^- channels could be activated by PKA in guinea pig ventricle under identical experimental conditions, IBMX (500 µmol/L) was added to the bath solution and 3 of 5 (60%) patches exhibited unitary Cl^- currents within 3 minutes of application. Fig 3 shows representative channel currents at various transmembrane potentials (Fig 3A) and a composite I-V (Fig 3B). These channels reversed at a mean potential of -33±3 mV (n=3), giving an estimated intracellular Cl^- concentration of 40 mmol/L, and were slightly outwardly rectifying under these conditions. The mean single-channel conductance was 8.76±0.067 pS. In one patch containing two channels of the same amplitude and held at a transmembrane potential of -65 mV, the open-time histogram could be fitted to one exponential and showed a _open of 493 milliseconds. At negative potentials channel openings were also flickery. These Cl^- channels were also voltage independent, with NP_o showing no obvious dependence on voltage over the range of +15 to +60 mV; mean NP_o=0.47±0.06 (n=3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Voltage dependence of single-channel currents activated by IBMX in cell-attached patches. A, Schematic diagram showing conditions used to activate PKA-dependent Cl- channels and below, representative channels of PKA-activated currents at various estimated transmembrane potentials. The dotted line indicates the closed level; only one channel is active in this patch. Frequencies are the same as Fig 1. At very negative potentials, channel openings were flickery. B, Mean I-V relation obtained from three patches. Standard error bars are indicated at each transmembrane potential. Mean single-channel conductance ()=8.76±0.67 pS and channels showed a slight degree of outward rectification.

The effects of the disulfonic stilbene compound DIDS, which blocks a variety of Cl^- channels but not CFTR Cl^- channels³ or cardiac PKA-regulated Cl^- channels,¹⁹ were tested on PKC-activated Cl^- channels. DIDS has been shown to be effective when applied to the cytosolic or external membrane surface and can also block channels recorded from cell-attached patches.²⁰ In macroscopic studies, DIDS applied to the bath effectively blocks Ca²⁺-activated Cl^- channels within 5 minutes.²¹ We used a relatively high concentration of DIDS (500 µmol/L) in one patch and the effect of bath-applied DIDS in the presence of PDBu was monitored over a period of 15 minutes. Fig 4 shows representative channel-current data from this patch, recorded at a membrane potential of -65 mV in the presence of PDBu (Fig 4A) and after 15 minutes in the presence of DIDS+PDBu (Fig 4B). The bar graph (Fig 4C) summarizes the effect of DIDS on NP_o at 3-minute intervals. NP_o in the presence of DIDS varied slightly over time and during such long channel recordings, PKC-activated Cl^- channels were seen to exhibit some bursting activity. However, PKC-activated Cl^- channels were relatively insensitive to DIDS, since little consistent effect on NP_o was observed over 15 minutes (NP_o was 0.29 in PDBu alone and 0.28 after 15 minutes in PDBu+DIDS). A similar DIDS insensitivity was observed in other patches for Cl^- channels activated by PKA (IBMX; data not shown), consistent with earlier reports.^{3 19}

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of DIDS on PKC-activated Cl- channels. Tracings show representative channel currents activated with PDBu (A) and in the presence of DIDS for 15 minutes (B), at an estimated transmembrane potential of -65 mV. The dotted line indicates the closed level and one channel is active in this patch. C, Bar graph represents the change in NPo (calculated from 3-minute segments of data) at 3-minute intervals in the presence of DIDS.

Consensus phosphorylation sites for both PKA and PKC have been identified in the R domain of both cardiac¹⁴ and epithelial CFTR,¹³ and peptide mapping studies have identified specific serines phosphorylated by PKA and PKC in epithelial CFTR.²² It is conceivable that CFTR channels phosphorylated by PKC might be further phosphorylated by PKA at different sites. Alternatively, PKC and PKA might activate different types of Cl^- channels. In guinea pig ventricle, the effect of PKA on PKC-activated whole-cell current is not additive when maximal concentrations of agonists are used, but with submaximal concentrations of agonists, the currents are roughly additive.⁹ We tested whether PKC-activated Cl^- channels could be modulated by PKA or whether PKA might activate a class of anion channels with a different unitary conductance. In these experiments, patches which exhibited PDBu-activated Cl^- channels were subsequently exposed to IBMX+PDBu added to the bath. Fig 5 shows representative channel currents (patch potential -65 mV) and corresponding amplitude histograms from one experiment. Fig 5A shows PDBu-activated Cl^- channels, and the effect of subsequent addition of IBMX to the bath is shown in Fig 5B. The amplitude histograms clearly show an increase in channel activity in the presence of IBMX, without any noticeable change in channel amplitude or the number of active channels in the patch (two active channels were present in PDBu alone and after PDBu+IBMX). The single-channel conductance of these channels in the presence of PDBu alone and after PDBu+IBMX was 8.04 pS and 8.06 pS, respectively. In this cell, NP_o was dramatically increased from 0.12 in the presence of PDBu alone to 0.71 following addition of IBMX in the continued presence of PDBu. NP_o in PDBu alone was increased from 0.2±0.046 to 0.49±0.11 by PDBu+IBMX (n=3; patch potential -65 mV), without any significant changes in unitary conductance.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of PKA on PKC-activated Cl- channels. Tracings show representative channel currents activated with PDBu (A) and in the presence of IBMX (B), at an estimated transmembrane potential of -65 mV. The dotted line indicates the closed level. Filtered and digitized as Fig 1. Right, Corresponding amplitude histograms fitted with a gaussian distribution. As in Fig 2, the peak at zero current amplitude represents the time spent in the closed state. Open-channel levels in this case are shown to the left of the zero peak (as channels are recorded at a negative transmembrane potential). With the addition of IBMX, channel activity becomes greatly increased reflected in the increase in the number of events of open peak levels. In this patch, NPo was increased from 0.12 to 0.71 in the presence of PDBu and PDBu+IBMX, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
While previous studies have identified and characterized the properties of whole-cell Cl^- currents activated by PKC in guinea pig^{6 8} and feline cardiac myocytes,⁹ there is little information presently available on the identity of the single Cl^- channels activated by PKC in heart and whether these channels are similar or distinct from those activated through the PKA pathway. In cell-attached patches in guinea pig ventricular myocytes, we have identified small-conductance Cl^- channels which are activated by the phorbol esters PDBu and PMA and have many properties in common with CFTR Cl^- channels, which are activated by PKA.

The single-channel conductance of PKA-activated Cl^- channels reported previously in guinea pig ventricle,^{2 4 17} native epithelial tissue,³ and in systems expressing CFTR^{23 24} ranges between 7 and 13 pS. We also found that the single-channel conductance for the PKA-activated Cl^- channel was within this range and interestingly, the single–Cl^- channel conductance activated by PKC was nearly the same. Furthermore, addition of IBMX to PDBu-activated Cl^- channels increased NP_o of channels without changing channel current amplitude. This may suggest that both PKA and PKC activate the same population of Cl^- channels.

With 150 mmol/L external Cl^-, both PKA- and PKC-activated Cl^- channels exhibited some degree of outward rectification in cell-attached patches. Despite uncertainties regarding the actual value of [Cl^-]_i in nondialyzed myocytes, estimates of Cl^- content in mammalian cells²⁵ would suggest that an asymmetrical Cl^- gradient was present in these experiments, and measurements of the reversal potential of PKA- and PKC-activated Cl^- channels place [Cl^-]_i in the range of 40 to 60 mmol/L. A small degree of outward rectification would be expected under these conditions for cardiac CFTR Cl^- channels,⁵ which also was observed for PKC-activated Cl^- channels. When conditions were used to closely mimic symmetrical Cl^- across the cell membrane (pipette contained 40 mmol/L Cl^-), PKC-activated Cl^- channels were more linear and reversal potentials shifted to more positive potentials. However, obvious limitations of the cell-attached method precluded any rigorous comparison of the rectification properties of PKA- and PKC-activated Cl^- channels.

The PKC- and PKA-activated Cl^- channels were voltage independent, reversal potentials were close to predicted E_Cl, and both PKC- and PKA-activated Cl^- channels had similar mean open probabilities. Channel kinetics for both PKC- and PKA-activated Cl^- channels were also similar: _open was 353 and 493 milliseconds, respectively, and at least two closed states were described for PKC-activated Cl^- channels where _closed1=429 milliseconds and _closed2=3058 millliseconds. These values of _open and _closed are within the range reported earlier for PKA-activated Cl^- channels in guinea pig ventricular myocytes.^{2 17} Furthermore, PKC-activated Cl^- channels displayed bursting activity with a mean burst duration of 1.0 second, which is comparable to the value of 0.8 second described for CFTR expressed in immortalized human airway cells.²⁶

The biophysical properties of PKA- and PKC-activated Cl^- channels examined were found to be very similar. This suggests that as with epithelial CFTR, the cardiac isoform of CFTR can also be regulated through different second-messenger pathways. However, based on macroscopic Cl^- currents in guinea pig ventricle, it has been suggested that PKC activates a Cl^- channel with properties similar but not identical to PKA-activated Cl^- channels.⁶ In our study, addition of PDBu did not activate Cl^- channels in some patches and in some instances PKA stimulation did activate Cl^- channels in these patches. This may suggest that separate Cl^- channels are phosphorylated by PKA and PKC. Alternatively, higher levels of endogenous PKA than PKC in cardiac myocytes may determine the success rate of activating Cl^- channels in cell-attached patches.

In the presence of asymmetrical Cl^- (54 mmol/L [Cl^-]_i/146 mmol/L [Cl^-]_o), phorbol esters activated a mean whole-cell current of 275±35 pA at +60 mV.⁸ By extrapolation from Fig 1B, where external Cl^- is 150 mmol/L and internal Cl^- is estimated to be between 40 and 60 mmol/L, the single-channel current amplitude is approximately 0.8 pA at +60 mV. The whole-cell and single-channel data suggest that there are 344 open channels per cell. Using a mean open probability of 0.5 (taking the mean value of NP_o and assuming that all channels have been activated in a patch), the channel density per cell is estimated to be 688 channels. Assuming a specific membrane capacitance of 1 µF · cm^-2 and a cell capacitance of 180 pF²⁷ (cell surface area equal to 18 000 µm²), the average channel density would be approximately 0.04 µm^-2. This closely matches the average channel-density calculations for cAMP-activated Cl^- channels reported previously in guinea pig ventricle (0.06 µm^-2 and 0.1 µm^-2).^{4 2} With pipette tip diameters of 9 to 10 µm, like those used in our studies, and assuming a planar circular patch of membrane, a patch would be expected to contain approximately 3 channels, which we have observed in patches which did respond to PDBu. However, our assumptions that NP_o is an accurate estimate of P_o and that the area of a patch of membrane at the pipette tip can be described as a planar circle may overestimate P_o or underestimate the number of channels present.

The possibility that PKC activates a different Cl^- channel in heart was considered. Five other macroscopic Cl^- currents have been reported in heart: a cAMP-dependent current attributable to CFTR,^{28 29} a transient Ca²⁺-activated current,^{21 30} a swelling-induced current,^{31 32 33 34 35} an ATP-activated current,^{36 37} and a basally active current.³⁸ All of these currents would display outward rectification under the conditions used in this study. Other than CFTR, it seems unlikely that the PKC-activated Cl^- channels may represent one of these currents. Macroscopic studies have shown that the Ca²⁺-activated Cl^- current,^{21 30} the swelling-induced Cl^- current,^{3 35} and the ATP-activated Cl^- current^{37 39} were all sensitive to block by DIDS, unlike the PKA- or PKC-activated Cl^- channels in our study. Additionally, the swelling-induced currents remain active in the presence of protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)³³ and N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8).³¹ Staurosporine dramatically reduced NP_o in the present study, and in addition, the solution used in this study would not be expected to cause osmotic swelling. Furthermore, our solutions were nominally Ca²⁺ free and channel activity was not observed using these solutions in the absence of agonists.

Taken together, the results of this study suggest that PKC can activate a population of Cl^- channels with properties nearly identical to those due to the cardiac isoform of CFTR. One explanation is that both PKC and PKA activate CFTR channels. Another possibility is that PKC activates a separate population of Cl^- channels with a unitary conductance similar to CFTR Cl^- channels. In our study, PKA stimulation further increased the NP_o of PKC-activated Cl^- channels. In one patch, channel activity was increased sixfold, whereas in other patches P_o was doubled by PKA stimulation. This suggests that the potentiating effect of PKA on PKC-activated Cl^- channels may vary. PKC is known to stimulate epithelial CFTR Cl^- channels to a lesser degree than PKA.^{23 24} In heart, macroscopic currents activated with phorbol esters in the absence of exogenous PKC were significantly smaller than PKA-activated Cl^- currents in the same tissues.⁶ The mechanism for potentiation by PKA is unknown; however, peptide mapping studies have demonstrated that epithelial CFTR is phosphorylated to a low level by PKC in vivo,²² suggesting that PKC may facilitate the phosphorylation of other PKA sites. There is some evidence that basal phosphorylation of some of the consensus sequences of CFTR is necessary for activation of CFTR through PKA,⁴⁰ suggesting that partial phosphorylation by PKC may expose more sites to become phosphorylated by PKA. It is noteworthy that the cardiac CFTR isoform has been shown to exhibit a high degree of conservation of PKA and PKC consensus phosphorylation sites.⁴¹

In conclusion, we have identified a population of unitary Cl^- channels activated by PKC in guinea pig ventricular myocytes. These channels have many of the same properties as cAMP-activated Cl^- channels, suggesting that both PKA and PKC may activate the cardiac isoform of CFTR. Our experiments rule out the possibility that PKC activates a population of Cl^- channels with conductance properties distinct from those activated by PKA, but we do not rule out the possibility that PKC and PKA activate different populations of Cl^- channels with similar conductance properties or that PKC activates small-conductance channels which are below the limits of our resolution. Such a distinction may require the use of CFTR antisense oligonucleotides in future studies.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-30143. Dr Collier is supported by a fellowship from the American Heart Association, Nevada Affiliate. We thank Mike Sokoloff for his technical assistance and Dr Craig Gelband for advice regarding single-channel data analysis.

Received June 30, 1994; accepted November 2, 1994.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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