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(Circulation Research. 1996;78:936-944.)
© 1996 American Heart Association, Inc.

Articles

Unitary Cl^- Channels Activated by Cytoplasmic Ca²⁺ in Canine Ventricular Myocytes

Mei Lin Collier, Paul C. Levesque, James L. Kenyon, Joseph R. Hume

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

Correspondence to Dr J.R. Hume, Department of Physiology and Cell Biology/351, University of Nevada School of Medicine, Reno, NV 89557-0046.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Recent whole-cell studies have shown that Ca²⁺-activated Cl^- currents contribute to the Ca²⁺-dependent 4-aminopyridine–insensitive component of the transient outward current and to the arrhythmogenic transient inward current in rabbit and canine cardiac cells. These Cl^--sensitive currents are activated by Ca²⁺ release from the sarcoplasmic reticulum and are inhibited by anion transport blockers; however, the unitary single channels responsible have yet to be identified. We used inside-out patches from canine ventricular myocytes and conditions under which the only likely permeant ion is Cl^- to identify 4-aminopyridine–resistant unitary Ca²⁺-activated Cl^- channels. Ca²⁺ applied to the cytoplasmic surface of membrane patches activated small-conductance (1.0 to 1.3 pS) channels. These channels were Cl^- selective, with rectification properties that could be described by the Goldman-Hodgkin-Katz current equation. Channel activity exhibited time independence when cytoplasmic Ca²⁺ was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca²⁺ (ranging from pCa 6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NP_o, where N is the number of channels opened and P_o is open probability. At negative membrane potentials (-60 to -130 mV), an estimate of the dependence of NP_o on cytoplasmic Ca²⁺ yielded an apparent K_d of 150.2 µmol/L. At pCa 3, an average channel density of 3 µm^-2 was estimated. Calculations based on these estimates of cytoplasmic Ca²⁺ sensitivity and channel current amplitude and density suggest that these small-conductance Cl^- channels contribute significant whole-cell membrane current in response to changes in intracellular Ca²⁺ within the physiological range. We suggest that these small-conductance Ca²⁺-activated Cl^- channels underlie the transient Ca²⁺-activated 4-aminopyridine–insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca²⁺ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.

Key Words: Ca²⁺-activated transient outward current • canine ventricles • cardiac electrophysiology • Cl^- channels

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In many types of cardiac cells, a transient outward current, I_to, contributes to the initial phase of repolarization during the action potential. This current has at various times been referred to as an "early outward current," "initial outward current," or "positive dynamic current," and the charge carrier for I_to has been the subject of intensive investigation for >30 years.¹ Kenyon and Gibbons^{2 3} have provided compelling evidence that I_to in sheep Purkinje fibers consists of a voltage-activated 4-aminopyridine–sensitive K⁺ current as well as a smaller Cl^--sensitive current. Subsequent studies revealed that in many cardiac cells, I_to is composed of both Ca²⁺-insensitive and Ca²⁺-sensitive components (referred to as I_to1 and I_to2, respectively), with the Ca²⁺-insensitive component exhibiting sensitivity to block by aminopyridines.^{4 5 6} Recent molecular studies have reported the cloning of a K⁺ channel cDNA from human⁷ and ferret⁸ ventricle that expresses a Ca²⁺-insensitive I_to-like K⁺ current.

In contrast, the identification of the charge carrier for the Ca²⁺-sensitive component of I_to has remained more elusive. It has recently been shown in whole-cell current experiments that this current may be due to the activation of Ca²⁺-sensitive Cl^- channels in rabbit ventricular⁹ and atrial¹⁰ myocytes and Purkinje cells.¹¹ In canine ventricular myocytes, a similar Ca²⁺-sensitive Cl^- current has been shown to be activated by Ca²⁺ released from the sarcoplasmic reticulum and blocked by anion transport inhibitors.¹² At this time, it is not known whether or not the properties of Ca²⁺-activated Cl^- channels in heart are similar to those described in a variety of other types of tissues, including smooth muscle cells,¹³ endocrine cells,¹⁴ epithelial cells,¹⁵ secretory glands,¹⁶ and Xenopus oocytes.¹⁷

Despite the potential importance of Ca²⁺-activated Cl^- channels for cardiac repolarization and as a potential charge carrier for the arrhythmogenic transient inward current,^{12 18} unitary Ca²⁺-activated Cl^- channels have yet to be identified in heart. In the present study, we report the presence of small-conductance (1.0 pS) Cl^--sensitive unitary channels that are activated by cytoplasmic Ca²⁺ in inside-out membrane patches from canine ventricular myocytes and inhibited by niflumic acid and DIDS.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adult mongrel dogs of either sex were anesthetized with pentobarbital sodium (45 mg/kg), and their hearts were quickly removed and placed in a PSS containing (mmol/L) NaCl 126, KCl 4.4, MgCl₂ 5, CaCl₂ 1.5, dextrose 22, HEPES 12, taurine 20, creatine 5, sodium pyruvate 5, and NaH₂PO₄ 1, pH 7.4 at 22°C. All cell-dispersion steps were carried out at room temperature. A section of the right ventricle was removed, and 1- to 2-mm-thick shavings of tissue were dissected from the epicardial region. Shavings of tissue were gently stirred in Ca²⁺-free PSS for 15 minutes, after which the tissue was minced. Minced tissue was stirred in fresh PSS containing 0.1 mmol/L CaCl₂, collagenase (Worthington type 2, 0.6 mg/mL), and protease (Sigma type XXIV, 0.03 mg/mL) for 40 minutes. The pieces were then washed free of enzyme and resuspended in PSS containing 0.1 mmol/L CaCl₂. Cells released from the tissue by stirring were slowly resuspended in PSS containing 1.5 mmol/L CaCl₂. All cells studied were rod-shaped and exhibited clear striations.

Conventional patch-clamp techniques were used to record whole-cell and single-channel currents from inside-out patches.¹⁹ Patch pipettes were made using borosilicate glass capillary tubing (outer diameter, 1.5 mm) with resistances of 1 to 3 M. The tips of the pipettes used for single-channel recordings were coated with a silicone elastomer (Sylgard, Dow Corning). Junction potentials were nulled before formation of a seal, and a 3-mol/L KCl agar salt bridge between the bath and an Ag/AgCl reference electrode was used to minimize changes in liquid junction potential. Whole-cell voltage-clamp recordings were obtained using an Axopatch 1-B patch-clamp amplifier (Axon Instruments). Data were filtered at a frequency of 2 kHz and digitized on-line at 5 kHz using an IBM PC/AT computer and pCLAMP 5.5.1 software. Single-channel currents were recorded using a Dagan 3900A amplifier (Dagan Corp). Channel activity was recorded at a gain of 500 mV/pA, filtered with a four-pole Bessel filter at 50 Hz, stored on videotape, and later digitized at 1 kHz and analyzed on an IBM PC/AT computer. Under these conditions, single-channel events shorter than 20 milliseconds will be missed or distorted. For some experiments, single-channel currents were digitized on-line. pCLAMP 6.0.1 software was used for single-channel analysis.

For whole-cell current recordings, the extracellular (bath) solution contained (mmol/L) NaCl 140, CsCl 5.4, MgCl₂ 1, CaCl₂ 2.5, HEPES 5.5, glucose 10, and 4-AP 2, pH 7.4 (NaOH). The intracellular (pipette) solution contained (mmol/L) cesium aspartate 110, TEA-Cl 20, CsCl 20, MgCl₂ 1.0, MgATP 5, and HEPES 5, pH 7.2 (CsOH). Cesium and TEA were used to block K⁺ channels. Aspartate was used as the replacement anion in experiments in which [Cl^-]_o was changed. The liquid junction potential between standard intracellular and extracellular solutions was 10 mV, and the whole-cell voltages were corrected accordingly. For single-channel experiments, the standard bath and pipette solutions contained (mmol/L) NMG-Cl 140, CsCl 5, MgCl₂ 2.3, EGTA 1, HEPES 10, and dextrose 10, pH 7.4 (HCl). In experiments in which [Cl^-] was varied, NMG-Cl was replaced with NMG-aspartate, and MgCl₂ was replaced with MgSO₄. In some experiments, HEPES was replaced with Tris base, and MgCl₂ was omitted by replacement with NMG-Cl to prevent the possibility of divalent block at strong positive potentials.²⁰ To encourage tight seal formation, the pipette solution had a pCa of 5. To exclude the possibility of Cs⁺ conducting through the transient outward K⁺ channel, all pipette solutions also contained 10 mmol/L 4-AP and 2 mmol/L TEA.¹¹ To achieve a pCa of 9, the standard bath solution contained 10 mmol/L EGTA, and a Ca²⁺ titration program was used to obtain a range of pCa levels (from 9 to 3). All command voltages and single-channel current data were displayed in the standard convention.

Amplitudes of single-channel currents at various voltages were manually reviewed; the smallest current amplitude that could be reliably measured was 0.03 pA. NP_o at any one potential was obtained from the following equation:

(1)

where N is the number of channels in a patch, P_o is the open probability for a single channel, t_s is total time spent at each current level (corresponding to s=1,2, . . . n channels open), and T is total time of recording. In some experiments, to estimate channel density with pCa 3, N was estimated by measuring the maximal number of open-channel current levels from the zero current baseline previously determined at pCa 9.

I-V relations were plotted for each experiment, and conductance of the channel was calculated by fitting a straight line to the linear portion of the graph; in symmetrical Cl^- conditions (150 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o), conductance was measured between -90 and +90 mV; in asymmetrical conditions; conductance was measured between -80 and -140 mV (150 mmol/L [Cl^-]_i/5 mmol/L [Cl^-]_o) or between +50 and +100 mV (5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o, 77.5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o). All grouped data are expressed as mean±SEM. I-V relations were fitted to the current equation as follows^{21 22} :

(2)

where I is the measured single-channel current; V_m is membrane potential, z is valence; R, T, and F are the usual thermodynamic constants; and P is permeability. Distributions of all-points amplitude histograms were fitted by gaussian curves. In order to summarize and provide a quantification of the effect of niflumic acid on the kinetics of channel gating, we compared autocorrelations of current recordings obtained under control and test conditions. The autocorrelations were calculated from the covariance as described by Pallotta et al²³ :

where C(T) is the covariance function, I(t) is the channel current at time t, I(t+T) is the current at a time interval T later, is the mean current, and L is the time interval over which the autocorrelation is calculated. The covariance was obtained from a fast Fourier transform of the current spectrum, and the autocorrelation was obtained by normalizing the covariance so that C(0)=1.

Niflumic acid was prepared as a 50 mmol/L stock solution in ethanol, and the disulfonic stilbene Cl^- transport blocker DIDS was added directly to the bath solution. To prevent degradation of the compounds, solutions were made on the day of experimentation and kept in the dark. All compounds were purchased from Sigma Chemical Co. All experiments were performed at room temperature (22°C to 24°C).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In order to detect unitary Cl^- channels from inside-out membrane patches, current flow through Na⁺ and K⁺ channels was minimized using Na⁺- and K⁺-free solutions and 4-AP (10 mmol/L) and TEA (2 mmol/L) in the pipette. Under these conditions, single-channel currents could be recorded from membrane patches from canine ventricular myocytes excised into a bath solution having a pCa of 6. Channel activity usually did not run down after patch excision and was never detected in patches exposed to a bath solution with a pCa of 9. In a typical experiment, channel activity was detected in a patch excised into a bath pCa of 3 (Fig 1A). Subsequent exposure of the patch to a bath solution with a pCa of 9 (Fig 1B) caused all apparent channel activity to disappear; the residual current baseline representing a small background leak conductance was used to establish the closed-channel current baseline for this patch at -130 mV (solid lines in insets). After this procedure, the patch was subsequently exposed to a bath solution of pCa 4 (Fig 1C). With pCa 3, up to 16 individual channel openings were estimated (Fig 1A). Further exposure of the patch to a bath pCa of 4 resulted in an estimated opening of nine individual channels (Fig 1C). NP_o, calculated from 3-minute recordings, was 4.08 and 13.73 in the presence of bath pCa values of 4 and 3, respectively. In this experiment, pipette resistance was 1 M. Assuming maximal activation at pCa 3 and a free membrane patch area of 4 µm², this experiment gives a rough estimate of channel density of 4 µm^-2, suggesting that these Ca²⁺-activated channels are expressed at a relatively high density in canine ventricle.

View larger version (21K): [in this window] [in a new window]   Figure 1. Small-conductance Ca2+-dependent channels from an inside-out patch held at -130 mV. Representative single-channel currents and corresponding amplitude histograms are shown at various bath pCa levels (150 mmol/L [Cl-]i/5 mmol/L [Cl-]o). Channels were observed when the patch was excised into a bath solution of pCa 3 (A). Solid lines indicate closed levels (determined at pCa >9); dotted lines, open levels. No channels were detected at bath pCa >9, and the peak at zero amplitude is the closed level (B). At pCa 3, up to 16 channels were activated in this patch. With bath pCa 4, the corresponding amplitude histogram from 3 minutes of channel recording shows nine open-channel peaks (C). Amplitude histograms were fit with gaussian distributions. Currents were filtered at 50 Hz and digitized at a frequency of 1 kHz.

To determine whether or not Cl^- was the major charge carrier for these Ca²⁺-activated channels, we examined the effects of altering the Cl^- transmembrane gradient on recorded channel activity. Fig 2A shows representative single-channel currents recorded at various membrane potentials in the presence of a symmetrical Cl^- gradient (150 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o, pCa 4). Channel activity was absent at 0 mV, which is consistent with the predicted reversal potential for Cl^- under these conditions. Channel openings could be easily detected at potentials beyond -40 and +40 mV in this patch, and channel openings were usually long under these recording conditions, on the order of hundreds of milliseconds. Patches with just one channel present (at pCa 3) were never observed, which precluded a detailed kinetic analysis. Fig 2B shows I-V relations of channel activity accumulated from a number of membrane patches in the presence of different Cl^- gradients and with bath pCa values of 4 or 3. With a symmetrical Cl^- gradient (150 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o), channels could be detected at both positive and negative membrane potentials (open circles in Fig 2B) and exhibited a reversal potential near the predicted value of the Cl^- reversal potential (0 mV). In these experiments, [Cl^-] in the bath was subsequently changed from 150 mmol/L to 77.5 mmol/L (solid circles in Fig 2B, 77.5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o), which had little effect on the amplitude of unitary events at positive membrane potentials but decreased these amplitudes over the negative range of potentials. In other patches with 5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o, unitary currents could be detected only at positive potentials (open squares in Fig 2B), and in the presence of 150 mmol/L [Cl^-]_i/5 mmol/L [Cl^-]_o, unitary currents could be detected only at negative potentials (solid squares in Fig 2B). To determine if these channels exhibited properties consistent with a significant Cl^- permeability, data were fit to I-V relations predicted by assuming a Cl^--sensitive channel exhibiting GHK properties for the two Cl^- gradients in which both inward and outward currents could be detected. GHK fits are shown as solid lines in Fig 2B. These were well described by the GHK fits. Changing from a symmetrical Cl^- gradient (150 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o) to an asymmetrical gradient (77.5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o) resulted in a slightly outwardly rectifying I-V relation, and a shift in reversal potential of -18 mV, close to the predicted -17-mV shift expected for a Cl^--selective channel under these conditions. In patches in which channels were measured using large asymmetrical Cl^- gradients, complete I-V relations and reversal potentials could not be accurately determined because of the small unitary conductance and expected rectification, but the data were consistent with GHK theory (dotted lines in Fig 2B). The mean slope conductance (estimated from linear regression; see "Materials and Methods") was 1.03±0.08 pS (n=5), 0.77±0.06 pS (n=5), 0.91±0.12 pS (n=4), and 1.3±0.13 pS (n=11) for Cl^- gradients of 150 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o, 77.5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o, 5 mmol/L [Cl^-]_i/150 mmol/L [Cl^-]_o, and 150 mmol/L [Cl^-]_i/5 mmol/L [Cl^-]_o, respectively. These data suggest that the observed Ca²⁺-dependent channels exhibit behavior consistent with GHK theory and confirm that Cl^- is, indeed, the major charge carrier under these conditions.

View larger version (25K): [in this window] [in a new window]   Figure 2. Cl- dependence of single-channel currents in inside-out membrane patches. A, Representative single-channel currents recorded in symmetrical Cl- (150 mmol/L [Cl-]i/150 mmol/L [Cl-]o) with bath pCa 4. Solid lines indicate closed levels; dotted lines, open levels. Upward current deflections indicate the flow of Cl- from the pipette to the bath solution. Data were filtered and digitized as in Fig 1. B, Mean I-V relations of currents recorded in 150 mmol/L [Cl-]i/150 mmol/L [Cl-]o (, n=5), 77.5 mmol/L [Cl-]i/150 mmol/L [Cl-]o (, n=5), 5 mmol/L [Cl-]i/150 mmol/L [Cl-]o (, n=4), and 150 mmol/L [Cl-]i/5 mmol/L [Cl-]o (, n=11). With the exception of 150 mmol/L [Cl-]i/5 mmol/L [Cl-]o, Mg2+-free solutions were used (see "Materials and Methods"). Standard error bars are indicated at each potential when larger than the symbol size. In the case of 150 mmol/L [Cl-]i/150 mmol/L [Cl-]o and 75 mmol/L [Cl-]i/150 mmol/L [Cl-]o, data were fit to the GHK current equation (solid lines). Predicted GHK I-V relations are shown as dotted lines for 5 mmol/L [Cl-]i/150 mmol/L [Cl-]o and 150 mmol/L [Cl-]i/5 mmol/L [Cl-]o. The data obtained in 77.5 mmol/L [Cl-]i/150 mmol/L [Cl-]o were corrected for a mean -5-mV change in liquid junction potential, which occurred on changing from 150 mmol/L [Cl-]i/150 mmol/L [Cl-]o (as determined in separate patch-free experiments).

Since these channels were of small amplitude, large potentials were usually required to improve resolution. In addition, in symmetrical solutions, membrane seals were less stable at very positive potentials. In order to promote longer recordings, subsequent experiments designed to characterize the Ca²⁺ sensitivity and kinetic behavior of the channels in more detail were conducted in the presence of 150 mmol/L [Cl^-]_i/5 mmol/L [Cl^-]_o, which enabled longer stable recordings of channel activity at negative potentials.

To determine the effects of cytosolic Ca²⁺ on channel activity, the effects of different levels of bath pCa on NP_o were examined in a number of membrane patches. Fig 3A shows representative single-channel currents recorded at various levels of bath pCa for a membrane patch held continuously at -70 mV. With a bath pCa of 9, the patch was silent, with no detectable channel openings observed (Fig 3A, recording a). When the bath pCa was changed to 6, the current tracing became noisier, and some individual transitions corresponding to channel openings were observed (Fig 3A, recording b). However, because most of these openings were very brief in duration, many events were likely missed or distorted because of the frequency limitations of the recording conditions used (see "Materials and Methods"). At a bath pCa 5 or 4, the duration of channel openings was prolonged, and individual transitions could be more easily resolved (Fig 3A, recordings c and d). NP_o, calculated from 3-minute recordings, was 0, 0.19, 2.82, and 3.91 in the presence of bath pCa values of 9, 6, 5, and 4, respectively. These data show that small-conductance Cl^- channels are activated by cytoplasmic Ca²⁺ within a physiological range; however, estimates of NP_o with a pCa of 6 will be underestimated because of the frequency limitations of the recording conditions (50 Hz). Despite these limitations, we attempted to estimate the effects of bath pCa on NP_o, and data were collected from a total of 15 patches in which channel activity was measured over the range of -60 to -130 mV. Although we did not examine whether or not the activity of these small-conductance Ca²⁺-activated Cl^- channels exhibited any intrinsic voltage dependence over a wider range of potentials, voltage dependence over the range of -60 to -130 mV was expected to be minimal, and the data were pooled. Fig 3B summarizes the effects of bath pCa on NP_o. Assuming saturation at pCa 3, the data were fit to a simple binding equation with an estimated K_d of 150.2 µmol/L for cytoplasmic Ca²⁺. Again, some caution in interpreting this value for K_d is warranted, since these data will underestimate the actual value of NP_o at pCa 6. In four patches with pCa 3, the mean number of channels detected was 11.25±2.5. Assuming maximal channel activation at pCa 3 and a membrane patch area of 4 µm², mean channel density is 2.8 µm^-2.

View larger version (21K): [in this window] [in a new window]   Figure 3. Effect of cytoplasmic pCa on small-conductance Ca2+-activated Cl- channels. A, Patch was held at -70 mV, and the bath solution (cytoplasmic surface) contained pCa levels of 9 (recording a), 6 (recording b), 5 (recording c), or 4 (recording d). Solid lines represent the closed-channel level. In this experiment, a 150 mmol/L [Cl-]i/5 mmol/L [Cl-]o gradient was used. B, Effect of bath pCa on NxPo of small-conductance Ca2+-activated Cl- channels is shown. Patches were held at potentials between -60 and -130 mV, and channel activity was measured over a range of bath pCa levels. Mean data points (±SEM) were fit to the relation (solid line): NxPo=NxPo(max)/(1+Kd/[Ca2+]), where N is the number of channels, Po is open probability, NxPo(max) is Nxmaximum open probability at pCa 3 (6.14±1.75), Kd is 150.2 µmol/L, and [Ca2+] is bath Ca2+ concentration. Data were accumulated from a total of 15 inside-out membrane patches.

Experiments were also conducted to examine the kinetic behavior of the ensemble-averaged current with bath pCa kept constant. Currents were recorded during 4-second steps from a holding potential of 0 to -100 mV. Current was first recorded with a bath pCa of 9, in which no channel openings occurred, as a control to determine the closed-channel current level. Bath solution was then changed to one with a pCa of 3. Single-channel currents recorded under this condition were leak-subtracted from control tracings, and representative single-channel currents are shown in Fig 4. For this experiment, the pipette had a resistance of 4M. The averaged ensemble current calculated from a total of 16 episodes is shown at the bottom of Fig 4, and it exhibited no obvious kinetic behavior with pCa held constant at 3. This result is consistent with earlier results obtained in the same preparation with whole-cell currents¹² and confirms that Ca²⁺-activated Cl^- currents are essentially time independent when cytoplasmic Ca²⁺ is held constant.

View larger version (32K): [in this window] [in a new window]   Figure 4. A, Inside-out patch recordings of Ca2+-activated Cl- channels during voltage steps (150 mmol/L [Cl-]i/5 mmol/L [Cl-]o, bath pCa 3). Representative 4-second recordings of single-channel currents are shown. Linear leak and capacity currents have been subtracted. The averaged current from a total of 16 episodes is shown at the bottom.

To further establish that these small-conductance Ca²⁺-activated channels are carried by Cl^-, the effects of the anion transport blockers, DIDS (a stilbene disulfonic acid derivative) and niflumic acid (a carboxylic acid derivative), were examined. Although DIDS has previously been shown to block whole-cell Ca²⁺-activated Cl^- currents in canine ventricular myocytes,^{9 10 12} the effects of niflumic acid on Ca²⁺-activated Cl^- currents in heart have not been previously tested. Therefore, the effects of DIDS and niflumic acid on whole-cell aminopyridine-insensitive Ca²⁺-activated Cl^- currents in canine ventricular myocytes were compared. The voltage-clamp protocol used was similar to that previously described¹⁰ and relies on the activation of sarcolemmal Ca²⁺ channels to trigger Ca²⁺-induced Ca²⁺ release, which activates Ca²⁺-activated Cl^- currents. EGTA or BAPTA were not used in the pipette solutions so that normal endogenous Ca²⁺ buffering and Ca²⁺ extrusion mechanisms would not be disturbed. Under these conditions, a small outward current deflection was observed during the decay of the Ca²⁺ current activated during a voltage-clamp step from -40 to 10 mV (Fig 5, top recordings). I_to was inhibited within 2 to 3 minutes after adding 300 µmol/L DIDS to the bath solution (Fig 5A). The superimposed current tracings were obtained in the same cell before and 3 minutes after DIDS. The DIDS-sensitive current, obtained by subtracting the current after DIDS from control, is shown at the bottom of panel A. The shape and magnitude of this conductance is similar to the Ca²⁺-activated Cl^- conductance previously studied in this tissue and is believed to be activated by the intracellular Ca²⁺ transient.¹² Inhibition of the current by DIDS was reversible upon washout in this cell and in four additional cells. The DIDS-sensitive conductance is likely carried by Cl^-, since replacing 80% of extracellular Cl^- with aspartate (Cl^- reversal potential, 8.4 mV) also abolished this conductance, leaving only the Ca²⁺ current during the voltage step to 10 mV (not shown). These results confirm that a DIDS-sensitive, transient, Ca²⁺-dependent Cl^- conductance is present in canine ventricle. Like DIDS, niflumic acid inhibited I_to, which overlaps the decay of the Ca²⁺ current (Fig 5B). The superimposed current tracings were recorded from the same cell before and 3 minutes after bath application of 50 µmol/L niflumic acid. The niflumic acid–sensitive difference current is shown at the bottom of panel B. The effect of niflumic acid was reversible and observed in three other cells. These results suggest that niflumic acid, which has previously been shown to be an effective inhibitor of Ca²⁺-activated Cl^- current in vascular smooth muscle,²⁴ is also an effective inhibitor of macroscopic Ca²⁺-activated Cl^- currents in heart.

View larger version (12K): [in this window] [in a new window]   Figure 5. Effects of DIDS and niflumic acid (NFA) on whole-cell currents. Ca2+ currents, along with overlapping Ca2+-activated Cl- current, were elicited by applying 125-millisecond voltage steps from a holding potential of -40 to 10 mV. A conditioning train of three 250-millisecond voltage steps from -80 to 10 mV was applied 10 seconds before each test step to maintain sarcoplasmic reticular Ca2+ stores. Superimposed current tracings were recorded from the same cell before and 3 minutes after exposure to 300 µmol/L DIDS (A) or after exposure to 50 µmol/L NFA (B). Difference currents, obtained by subtracting current in the presence of inhibitor from control current, are shown below each set of tracings. Similar effects of DIDS and NFA were observed in four and three additional cells, respectively.

The effects of 50 µmol/L niflumic acid on single Ca²⁺-activated Cl^- channels in an excised membrane patch (bath pCa 4, -80 mV) are shown in Fig 6A. Within 1 minute of niflumic acid application to the bath, the channel current became noisy, with individual openings difficult to resolve (recording b) compared with control (recording a), and this effect reversed upon washout of the drug (recording c). In order to quantify the changes in kinetics of channel gating, we examined autocorrelations of 160-second recordings obtained in the control condition and 65-second recordings obtained in the presence of niflumic acid. As shown in Fig 6B, both autocorrelations showed fast and slow components but were quantitatively different. The addition of niflumic acid reduced the time constants of both the fast and slow relaxations (see Fig 6 legend) and greatly increased the relative amplitude of the fast component at the expense of the slow component. These changes are consistent with the induction of brief channel openings and closings (flicker) shown in Fig 6A.

View larger version (31K): [in this window] [in a new window]   Figure 6. Effects of niflumic acid and DIDS on unitary Ca2+-activated Cl- currents. Ca2+-activated Cl- channels were recorded in inside-out patches held at -80 mV (150 mmol/L [Cl-]i/5 mmol/L [Cl-]o; bath pCa 4; A, C, and D). Representative single-channel currents are shown before (A, recording a), in presence of (A, recording b), and after washout of (A, recording c) 50 µmol/L niflumic acid. Solid lines indicate closed levels; dotted lines, open levels. Autocorrelations calculated from recordings in control and in the presence of niflumic acid fit with double-exponential curves (B). The time constants (1 and 2) determined by the curve fits are 1=0.03 second and 2=27 seconds in the control condition and 1=0.01 second and 2=4 seconds in the presence of niflumic acid. The effects of 300 µmol/L DIDS on unitary Ca2+-activated Cl- currents are also shown (C and D). Three channels (reflected in the number of open-channel peaks) were recorded before DIDS (C). After 4 minutes of bath-applied DIDS, only one opening channel was detected (D). All currents were filtered and digitized as in Fig 1. Qualitatively similar results were observed in two other patches exposed to niflumic acid and DIDS, respectively.

The effect of 300 µmol/L DIDS on single-channel currents (bath pCa 4, -80 mV) in a separate patch is shown in Fig 6C and 6D. Three current levels were detected in this patch under control conditions (Fig 6C). The effect of DIDS was gradual, and after a 4-minute bath application, only one channel was observed open (Fig 6D). Within 4 minutes, DIDS reduced NP_o from 0.722 to 0.089.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study provides the first identification of small-conductance, Ca²⁺-activated, Cl^--selective channels that may be responsible for the Ca²⁺-dependent (4-AP–insensitive) component of I_to (I_to2) in heart. A variety of evidence presented is consistent with Cl^- being the major permeant ion for these channels. The observed change in reversal potential of the currents with changes in the Cl^- gradient indicated significant Cl^- selectivity, and rectification properties could be described by the GHK current equation. The possibility that these channels could be Ca²⁺-activated K⁺ channels²⁵ or Ca²⁺-activated nonspecific cation channels²⁶ is unlikely under the experimental conditions used in the present study. With an asymmetrical Cl^- gradient, the currents changed in a manner consistent with a significant Cl^- permeability and inconsistent with a significant cesium or NMG permeability (symmetrical gradients). Finally, the channels exhibited a similar sensitivity to block by the anion transport blockers, DIDS and niflumic acid, as whole-cell Ca²⁺-activated Cl^- currents in the same preparation.

Channel activity in the presence of asymmetrical Cl^- gradients exhibited outward rectification in the presence of low [Cl^-]_i/high [Cl^-]_o and inward rectification in the presence of high [Cl^-]_i/low [Cl^-]_o. In the presence of a symmetrical Cl^- gradient, channel conductance was linear. These properties resemble those of cAMP-dependent Cl^- channels in heart²⁷ and are consistent with GHK theory. Similarly, it has been reported that whole-cell Ca²⁺-activated Cl^- currents in Purkinje cells exhibit a linear I-V relation between -50 and +50 mV in the presence of symmetrical Cl^-.¹¹ However, these channels, like most other types of Cl^- channels, will usually exhibit outward rectification because of the asymmetrical Cl^- gradient present under normal physiological conditions.^{9 11 12}

The unitary conductance of the channels is between 1.0 and 1.3 pS. It is noteworthy that a preliminary report suggested larger conductance, unitary Ca²⁺-activated Cl^- channels (18 to 43 pS) in rabbit cardiac myocytes.²⁸ The reason for this apparent discrepancy is not clear at this time; however, the small-conductance channels reported here compare well with low-conductance Ca²⁺-activated Cl^- channels recorded in Xenopus oocytes,²⁹ endocrine cells,¹⁴ and smooth muscle cells.¹³ Noise analysis of a small Ca²⁺-dependent Cl^- conductance in cells from lacrimal glands suggested that channels underlying the current had a unitary conductance of 1 to 2 pS.¹⁶ Similarities in the properties of whole-cell Ca²⁺-activated Cl^- currents and low-conductance Ca²⁺-activated Cl^- channels in a variety of diverse preparations has prompted the recent suggestion that a single type of Ca²⁺-sensitive Cl^- channel may be expressed in mammalian tissues.³⁰

In heart, whole-cell experiments have shown that Ca²⁺-activated Cl^- currents are preferentially activated after sarcoplasmic reticular Ca²⁺ release, since these currents are abolished by caffeine or ryanodine treatment, without affecting Ca²⁺ current activation.^{9 12 31} In fact, the relative insensitivity of Cl^- currents to activation by Ca²⁺ entering through voltage-dependent Ca²⁺ channels demonstrated in these experiments might be explained by a lower intrinsic Ca²⁺ sensitivity of these channels compared with other types of Ca²⁺-sensitive sarcolemmal channels, as has already been suggested in other types of tissues (see Reference 30³⁰ ). The small-conductance unitary Ca²⁺-activated Cl^- currents reported in the present study exhibited a relatively low Ca²⁺ sensitivity that is rather similar to that reported for low-conductance Cl^- channels from cells of lacrimal glands,¹⁶ Xenopus oocytes,²⁹ and pars intermedia.¹⁴ Despite this relatively low Ca²⁺ sensitivity, these channels may still carry significant current within physiological ranges of intracellular Ca²⁺, since they are expressed in relatively high density. For example, assuming a cell surface area of 13 000 µm², a channel density of 3 µm^-2, and an estimated single channel current amplitude of 0.05 pA at 10 mV, maximal activation (P_o of 1) would result in a macroscopic current of 1.9 nA. A P_o of <0.1 could easily account for the macroscopic Ca²⁺-activated Cl^- currents of the magnitude illustrated in Fig 5. However, it should be emphasized that our results do not preclude the possibilities that the Ca²⁺ sensitivity of the channels is voltage dependent and higher at more physiological membrane potentials or that some channel properties, including Ca²⁺ sensitivity, may become altered in excised patches because of the loss of a cytosolic component, such as G proteins, or phosphorylation. Alternatively, it is also possible that the [Ca²⁺] near the channel may be different from the bulk cytoplasmic [Ca²⁺] in intact cells.^{32 33} However, modulation and regulation of Ca²⁺-activated Cl^- channels may be even more complicated in heart. Two components of whole-cell Ca²⁺-activated Cl^- current have recently been identified in rabbit Purkinje cells.³⁴ The first component quickly activated and relaxed before intracellular Ca²⁺ reached a peak, whereas the second component showed slower activation and lower Ca²⁺ sensitivities. Future single-channel studies should reveal whether or not multiple types of unitary Cl^- channels with different Ca²⁺ sensitivities may explain these observations.

We conclude that these low-conductance Ca²⁺-activated Cl^- channels are responsible for the Ca²⁺-dependent I_to previously described in rabbit and dog myocytes.^{9 10 11 12} The channels are (1) Cl^- selective, (2) dependent on intracellular Ca²⁺ for activation, (3) 4-AP resistant, (4) time independent with constant intracellular Ca²⁺, and (5) blocked by the anion transport blockers, niflumic acid and DIDS. Channel activity is greatly increased by elevation of intracellular Ca²⁺, suggesting that these channels may normally contribute to phase-1 repolarization and, under conditions of Ca²⁺ overload, also contribute to I_to and to the development of cardiac arrhythmias.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine GHK = Goldman-Hodgkin-Katz I-V = current-voltage Ito = transient outward current NMG = N-methyl-D-glucamine NPo = open probability (Po), where N is the number of channels PSS = physiological saline solution TEA = tetraethylammonium

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-52803. Dr Collier was supported by a fellowship and subsequently a Grant-in-Aid from the American Heart Association, Nevada Affiliate, Inc. We thank Dr R.J. Bauer for the computer program used to calculate the autocorrelations.

	Footnotes

 
Previously published as a preliminary report in abstract form (J Physiol [Lond]. 1995;487:146P).

Received December 15, 1995; accepted January 25, 1996.

	References

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