Involvement of anion channel(s) in the modulation of the transient outward K+ channel in rat ventricular myocytes

Xiao-Gang Lai,1 Jun Yang,1 Shi-Sheng Zhou,1,2 Jun Zhu,1 Gui-Rong Li,3 and Tak-Ming Wong3

1Department of Physiology, The Fourth Military Medical University, Xi'an 710032; 2Institute of Basic Medical Sciences, Medical College, Dalian University, Dalian 116622; and 3Faculty of Medicine, Department of Physiology, The University of Hong Kong, Hong Kong, China

Submitted 11 July 2003 ; accepted in final form 17 February 2004


    ABSTRACT
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 ABSTRACT
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The cardiac Ca2+-independent transient outward K+ current (Ito), a major repolarizing ionic current, is markedly affected by Cl substitution and anion channel blockers. We reexplored the mechanism of the action of anions on Ito by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs+ substitution for intracellular K+. Replacement of most of the extracellular Cl with less permeant anions, aspartate (Asp) and glutamate (Glu), markedly suppressed the current. Removal of external Na+ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on Ito. In contrast, the permeant Cl substitute Br did not markedly affect the current, whereas F substitution for Cl induced a slight inhibition. The Ito elicited during Br substitution for Cl was also sensitive to blockade by 4-AP. The ability of Cl substitutes to induce rightward shifts of the steady-state inactivation curve of Ito was in the following sequence: NO3 > Cl {approx} Br > gluconate > Glu > Asp. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of Ito similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp substitution for Cl was without significant effect on the intensity. These results suggest that the Ito channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.

transient outward potassium current; anion channel; actin cytoskeleton; myocyte; potassium ion


DEPOLARIZATION-ACTIVATED OUTWARD K+ currents play important roles in the regulation of the action potential plateau and duration in many mammalian hearts (3, 4). Two basic types of depolarization-activated outward K+ currents have been distinguished on the basis of differing time- and voltage-dependent properties and pharmacological sensitivities. One is a rapidly activating and inactivating transient outward current (Ito), which is sensitive to 4-aminopyridine (4-AP). The other is a slowly activating, tetraethylammonium-sensitive delayed-rectifier K+ current (3, 4). In rat cardiac ventricular myocytes, the Ito is a 4-AP-sensitive and intracellular Ca2+ (Cai2+)-independent K+ current (3, 14, 22), which is referred to as Ito in the present study. Considerable evidence suggests that the voltage-gated K+ channels Kv4.2 and Kv4.3 contribute to cardiac Ito (35).

Initial studies found that the Ito was reduced in Cl-free solution and concluded that this current was carried primarily by Cl (13, 15, 18). However, later studies revealed that K+, rather than Cl, was the main charge carrier of the current (24, 25). Inhibition of the current induced by less permeant Cl substitutes was thought to be probably the result of a decrease in free Ca2+ concentration, since the Cl substitutes used in those studies might chelate external Ca2+ (Cao2+; see Ref. 23). This notion was supported by the observation that the Ito was less sensitive to replacement of external Cl (Clo) when free Ca2+ was kept at a constant level (24, 25). Further studies also provided some evidence for the dependence of the Ito on Ca2+. Reducing Cai2+ by EGTA inhibits the Ito (41). On the other hand, other studies showed that the current in rat cardiac ventricular myocytes displays a Ca2+-independent property (3, 22), which is also inhibited by less permeant Cl substitutes and anion channel blockers (ACB; see Ref. 30). Heretofore, the mechanism of the influence of less-permeant Cl substitutes on cardiac Ito remains unclear.

In the mammalian heart, several anion channels have been functionally identified for over a decade (19). In the study of cardiac anion channels, a common phenomenon is that Cl substitutes and ACB have profound effects on other channels (1, 8, 16, 31, 43, 44), which is generally attributed to nonspecific effects. However, our primary study suggests that the nonspecific effects of Cl substitutes and ACB on cardiac Ca2+ channel may implicate a channel-channel interaction (46), an important regulating mode found in epithelia (27, 40). It is well accepted that the cystic fibrosis transmembrane conductance regulator (CFTR) may act both as a Cl channel and a regulator of the activity of other epithelial channels via a channel-channel interaction (27, 40), possibly mediated by the cytoskeleton (20). Moreover, studies have found that a variety of channels are regulated by the actin cytoskeleton (2, 7, 17, 20, 33, 34, 36, 45). The Kv4.2, which contributes to the Ito, is found to interact with filamin, a member of the {alpha}-actinin/spectrin/dystrophin family of actin-binding proteins. Absence of filamin results in suppression of Kv4.2 current (36). In mammalian cardiac ventricular myocyte, there is a possibility that one channel activity may influence other channel(s) via the actin cytoskeleton. Therefore, in the present study, to evaluate the hypothesis that channel-channel interaction might be implicated in the action of Cl substitutes, we reexamined the effects of Cl substitution on Ito. The present data indicate that the effects of anions on Ito are closely related to their permeability, and disruption of the actin microfilament by cytochalasin D (CytD) produced an effect similar to that of less permeant anions on the steady-state inactivation of Ito. The present results provide evidence for the hypothesis that the effects of anions on Ito involve a modulatory action of anion channel(s) on Ito channel, probably mediated by the actin cytoskeleton.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
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Cell preparations. Ventricular myocytes were enzymatically isolated from adult Sprague-Dawley rats (200~250 g), as reported previously (46). Briefly, the hearts were removed immediately after decapitation and retrogradely perfused at 37°C with the following solutions in turn: Tyrode solution (5 min), Ca2+-free Tyrode solution (5 min), Ca2+-free Tyrode solution with 0.5 mg/ml collagenase (type I; Sigma, St. Louis, MO) and 1 mg/ml BSA (35 min), and Kraftbrühe (KB; high K+) solution (5 min). After dissociation and collection, the cells were kept in KB solution at room temperature (23~25°C) for electrophysiological recordings. To study the role of the actin cytoskeleton, myocytes were incubated with either 100 µM CytD and 100 µM phalloidin or vehicle (DMSO) at 37°C for 2 h and then stored in 4°C KB solution.

Whole cell patch-clamp experiments. Aliquots of cell suspension were transferred to a perfusion chamber on the stage of an inverted microscope. Pipettes had tip resistances of 2–2.5 M{Omega} when filled with internal solution. Whole cell recordings were performed at room temperature (23~25°C) using a patch-clamp amplifier and Pulse software (HEKA Elektronik, Lambrecht, Germany). The offset potentials between both electrodes were zeroed before the pipette touched the cell. The liquid junction potential between the Asp-rich pipette and the standard bath solutions for recording Ito was calculated to be 15 mV by using the JPCalc program within Clampex 8.1 (Axon Instruments) and was corrected after the experiments. To minimize changes in liquid junction potentials caused by alteration in Clo, the Ag-AgCl bath ground electrode was placed in a separate pool of 3 M KCl, which was connected to the recording chamber by a 3 M KCl-agar bridge, as reported previously by Zygmunt and Gibbons (47). Whole cell Ito was elicited by 300-ms pulses from a holding potential of –65 mV (after correction of the junction potential) to test potentials ranging from –55 to +45 mV in 10-mV increments. Whole cell basal Cl currents were elicited from a holding potential of –40 mV to test potentials ranging from –100 to +100 mV in 20-mV increments. The pulses were 200 ms in duration and delivered at 1-s interval. The current signals were low-pass filtered at 5 kHz and stored in the hard disk of an IBM-compatible computer. Ito was calculated by subtracting the peak outward current from the current at the end of the test pulse.

Confocal imaging. Fluorescence labeling of F-actin was performed as previously described (29). Cell suspensions, pretreated with different protocols, were sedimented by centrifugation at 100 g for 1 min. The supernatant was discarded. Cells were then fixed in 4% paraformaldehyde-PBS solution for 10 min and washed three times with PBS by centrifugation. Cell suspensions were transferred to slides and kept in 4°C overnight. The cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. After three washes with PBS, cells were stained with FITC-labeled phalloidin (5 µM in PBS) to localize F-actin for 50 min in a dark room at room temperature and then were washed extensively with PBS. Slides were visualized using an Olympus Fluoview FV300 confocal microscope. FITC was excited at 490 nm and detected at 520 nm. Optical sections (0.6 µm thick) were taken of each sample to eliminate out-of-focus fluorescence of the intensely stained myocytes. To standardize the fluorescence intensity for all the experimental preparations, the time of image capture, the image intensity gain, the image enhancement, and the contrast and brightness settings were optimally adjusted at the outset and kept constant for all experiments.

Solutions. The Tyrode solution contained (in mM) 143 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 0.3 NaH2PO4, 5 glucose, and 5 HEPES-NaOH (pH 7.4). The nominally Ca2+-free Tyrode solution was made by omitting CaCl2 from the normal solution. The KB solution contained (in mM) 70 potassium glutamate, 25 KCl, 20 taurine, 10 KH2PO4, 3 MgCl2, 0.5 EGTA, 10 glucose, and 10 HEPES-KOH (pH 7.35).

The pipette solution for recording Ito contained (in mM) 110 potassium aspartate (unless specifically stated in the text), 20 KCl, 1 MgCl2, 5 Na2-phosphocreatine, 0.1 GTP, 5 MgATP, 5 EGTA, and 10 HEPES (pH was adjusted to 7.2 with KOH). In some experiments, intracellular K+ (Ki+; 130 mM) was replaced by equimolar Cs+ (pH = 7.2, adjusted with CsOH). The standard bath solution contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 0.4 KH2PO4, 1.8 CaCl2, 1 BaCl2, 0.5 CdCl2, 5 HEPES, and 10 glucose (pH 7.4). BaCl2 and CdCl2 were used to inhibit the inwardly rectifying K+ current, the L-type Ca2+ current, and the Ca2+-activated Cl current (48). In some experiments, external Na+ (Nao+) was replaced by equimolar N-methyl-D-glucamine (NMDG). In Cl substitution experiments, 140 mM Clo was replaced by equimolar Br, F, NO3, aspartate (Asp), glutamate (Glu), or gluconate (Glc). The free Ca2+ concentration in bath solutions was calculated using the CaBuf program (provided by Dr. G. Droogmans, Katholieke Universiteit Leuven, Belgium), as described previously (46). According to the calculation, the free Ca2+ in the bathing solutions after substitution of 140 mM Clo with equimolar Asp, Glu, or Glc was 1.76, 1.78, and 0.57 mM, respectively, when the total Ca2+ was 1.8 mM. Because the presence of other bivalent cations, Cd2+ and Ba2+, could not be taken into account in the calculation, the free Ca2+ concentration after replacement of Clo with less permeant anions may represent an underestimate.

The solutions for recording basal Cl current were as follows. The pipette solution contained (in mM) 135 NMDG-Cl, 2 EGTA, 5 Mg-ATP, 10 HEPES, and 10 mannitol, pH 7.2, with intracellular Cl (Cli) = 135 mM. The standard bath solution contained (in mM) 125 NaCl, 2.5 MgCl2, 2.5 CaCl2, 5 glucose, 10 HEPES, and 30 mannitol, pH 7.4, with Clo = 135 mM. Nifedipine (1 µM) was added to the bath solution to inhibit the L-type Ca2+ current. The membrane potential was depolarized from –70 to –40 mV, where it was held for 100 ms to inactivate the Na+ channels. K+ currents were eliminated by omission of K+ from pipette and bath solutions. In some experiments, 125 mM Clo was replaced by either equimolar Br or Asp.

Chemicals. 4-AP, nifedipine, CytD, phalloidin, and FITC-phalloidin were purchased from Sigma. Stock solutions of CytD (50 mM) and phalloidin (50 mM) in DMSO were diluted to the desired final concentrations immediately before use.

Statistical analysis. Data are presented as means ± SE. Statistical differences in the data were evaluated by Student's t-test or ANOVA as appropriate and were considered significant at values of P < 0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Effect of less permeant anions on Ito. The Ito, recorded in rat ventricular myocytes, was inhibited either by the specific Ito channel blocker 4-AP (n = 5, data not shown) or by Cs+ substitution for Ki+ (n = 4, data not shown), indicating that the current is the 4-AP-sensitive and Cai2+-independent Ito, as previously reported (3, 22). To explore the mechanism of action of Cl substitutes on Ito, we first observed the effects of replacing Cl with less permeant anions, Asp and Glu. Figure 1 shows the effects of substituting most of the Clo (140 mM) with equimolar Asp or Glu on Ito. In Na+-rich bath solution (Nao+ = 140 mM), substitution of Cl by Asp inhibited Ito (Fig. 1A), without discernible change in cell volume or morphology under the light microscope. The inhibition was 85.1 ± 3.9% at +45 mV (n = 9, P < 0.01). Similar effects were observed with Glu substitution for Cl (Fig. 1B). Replacement of Cl with Glu decreased the peak Ito at +45 mV by 57.7 ± 4.3% (n = 7, P < 0.01). Substitution of Clo by Asp or Glu did not significantly affect the sustained component of outward K+ current (Fig. 1). To examine whether Na+-K+-2Cl cotransport is involved in the inhibition of less permeant anions, we observed the effects of less permeant Cl substitutes on the Ito activated in 0 mM Nao+ (replacement of Nao+ with the impermeant monovalent cation NMDG+). Perfusing the cells with Na+-free solution did not prevent the inhibitory effect of the less permeant Cl substitutes Asp (Fig. 2A) or Glu (Fig. 2B) on Ito, indicating that Na+-K+-2Cl cotransport does not play a crucial role in this inhibitory effect. These results suggest that the regulation of Ito involves anion-related factor(s).



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Fig. 1. Effects of less permeant Cl substitutes on transient outward current (Ito) in Na+-rich solution. Outward currents were elicited by 300-ms depolarizing pulses from a holding potential of –65 mV to test potentials ranging from –55 to +45 mV in 10-mV increments. A: representative currents recorded in the control (a), after replacement of the majority of external Cl (Clo; 140 mM) with Asp (b), and after withdrawal of substitution (c). B: representative current traces recorded before (a) and after (b) replacement of the majority of Clo (140 mM) with Glu and after withdrawal of the substitution (c).

 


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Fig. 2. Effects of less permeant anions on Ito in external Na+ (Nao+)-free condition. Nao+ (140 mM) was replaced with equimolar N-methyl-D-glucamine (NMDG+). A: representative current traces of 4 experiments recorded in control (a), after replacement of the majority of Clo with Asp (b), and after withdrawal of substitution (c). B: representative current traces of 4 experiments recorded in control (a), after Glu-substitution for Cl (b), and after withdrawal of substitution (c).

 
Effects of permeant anions on Ito. We then determined the effect of the permeable anions, F and Br, on Ito. Replacement of most of Clo with F reduced the current slightly (Fig. 3A) compared with Asp or Glu substitution (Fig. 1). The inhibition by F substitution for Cl was 23.5 ± 3.6% at +45 mV, which was statistically significant (P < 0.01, n = 5). Replacement of the majority of Clo with equimolar Br also seemed to reduce Ito (Fig. 3B). At +45 mV the inhibition was 8.8 ± 3% (n = 6), which was not significant statistically. The Ito elicited by Br substitution was also sensitive to blockade by 4-AP (Fig. 3Bc, n = 4). The effects of a variety of anions on Ito are summarized in Fig. 4. The facilitative effect of different anions on the activation of Ito was in the following sequence: NO3 > Cl ≥ Br > F {approx} Glc > Glu > Asp. These results indicate that the activation of Ito is closely related to anionic permeability, i.e., Ito is more easily activated with increasing permeability of external anion. Thus it is likely that anionic permeability dependence of Ito may involve the activity of anion channel.



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Fig. 3. Effects of substitution of Clo with relatively permeant anions on cardiac Ito. Ito was elicited with the protocol as described in Fig. 1. A: representative current traces recorded before (a) and after (b) 140 mM Cl in the standard bath solution was replaced by equimolar F. B: representative current traces recorded before (a) and after (b) replacement of Clo (140 mM) with Br, in the presence of 4 mM 4-aminopyridine (4-AP) in Br substitution (c), and after withdrawal of 4-AP (d).

 


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Fig. 4. Effects of Cl substitutes on the current-voltage relationship of the Ito. The currents were elicited in the Na+-rich bath solution (Nao+ = 140 mM) by applying 300-ms depolarizing pulses from a holding potential of –65 mV in 10-mV increments, between –55 and +45 mV before (control) and after substitution of 140 mM Clo with NO3 (n = 6), Br (n = 6), F (n = 5), Glc (n = 7), Glu (n = 8), or Asp (n = 9). For each cell, Ito was measured by subtracting the peak outward current from the sustained current at each test potential and normalized to their respective amplitudes evoked at +45 mV in control (Clo = 140 mM).

 
Effect of different anions on the steady-state inactivation of Ito. To explore the mechanisms of action of Cl substitutes on Ito, we measured the voltage dependence of the steady-state inactivation of Ito in the Cl substitution conditions. The potential at which the current was completely inactivated shifted to a more positive potential (from –35 to –25 mV) after replacement of Cl with NO3 (Fig. 5A). In contrast, after replacement of Cl with Asp, the current was completely inactivated at a much more negative potential (–55 mV) than in control (–25 mV; Fig. 5B). Figure 5C shows the effects of a variety of anions on the steady-state inactivation-voltage relationships of Ito. NO3 substitution for Clo caused a shift in the potential of 50% inactivation of Ito (V0.5) toward a more positive potential by 5.7 ± 1.1 mV (P < 0.01, n = 7). In contrast, replacement of Clo with Glc, Glu, or Asp shifted the V0.5 toward a more negative potential by 5.2 ± 0.9 mV (P < 0.01, n = 6), 12.1 ± 0.8 mV (P < 0.01, n = 5), and 23.2 ± 1.8 mV (P < 0.01, n = 8), respectively. Br substitution for Clo did not significantly influence the steady-state inactivation curve of Ito (n = 6). The ability of various anions to shift the steady-state inactivation curve of Ito to more positive potentials was in the following sequence: NO3 > Cl {approx} Br > Glc > Glu > Asp.



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Fig. 5. Effects of Cl substitutes on the steady-state inactivation of Ito in rat ventricular myocytes. Original current traces recorded before (A and B, left) and after replacement of the majority of Clo (140 mM) with NO3 and Asp in A and B, right, respectively. Ito was activated by 300-ms test pulses to +45 mV from holding potentials ranging from –125 to –15 mV in 10-mV increments. Nos. in A and B indicate the membrane potentials at which the currents were recorded. C: effects of Cl substitutes on the steady-state inactivation curve of Ito. Currents in C were expressed as fractions of maximal Ito (I/Imax) and plotted as a function of voltage. Curves were fitted to experimental data using the Boltzmann equation: 1/{1 + exp[(VmV0.5)/k]}, where V0.5 and k are the potentials of half-maximal inactivation and the slope factor, respectively.

 
Effect of CytD treatment on the steady-state inactivation of Ito. Both voltage-gated K+ channels (17, 33, 34, 36) and anion channels (e.g., the ClC-2 Cl channel, the CFTR channel, and the swelling-activated Cl channel; see Refs. 2, 7, and 45) are linked to the actin cytoskeleton and are regulated by it. Because the effects of anions on Ito may involve an interaction between the Ito channel and anion channel(s), we investigated whether the actin cytoskeleton was involved in the actions of anions by disrupting actin microfilaments with CytD, a fungal toxin known to specifically break down F-actin fibers (9). In DMSO treatment myocytes, substitution of Cl with Asp caused a 19.5 ± 1.9 mV leftward shift of the V0.5 of Ito (n = 19, P < 0.01). In contrast, the same substitution in CytD-treated myocytes only caused a 7.4 ± 1.4 mV leftward shift of the V0.5 (n = 25). In some CytD-pretreated myocytes (4 of 25 cells), replacement of Clo with Asp did not produce significant shift of the Ito inactivation curve (Fig. 6, B and C). These data indicate that disruption of actin filaments produces effects mirroring those less permeant anions, suggesting that the effects of anions on the Ito channel may involve the actin cytoskeleton.



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Fig. 6. Effects of phalloidin and cytochalasin D (CytD) treatment on Ito of rat ventricular myocytes. A: representative current traces recorded before (left) and after (right) replacement of the majority of Clo (140 mM) with Asp in a myocyte pretreated with 100 µM phalloidin. B: original current traces recorded before (left) and after (right) replacement of the majority of Clo (140 mM) with Asp in a CytD-treated myocyte. Ito was activated by 300-ms test pulses to +45 mV from holding potentials ranging from –125 to –15 mV in 10-mV increments. C: relationship between voltage and the steady-state inactivation of Ito in vehicle (DMSO)- or CytD-treated myocytes before and after Asp substitution for Clo. The current was expressed as a fraction of maximal Ito (I/Imax) and plotted as a function of voltage. The curves were fitted by the Boltzmann equation.

 
Imaging of F-actin in myocytes pretreated either with Asp or with CytD. To assess the possibility that less permeant Cl substitutes may induce F-actin disruption, we observed the structural changes in the actin cytoskeleton of cardiac myocytes pretreated with Asp-rich external solution. Confocal images showed that cells pretreated with Asp-rich external solution did not induce significant changes in the average pixel intensity of FITC-phalloidin staining of F-actin (Fig. 7B) compared with the images of myocytes pretreated with control solution (Fig. 7A). In contrast, CytD pretreatment induced a significant decrease in the FITC-phalloidin staining intensity of F-actin (Fig. 7D) compared with the control cells treated only with the vehicle (Fig. 7C). Moreover, stabilization of actin cytoskeleton with phalloidin did not prevent Asp substitution-induced inhibition of Ito (Fig. 6A, n = 4). These results indicate that suppression of Ito by less permeant anions is not by a direct influence of actin cytoskeleton.



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Fig. 7. Effects of pretreatment with CytD and Asp substitution for Cl on cardiac ventricular F-actin. A and B: representative images from the myocytes pretreated with normal Clo solution (A) or Asp-rich solution (B). Cells were transferred from Kraftbrühe solution to either control bath solution (normal Clo) or Asp substitution for Clo solution for 30 min before formaldehyde fixation. C and D: representative images from vehicle (C)- and CytD-treated (D) cells. For each condition, F-actin was stained with FITC-labeled phalloidin and examined by confocal microscopy. The images are from at least 4 separate experiments. Scale bar = 10 µm.

 
Basal activity of Cl channels in rat ventricular myocytes. In a symmetrical Cl gradient (Clo = Cli = 135 mM), a basal time-independent current was observed (Fig. 8A). The reversal potential of the basal current was –1.1 ± 1.2 mV (n = 4). This was close to the Cl equilibrium potential (ECl = 0 mV) predicted by the Nernst Equation. Replacement of 125 mM Clo with Asp shifted the reversal potential from –1.1 ± 1.2 to +62.5 ± 3.8 mV (n = 4), close to the new ECl (+67 mV), whereas Br substitution for Cl did not induce significant shift of the reversal potential (–0.5 ± 1.3 mV, n = 4). These results suggest that the basal current is carried by Cl. The anion permeability sequence of the channel, obtained from the shifts of the reversal potentials of the basal Cl current, was Cl {approx} Br >> Asp (Fig. 8B). These data indicate that there is a basal activity of Cl channels in rat cardiac ventricular myocytes under basal conditions.



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Fig. 8. Basal Cl current in rat ventricular myocytes. Whole cell basal Cl currents were elicited from a holding potential of –40 mV to test potentials ranging from –100 to +100 mV in 20-mV increments. A: representative whole cell currents recorded under a symmetrical Cl gradient condition (Clo = 135 mM; a) and after replacement of 125 mM Clo with equimolar Br (b) and Asp (c). Arrows indicate 0 current level. B: current (I)-voltage relationships of the currents in A.

 

    DISCUSSION
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 MATERIALS AND METHODS
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 DISCUSSION
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The main findings in the present study are as follows: 1) The activity of Ito in rat ventricular myocytes displays an anionic permeability-dependent property, i.e., Ito is facilitated in permeant anion-rich extracellular solution, but it is suppressed by less permeant Cl substitutes. The effects of the anions on Ito are the result of a shift of the steady-state inactivation curve. 2) Depolymerization of actin microfilaments with CytD, like less permeant anions, shifts the steady-state inactivation curve to a more negative potential.

The cardiac Ito was initially thought to be carried by Cl because it decreased in Cl-free solution (13, 15, 18). However, subsequent studies indicated that this current was primarily carried by K+ (24). The effects of Cl substitution on the Ito are usually attributed to altered Cao2+, since less permeant Cl substitutes may reduce the free Ca2+ concentration (23) and consequently inhibit a Ca2+-activated Cl current (47). However, in rat cardiac ventricular myocytes, the Ito, as a matter of fact, is a sole current carried by K+ (Ito), which is independent of Cai2+ and sensitive to blockade by 4-AP (3, 14, 22). Both biochemical and functional evidence shows that Kv4.2 and Kv4.3 contribute to the cardiac Ito (10, 11, 35). Although rat cardiac Ito is also inhibited by less permeant anions, Lefevre et al. (30) have demonstrated that the inhibitory effect of the anions is not significantly altered when Cao2+ is removed and Cai2+ is buffered with a high concentration of EGTA. In the present study, it is unlikely that the effect of less permeant anions was the result of changes in free Cao2+ because Asp and Glu, which only cause a slight decrease in free Cao2+ (<2.3%, see MATERIALS AND METHODS), induce a more markedly inhibitory effect than Glc does. The latter markedly decreased the free Cao2+ by 68.3%. These unparallel effects of the less permeant anions on the free Cao2+ and Ito suggest that their action may involve a mechanism other than chelation of Ca2+. It may be argued that a fraction of Ito may be carried by Cl. If Cl can pass through the Ito channel, then the residual Ito carried by Cl should remain after removing Ki+. However, when the myocytes were dialyzed with the pipette solution in which 130 mM K+ was replaced by equimolar Cs+, Ito decreased progressively and disappeared within 4 min, although the concentrations of both Clo and Cli remained unchanged (n = 4, data not shown). This is similar to the observation reported by Lefevre et al. (30). Therefore, it is unlikely that Cl is a carrier of Ito. In the present study, a KCl-agar bridge electrode was used to minimize the junction potential caused by alteration in Clo concentration. If the KCl-agar bridge electrode failed to effectively reduce the liquid junction potentials, changes in liquid junction potentials should alter the activation of both Ito and the sustained component of outward K+ current. However, less permeant anions, like the specific Ito inhibitor 4-AP, only inhibited Ito, whereas the sustained component of outward K+ current was not significantly affected. Thus it is unlikely that inhibition of Ito by less permeant anions is caused by changes in liquid junction potentials. The present data suggest that the effects of Cl and its substitutes on cardiac Ito involve an anion-related factor.

In cardiac myocytes, the physiological K+ gradient is maintained by a combination of outward K+ movement through K+ channels and an inward movement via the Na+-K+-2Cl cotransporter (32) and the activity of Na+-K+-ATPase. Inhibition of Na+-K+-2Cl cotransport may cause accumulation-depletion of K+ in situ, i.e., an increase in the external K+ (Ko+) and a decrease in the Ki+, thereby reducing the driving force for K+. Thus the inhibition of Ito by less permeant anions may be the result of an inhibition of Na+-K+-2Cl cotransport, as proposed by Lefevre et al. (30). However, in contrast to the less permeant anions, substitution of Cl by some permeant anions, which should also impair Na+-K+-2Cl cotransport, was found to facilitate Ito rather than inhibit it. Moreover, the driving force for K+ is not expected to be significantly altered in Cl substitution conditions because the Ki+ concentration is clamped at a constant level in the whole cell patch-clamp configuration, and the Ko+ is maintained constant by continual perfusion. Na+ is also an important factor for stimulating Na+-K+-2Cl cotransport and Na+-K+-ATPase; substitution of Nao+ with an impermeant cation should eliminate Na+-K+-2Cl cotransport. However, removal of Nao+ does not affect the inhibitory action of less permeant anions (Fig. 2). Furthermore, intracellular Na+ has also been found to have no effects on Ito (14). Inhibition of the Na+-K+-ATPase with its specific inhibitor ouabain (0.5 µM) does not markedly influence the action of the less permeant anion Asp (n = 5, data not shown). Therefore, it seems unlikely that the inhibitory effect of less permeant anions on Ito is caused by either an impairment of Na+-K+-2Cl cotransport or alteration of the activity of Na+-K+-ATPase.

Anion channels exist ubiquitously in cells, including rat ventricular myocytes (12, 26, 42), and display distinct anionic selectivity and permeability sequences (21). Studies have revealed that external anions may modulate the gating of some anion channels (38, 39). In the present study, a basal Cl channel activity was observed. The Cl channel had an anion permeability sequence of Cl {approx} Br >> Asp, which matched the action of these anions on Ito. Thus the present results could not rule out the possibility that the Ito channel may be modulated by anion channel(s) via a channel-channel interaction. Recent evidence reveals that the actin cytoskeleton is implicated in the regulation of a variety of ion channels, including voltage-gated K+ channels (17, 33, 34, 36) and anion channels (2, 6, 7, 37, 45). A previous study on epithelial anion channels has suggested that actin plays a role in channel-channel interaction (20). The present study found that disrupting the actin microfilaments with CytD produced an effect similar to that induced by less permeant anions. Confocal imaging revealed that CytD induced a disruption of actin microfilaments. In contrast, less permeant anion Asp did not affect the structure of the actin cytoskeleton. It is unlikely that less permeant anions affect Ito by directly acting on the actin cytoskeleton. These results suggest that the actin cytoskeleton may play a linkage role in the interaction between the anion channel and the Ito channel. Although the present study suggests that Cl channel may be involved in the modulation of the Ito channel in rat cardiac ventricular myocytes, direct evidence for the interaction between the Cl channel and Ito channel is lacking. Further work is required to test this hypothesis.

In many mammalian hearts, including humans, Ito is mainly responsible for the initial rapid phase of the action potential repolarization. Defects of the Ito channel may result in both abnormal electrical activities (28, 35) and morphological changes in the heart (5, 28). Therefore, investigation into the regulation of cardiac Ito is of clinical importance. The present study provides new insight into the regulation of cardiac Ito.


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 ABSTRACT
 MATERIALS AND METHODS
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This research was supported by National Natural Science Foundation of China Grants 39870318 and 30270602 and the Sun Yat Sen Foundation Fund from the University of Hong Kong.


    ACKNOWLEDGMENTS
 
We thank Dr. G. Droogmans for the kind gift of the CaBuf program.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. S. Zhou, PhD, Dept. of Physiology, The Fourth Military Medical Univ., No.17, West Chang-Le Road, Xi'an 710032, China.

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Accili EA and DiFrancesco D. Inhibition of the hyperpolarization-activated current (if) of rabbit SA node myocytes by niflumic acid. Pflügers Arch 431: 757–762, 1996.[CrossRef][ISI][Medline]

2. Ahmed N, Ramjeesingh M, Wong S, Varga A, Garami E, and Bear CE. Chloride channel activity of ClC-2 is modified by the actin cytoskeleton. Biochem J 352: 789–794, 2000.[CrossRef][ISI][Medline]

3. Apkon M and Nerbonne JM. Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes. J Gen Physiol 97: 973–1011, 1991.[Abstract]

4. Barry DM and Nerbonne JM. Myocardial potassium channels: electrophysiological and molecular diversity. Annu Rev Physiol 58: 363–394, 1996.[CrossRef][ISI][Medline]

5. Barry DM, Xu H, Schuessler RB, and Nerbonne JM. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 {alpha} subunit. Circ Res 83: 560–567, 1998.[Abstract/Free Full Text]

6. Cantiello HF. Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator. Exp Physiol 81: 505–514, 1996.[Abstract]

7. Chasan B, Geisse NA, Pedatella K, Wooster DG, Teintze M, Carattino MD, Goldmann WH, and Cantiello HF. Evidence for direct interaction between actin and the cystic fibrosis transmembrane conductance regulator. Eur Biophys J 30: 617–624, 2002.[CrossRef][ISI][Medline]

8. Conforti L, Sumii K, and Sperelakis N. Diphenylamine-2-carboxylate blocks voltage-dependent Na+ and Ca2+ channels in rat ventricular cardiomyocytes. Eur J Pharmacol 259: 215–218, 1994.[CrossRef][ISI][Medline]

9. Cooper JA. Effects of cytochalasin and phalloidin on actin. J Cell Biol 105: 1473–1478, 1987.[ISI][Medline]

10. Dixon JE and McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res 75: 252–260, 1994.[Abstract]

11. Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, and McKinnon D. Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res 79: 659–668, 1996.[Abstract/Free Full Text]

12. Duan D, Ye L, Britton F, Horowitz B, Hume JR. A novel anionic inward rectifier in native cardiac myocytes. Circ Res 86: E63–E71, 2000.[ISI][Medline]

13. Dudel J, Peper K, Rudel R, and Trautwein W. The dynamic chloride component of membrane current in Purkinje fibers. Pflügers Arch 295: 197–212, 1967.

14. Dukes ID and Morad M. The transient K+ current in rat ventricular myocytes: evaluation of its Ca2+ and Na+ dependence. J Physiol 435: 395–420, 1991.[Abstract]

15. Fozzard HA and Hiraoka M. The positive dynamic current and its inactivation properties in cardiac Purkinje fibres. J Physiol 234: 569–586, 1973.[ISI][Medline]

16. Frace AM, Maruoka F, and Noma A. Control of the hyperpolarization-activated cation current by external anions in rabbit sino-atrial node cells. J Physiol 453: 307–318, 1992.[Abstract]

17. Hattan D, Nesti E, Cachero TG, and Morielli AD. Tyrosine phosphorylation of Kv1.2 modulates its interaction with the actin-binding protein cortactin. J Biol Chem 277: 38596–38606, 2002.[Abstract/Free Full Text]

18. Hiraoka M and Hiraoka M. The role of the positive dynamic current on the action potential of cardiac Purkinje fibers. Jpn J Physiol 25: 705–717, 1975.[ISI][Medline]

19. Hume JR, Duan D, Collier ML, Yamazaki J, and Horowitz B. Anion transport in heart. Physiol Rev 80: 31–81, 2000.[Abstract/Free Full Text]

20. Ismailov II, Berdiev BK, Shlyonsky VG, Fuller CM, Prat AG, Jovov B, Cantiello HF, Ausiello DA, and Benos DJ. Role of actin in regulation of epithelial sodium channels by CFTR. Am J Physiol Cell Physiol 272: C1077–C1086, 1997.[Abstract/Free Full Text]

21. Jentsch TJ, Stein V, Weinreich F, and Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503–568, 2002.[Abstract/Free Full Text]

22. Josephson IR, Sanchez-Chapula J, and Brown AM. Early outward current in rat single ventricular cells. Circ Res 54: 157–162, 1984.[Abstract]

23. Kenyon JL and Gibbons WR. Effects of low-chloride solutions on action potentials of sheep cardiac Purkinje fibers. J Gen Physiol 70: 635–660, 1977.[Abstract/Free Full Text]

24. Kenyon JL and Gibbons WR. Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers. J Gen Physiol 73: 117–138, 1979.[Abstract]

25. Kenyon JL and Gibbons WR. 4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers. J Gen Physiol 73: 139–157, 1979.[Abstract]

26. Komukai K, Brette F, Pascarel C, Orchard CH. Electrophysiological response of rat ventricular myocytes to acidosis. Am J Physiol Heart Circ Physiol 283: H412–H422, 2002.[Abstract/Free Full Text]

27. Kunzelmann K. CFTR: interacting with everything? News Physiol Sci 16: 167–170, 2001.[Abstract/Free Full Text]

28. Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR, and Chien KR. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell 107: 801–813, 2001.[ISI][Medline]

29. Lader AS, Kwiatkowski DJ, and Cantiello HF. Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels. Am J Physiol Cell Physiol 277: C1277–C1283, 1999.[Abstract/Free Full Text]

30. Lefevre T, Lefevre IA, Coulombe A, and Coraboeuf E. Effects of chloride ion substitutes and chloride channel blockers on the transient outward current in rat ventricular myocytes. Biochim Biophys Acta 1273: 31–43, 1996.[ISI][Medline]

31. Liu J, Lai ZF, Wang XD, Tokutomi N, and Nishi K. Inhibition of sodium current by chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in guinea pig cardiac ventricular cells. J Cardiovasc Pharmacol 31: 558–567, 1998.[CrossRef][ISI][Medline]

32. Liu S, Jacob R, Piwnica-Worms D, and Lieberman M. Na+-K+-2Cl cotransport in cultured embryonic chick heart cells. Am J Physiol Cell Physiol 253: C721–C730, 1987.[Abstract/Free Full Text]

33. Mason HS, Latten MJ, Godoy LD, Horowitz B, and Kenyon JL. Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton. Mol Pharmacol 61: 285–293, 2002.[Abstract/Free Full Text]

34. Nakahira K, Matos MF, and Trimmer JS. Differential interaction of voltage-gated K+ channel {beta}-subunits with cytoskeleton is mediated by unique amino terminal domains. J Mol Neurosci 11: 199–208, 1998.[CrossRef][ISI][Medline]

35. Oudit GY, Kassiri Z, Sah R, Ramirez RJ, Zobel C, and Backx PH. The molecular physiology of the cardiac transient outward potassium current (Ito) in normal and diseased myocardium. J Mol Cell Cardiol 33: 851–872, 2001.[CrossRef][ISI][Medline]

36. Petrecca K, Miller DM, and Shrier A. Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin-binding protein filamin. J Neurosci 20: 8736–8744, 2000.[Abstract/Free Full Text]

37. Prat AG, Cunningham CC, Jackson GR Jr., Borkan SC, Wang Y, Ausiello DA, and Cantiello HF. Actin filament organization is required for proper cAMP-dependent activation of CFTR. Am J Physiol Cell Physiol 277: C1160–C1169, 1999.[Abstract/Free Full Text]

38. Pusch M, Jordt SE, Stein V, and Jentsch TJ. Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol 515: 341–353, 1999.[Abstract/Free Full Text]

39. Pusch M, Ludewig U, Rehfeldt A, and Jentsch TJ. Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion. Nature 373: 527–531, 1995.[CrossRef][ISI][Medline]

40. Schwiebert EM, Benos DJ, Egan ME, Stutts MJ, and Guggino WB. CFTR is a conductance regulator as well as a chloride channel. Physiol Rev 79, Suppl 1: S145–S166, 1999.

41. Siegelbaum SA and Tsien RW. Calcium-activated transient outward current in calf cardiac Purkinje fibres. J Physiol 299: 485–506, 1980.[Abstract]

42. Thiemann A, Grunder S, Pusch M, and Jentsch TJ. A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356: 57–60, 1992.[CrossRef][ISI][Medline]

43. Walsh KB and Wang C. Effect of chloride channel blockers on the cardiac CFTR chloride and L-type calcium currents. Cardiovasc Res 32: 391–399, 1996.[CrossRef][ISI][Medline]

44. Wang HS, Dixon JE, and McKinnon D. Unexpected and differential effects of Cl channel blockers on the Kv4.3 and Kv4.2 K+ channels. Implications for the study of the Ito2 current. Circ Res 81: 711–718, 1997.[Abstract/Free Full Text]

45. Zhang J, Larsen TH, and Lieberman M. F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes. Am J Physiol Cell Physiol 273: C1215–C1224, 1997.[Abstract/Free Full Text]

46. Zhou SS, Gao Z, Dong L, Ding YF, Zhang XD, Wang YM, Pei JM, Gao F, and Ma XL. Anion channels influence excitation-contraction coupling by modulating L-type Ca2+ channel in ventricular myocytes. J Appl Physiol 93: 1660–1668, 2002.[Abstract/Free Full Text]

47. Zygmunt AC and Gibbons WR. Calcium-activated chloride current in rabbit ventricular myocytes. Circ Res 68: 424–437, 1991.[Abstract]

48. Zygmunt AC, Goodrow RJ, and Antzelevitch C. Sodium effects on 4-aminopyridine-sensitive transient outward current in canine ventricular cells. Am J Physiol Heart Circ Physiol 272: H1–H11, 1997.[Abstract/Free Full Text]