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Am J Physiol Heart Circ Physiol 292: H2364-H2377, 2007. First published January 12, 2007; doi:10.1152/ajpheart.00409.2005
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W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition

Yu-Jie Qu, Vladimir E. Bondarenko, Chang Xie, Shimin Wang, Mouhamed S. Awayda, Harold C. Strauss, and Michael J. Morales

Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York

Submitted 23 April 2005 ; accepted in final form 2 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ca+-calmodulin (Ca2+-CaM)-dependent protein kinase II (Ca2+/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca2+-occupied CaM, and KN-93, an inhibitor of Ca2+/CaMKII, on the Kv4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC50 of 92.4 µM. The block was voltage dependent, with an effective electrical distance of 0.18 ± 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of Kv4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 µM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated Kv4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 µM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca2+/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca2+-CaM, as well as a change in activity of Ca2+-CaM-dependent enzymes, including Ca2+/CaMKII.

potassium channel; inactivation gating; KN-93; transient outward potassium current; calmodulin kinase II

CALMODULIN (CaM) is a ubiquitous Ca2+-binding protein that plays an important role in the Ca2+-signaling pathways of eukaryotic cells (37, 48, 59). Ca2+/CaM-dependent protein kinase II (Ca2+/CaMKII) is among the many kinases and phosphatases responsive to the Ca2+-bound CaM (Ca2+-CaM) (31a, 34, 49, 56, 64, 71). Recent studies have implicated increased Ca2+/CaMKII activity in the pathophysiology of different types of heart disease, including ventricular arrhythmias (4, 5, 26, 36, 45, 46). These data have shown that agents that lead to a decrease in Ca2+/CaMKII activity, such as W-7 and KN-93, are effective in different animal models of ventricular arrhythmias (4, 26, 36, 45, 46). Despite their inhibition of a common final target, Ca2+/CaMKII, W-7, which inhibits Ca2+-CaM, could also modulate additional enzymes and/or ion channels (4, 37, 38, 48, 59, 75). W-7 binds to CaM and blocks access to the activation site exposed when the Ca2+-binding sites in CaM are occupied (28, 56), thereby blocking the Ca2+-CaM effects. KN-93 competitively inhibits Ca2+-CaM binding to Ca2+/CaMKII (5, 7, 31a). Although prior studies examined the effects of W-7 and KN-93 on the native Ca2+ and K+ currents in cardiac myocytes, the effects of these two agents on the gating and permeation properties of Kv4 channels have not been analyzed in detail (5, 19, 38, 42, 60, 69).

One target of these drugs is the rapidly inactivating, voltage-dependent K+ current (Ito) in cardiac myocytes, which is regulated by Ca2+-dependent kinases (5, 16, 17, 53, 67, 69). Cardiac Ito makes important contributions to the early and subsequent stages of repolarization in cardiac, especially atrial, myocytes (12, 16). The consensus is that the K+ voltage-dependent Kv4.2 and Kv4.3 channels serve as pore-forming molecular substrates for the native Ito in cardiac myocytes (13, 22, 35, 52, 53, 77). In addition, the effects of KN-93 on the A-type current in the central nervous system and smooth muscle of the gastrointestinal tract have been studied (1, 2, 11, 35, 39, 40).

Ca2+/CaMKII activation and inhibition have been shown to modulate A-type current in colonic and gastric myocytes and in cardiac myocytes (1, 2, 19, 39, 40). Sergeant et al. (63) demonstrated that KN-93 and a constitutively active Ca2+/CaMKII modulated inactivation, recovery from inactivation, and the steady-state inactivation relation of the heterologously expressed Kv4.3 channel. However, although the role of CaM in modulation of the Kv4.3 channel is poorly understood, it is likely to be more complex, inasmuch as this ubiquitous Ca2+-binding protein modifies the function of numerous target enzymes (31a, 34, 37, 48, 49, 59, 71, 75). One approach that has helped answer this question has been the use of small organic molecules, such as W-7, which serve as inhibitors of Ca2+-CaM activity (56). Although W-7 has been shown to inhibit Ito (4, 5, 26, 36, 38, 42, 46), the underlying mechanism is not fully understood, as prior studies have not considered the possibility that W-7 might directly interact with Kv4.3 and cause open-channel block and use dependence. Therefore, elucidation of the mechanisms of the effects of W-7 on the Kv4.3 channel is highly desirable.

We set out to examine the effects of W-7 and KN-93 on the Kv4.3 channel. Our first goal was to establish the extent to which either or both agents affected the permeation and gating properties of the Kv4.3 channel. Second, we assessed the extent to which inhibition of Ca2+-CaM and inhibition of Ca2+-CaM binding to Ca2+/CaMKII produced similar effects on open-state inactivation and recovery from inactivation. Changes in open-state inactivation and recovery from inactivation in the presence of BAPTA were used as markers of changes of Ca2+/CaMKII activity (19, 63). We demonstrate that W-7 and KN-93 modified the gating properties of the Kv4.3 channel. W-7, but not KN-93, showed evidence of direct interaction with the inner pore region of the Kv4.3 channel, as only W-7 showed use-dependent block of Kv4.3. Both W-7 and KN-93 accelerated all three components of open-state inactivation and slowed recovery from inactivation in wild-type (WT) channels. However, our data indicate that specific concentrations of KN-93 had selective effects on open-state inactivation kinetics that are consistent with a decrease in Ca2+/CaMKII activity. On the other hand, W-7 had selective effects on recovery from inactivation but nonselective effects on inactivation, suggesting that its effects on inactivation kinetics were not solely mediated by a decrease in Ca2+/CaMKII activity. Hence, our data suggest that the proposed effects of W-7 on the Kv4.3 channel are mediated by a combination of channel pore blockade and change in activity of different Ca2+-CaM-dependent enzymes, including Ca2+/CaMKII.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
RNA preparation and channel expression. WT cDNA of Kv4.3 (short form) was a gift of Dr. David McKinnon (State University of New York at Stony Brook), and its use has been previously described (73). Synthetic mRNA was prepared using an mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX). A Kv4.3 channel with mutated Ca2+/CaMKII consensus phosphorylation sites (S516A and S550A) in the COOH terminus (Kv4.3[S516A, S550A]) was prepared by PCR-based site-directed mutagenesis, as previously described (63).

Xenopus laevis were handled in compliance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the University at Buffalo, State University of New York Institutional Animal Care and Use Committee. Mature female X. laevis (Xenopus One, Ann Arbor, MI) were anesthetized by immersion in a solution of ethyl 3-aminobenzoate methane sulfonate salt (Sigma Aldrich; 1.5 g/l), and ovarian lobes were removed through a small incision in the abdominal wall, as previously described (20). The lobes were placed in a collagenase-containing, Ca2+-free OR2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4, and 1–2 mg/ml collagenase; type II, Sigma, St. Louis, MO) for removal of the follicular layer. The solution containing the oocytes was gently agitated for ~1.5 h, and collagenase activity was arrested by bovine albumin, as previously described (20). Defolliculated oocytes (stage V–VI) were then injected with synthetic mRNA (up to 25 ng) using a microinjection system (Nanoject, Drummond Scientific, Broomall, PA) and incubated at 18°C for 24–72 h in an antibiotic-containing Barth's solution [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 10 mM HEPES, pH 7.4, and 1% antibiotic-antimycotic solution; Invitrogen].

Solution preparation. W-7 [HCl N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide], water-soluble KN-93 {2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine}, and water-soluble KN-92 {2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine}were obtained from Calbiochem and dissolved in ND-96 solution before use. KN-92 is an inactive structural analog of KN-93 (24). BAPTA-AM (Invitrogen) was dissolved in ethanol. The IC50 of KN-93 on Ca2+/CaMKII activity was reported to vary from 11.1 to 20 µM (33, 50).

To evaluate the effects of W-7 and KN-93 in the presence of BAPTA, we used BAPTA-AM dissolved in 100% ethanol and diluted in ND-96 solution to yield a final BAPTA-AM dilution of 1:1,000 (vol/vol).

Electrophysiological techniques. Oocytes were voltage clamped using a two-microelectrode "bath-clamp" amplifier (model OC-750A, Warner Instruments, Hamden, CT), as described in detail elsewhere (20). Microelectrodes were fabricated from 1.5-mm-OD borosilicate glass tubing (model TW150F-4, WPI) using a two-stage puller (model L/M-3 P-A, Adams & List, Great Neck, NY) filled with 3 M KCl; resistances were 0.6–1.5 M{Omega}. During recordings, oocytes were continuously bathed in control ND-96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 10 mM HEPES, with pH adjusted to 7.4 with NaOH). Currents were recorded at room temperature (21–23°C) and filtered at 2.5 kHz.

Experimental protocols. To evaluate the effects of W-7 and KN-93 on heterologously expressed Kv4.3 channels, we performed voltage-clamp protocols before and after exposure to W-7 or KN-93 in ND-96 solution. Oocytes were superfused with 100 µM BAPTA-AM for 30 min (58, 61, 65) or 500 µM BAPTA-AM for 60 min. Thereafter, the cells were washed and incubated in ND-96 solution for 30 min. Voltage-clamp protocols were then implemented to obtain experimental data after washout of BAPTA-AM. Parallel experiments were performed in WT and double-mutant Kv4.3 channels.

Data analysis. Digitized data were collected and analyzed using pCLAMP software (Axon Instruments) and stored in a computer. Unless otherwise stated, neither leakage nor capacitance was subtracted from the raw data traces from the two-microelectrode voltage-clamp recordings. The pulse protocols and the equations that best fit the data are presented in each case. Values are means ± SE. Confidence levels were calculated using Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of W-7 and KN-93 on peak Kv4.3 current. We established a dose-response relation from an analysis of changes in peak current at +50 mV during exposure to different concentrations of W-7 (Fig. 1). W-7 at >100 µM produced oocytes with a large leak conductance, rendering them unsuitable for use in lengthy experiments. The dose-response relation was fit to the following function: f(D) = 1/[1 + (D/IC50)n], where D is W-7 concentration. The optimal fit yielded an IC50 of 92.4 µM and a power coefficient (n) of 3.5 (Fig. 1B). The effects of 85 µM W-7 on peak current-voltage relations of Kv4.3 channels expressed in Xenopus oocytes were determined by comparison of the control data with the data in the presence of W-7 (Fig. 2). W-7 reduced the magnitude of peak Kv4.3 current (IKv4.3) and accelerated inactivation during the depolarizing pulses (Fig. 2, A–C). The peak current at +50 mV was reduced to 55.5 ± 4.3% of the control value at 85 µM W-7 (n = 7). In a separate set of experiments, we determined that the effects of W-7 on peak current were largely reversible (Fig. 3). W-7 at 85 µM decreased peak current to 54.2 ± 4.4% of the control value; peak current returned to 92.4 ± 0.5% of the control value at the end of a 30-min washout.


Figure 1
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  		Fig. 1. Dose-response relation for W-7 block of the Kv4.3 channel. A: current recordings from 2-electrode voltage-clamp experiments on Xenopus oocytes at 0 (control), 25, 75, and 100 µM W-7 during a 0.033-Hz train consisting of 2,000-ms depolarizing pulses to +50 mV from a holding potential (HP) of –90 mV. B: dose-response relation for W-7. bullet, Experimental data. In the fitting function, D represents W-7 concentration and n is power coefficient. Values are means ± SE.

 

Figure 2
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  		Fig. 2. W-7 block of Kv4.3 channel. A and B: current recordings during a 0.033-Hz pulse train consisting of 2,000-ms depolarizing pulses between –90 and +50 mV (HP = –90 mV) in control and at 85 µM W-7. W-7 decreased peak currents at potentials positive to –40 mV. C: normalized peak current-voltage (I-V) relations in control and at 85 µM W-7. D: voltage dependence of W-7 block of Kv4.3 channel. Normalized Kv4.3 currents (IKv4.3,W-7/IKv4.3,control) are plotted as a function of test-pulse voltage. Equation that best fits data is as follows: f(V) = KD,0 mV/{KD,0 mV + [D]exp({delta}zFV/RT)}, where z, F, R, and T have their usual meanings, D is W-7 concentration, {delta} is fraction of electrical field sensed by W-7 during the pulse train, and KD,0 mV is apparent dissociation constant at 0 mV ({delta} = 0.18 ± 0.05).

 

Figure 3
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  		Fig. 3. Reversibility of Kv4.3 channel block by W-7. Peak I-V normalized relations are shown in control, at 85 µM W-7, and after 30 min of washout. Effects of W-7 on peak current were almost completely reversible.

 
To ascertain whether the reduction in peak current was consistent with open-channel block, we examined the voltage dependence of block of peak current and calculated the fractional electrical distance of open-channel block (74, 76) as determined from the slope of the normalized current reduction-voltage relation. A nonzero value reflects the movement of a charged blocking compound in the permeation pathway and places W-7 at a binding site in the transmembrane electrical field. The fractional block of peak currents in the presence of W-7 plotted against test potential in Fig. 2D shows a relation with typical voltage dependence (74). The fraction of the electrical field ({delta}) sensed by W-7 during a pulse train is 0.18 ± 0.05 (n = 7; Fig. 2D). These data are consistent with open-channel block and the {delta} value that closely approximates the values reported for quinidine block of Kv1.4 and Kv1.5 channels (67, 69, 74), which suggests that W-7 interacts directly with the Kv4.3 channel through the binding site on the cytoplasmic side of the pore region.

Although the effects of KN-93 on the Kv4.3 channel were reported to modify kinetics of inactivation and recovery (19, 63), these studies did not address whether some of the effects of KN-93 on peak current were also due to open-channel block, i.e., were voltage dependent. KN-93 at 35, 50, and 100 µM reduced peak current at +50 mV by 4.9 ± 3.5%, 14.4 ± 5.9%, and 30.3 ± 3.5%, respectively (Fig. 4). However, there was no evidence for voltage-dependent block, as the calculated value of {delta} was close to 0 for each of the three concentrations of KN-93. Hence, although KN-93 decreased the peak current, this effect cannot be attributed to open-channel block on the cytoplasmic side of the channel. KN-92, the inactive congener of KN-93, at 100 µM was used as a control and had no effect on the peak current-voltage relation (data not shown).


Figure 4
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  		Fig. 4. KN-93 block of Kv4.3 channel. A: normalized current recordings during a 0.033-Hz pulse train consisting of 2,000-ms depolarizing pulses between –90 and +50 mV (HP = –90 mV) in control and at 35 and 100 µM KN-93. KN-93 accelerated open-state inactivation kinetics. B: normalized peak I-V relations in control and at 35, 50, and 100 µM KN-93.

 
Use dependence. The differences in voltage-dependent block with W-7 and KN-93 predicted that we would see use-dependent block during exposure to W-7, but not KN-93. Therefore, we examined the effects of W-7 and KN-93 on the Kv4.3 channel after the onset of a pulse train consisting of 8-ms depolarizing pulses to +50 mV at 2 Hz (Fig. 5). We selected an 8-ms pulse to minimize inactivation during the pulse and concentrations of W-7 and KN-93 that produced a clear-cut reduction in peak current (Figs. 1 and 4). As shown in Fig. 5A, after onset of a 2-Hz pulse train, the normalized currents showed nominal use-dependent block under control conditions. However, exposure to W-7 caused a progressive reduction in magnitude of the peak current until a new steady-state value was reached, and the magnitude of the reduction in the peak current under steady-state conditions was much greater than under control conditions. In contrast, 100 µM KN-93 did not progressively reduce the normalized peak currents after the first pulse, reflecting the absence of use dependence (Fig. 5B). With 35 µM KN-93, the observations were similar (data not shown). Hence, the use-dependent reduction in the peak current during exposure to W-7 is consistent with open-state block of the Kv4.3 channel, and the absence of similar use-dependent effects of KN-93 is consistent with the absence of open-state pore block.


Figure 5
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  		Fig. 5. Frequency dependence of block of Kv4.3 channel by W-7 and KN-93. A: 2.0-Hz pulse trains consisting of 8-ms pulses to +50 mV (HP = –90 mV) in control and at 75 µM W-7. Peak current was normalized to maximum control value in the absence and presence of W-7 at the same cycle length. B: pulse trains described in A in control conditions and at 100 µM KN-93. There is no evidence of use dependence in the presence of KN-93.

 
Steady-state inactivation. Next, we examined the effects of W-7 on steady-state inactivation (Fig. 6A). Steady-state inactivation relations were determined using a 2,000-ms first pulse (P1) followed by a second pulse (P2) to +50 mV. The values were as follows: half-maximal potential, V1/2 = –46.2 ± 1.1 and slope factor k = 6.0 ± 0.3 mV in the presence of 85 µM W-7 and V1/2 = –42.8 ± 3.2 and k = 5.5 ± 0.5 mV (n = 6) for control (Fig. 6A), and V1/2 = –44.4 ± 2.3 and k = 5.6 ± 0.4 mV in the presence of 100 µM KN-93 and V1/2 = –44.5 ± 2.6 and k = 5.2 ± 0.3 mV for control (n = 5; Fig. 6B). In contrast to W-7, 35 and 100 µM KN-93 had no effect on the steady-state inactivation relation. There was a 3.4-mV hyperpolarizing shift in V1/2 after exposure to W-7; however, there was no shift in V1/2 after exposure to KN-93. If the modest hyperpolarizing shift in steady-state inactivation in the presence of W-7 was mediated by a decrease in Ca2+/CaMKII activity, then one would expect the same degree of shift in V1/2 in the presence of W-7 and KN-93. However, since we observed the shift in V1/2 only in the presence of W-7, we conclude that the hyperpolarizing shift resulted from open-channel blockade superimposed on the inactivation induced by P1 during the pulse protocols. Similar shifts in the steady-state inactivation relation have been reported for open-channel blockers of Na+ channels (8, 29, 30, 31, 66). KN-92 had no effect on the steady-state inactivation relation (data not shown).


Figure 6
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  		Fig. 6. Effects of W-7 and KN-93 on steady-state inactivation. Steady-state inactivation was determined using a 2-pulse protocol. A 2,000-ms pulse between –120 and +50 mV (P1) was followed by a pulse to +50 mV (P2). W-7 at 85 µM caused a 3.4-mV hyperpolarizing shift in half-maximal potential (V1/2), while there is no change in steady-state inactivation relations in the presence of 100 µM KN-93.

 
Open-state inactivation kinetics. Agents that directly decrease the activity of Ca2+/CaMKII have been shown to accelerate inactivation kinetics of heterologously expressed Kv4.3 channels (19, 63). W-7 inhibits Ca2+-CaM effects, which in turn decrease the activity of Ca2+/CaMKII as well as other kinases and phosphatases. Therefore, the effects of W-7 on inactivation kinetics might reflect its influence on these other kinases and phosphatases and, therefore, could differ from the effects of KN-93. Using a 2,000-ms depolarizing pulse, as previously described (72), we examined the effects of W-7 on the kinetics of open-state inactivation. W-7 at 50 µM accelerated the kinetics of each of the three components of inactivation during a 2,000-ms pulse (Fig. 7A). The effects of W-7 on open-state inactivation were concentration dependent (Table 1). The time constants of the fast ({tau}1), intermediate ({tau}2), and slow ({tau}3) components of inactivation decreased dramatically by 2.6-, 3.1-, and 4.8-fold, respectively, at the highest concentration of W-7 (Table 1). As expected, 100 µM KN-93 also accelerated all three components of inactivation (Fig. 7B) (19, 63). Although the effects of KN-93 on all three components of open-state inactivation were similar to those of W-7, the effects of both agents might not result from a selective reduction in Ca2+/CaMKII activity in Xenopus oocytes (19, 63) (see below).


Figure 7
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  		Fig. 7. Effects of W-7 on kinetics of open-state inactivation of Kv4.3 channels. Inactivation kinetics were measured during 2,000-ms pulses between –20 and +50 mV (HP = –90 mV) under control conditions and in 50 µM W-7 or 100 µM KN-93. Fitting function for analysis of inactivation is as follows: f(t) = Formula Ajexp(–t/{tau}j), where N = 3. Data for inactivation time constants were obtained from 6 cells. W-7 and KN-93 caused an acceleration of all 3 components of inactivation.

 

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  		Table 1. Concentration-dependent effects of W-7 on open-state inactivation kinetics of Kv4.3 channel

 
Since we established that the effects of W-7 on peak current were largely reversible, we also performed a separate set of experiments to determine whether the effects of W-7 on Kv4.3 channel open-state inactivation kinetics were also reversible. We used a 1,000-ms depolarization pulse, which only results in a biexponential inactivation time course (72). During exposure to 85 µM W-7, {tau}1 and {tau}2 decreased from 47.1 ± 2.7 and 322.4 ± 27.1 ms to 24.8 ± 4.2 and 123.8 ± 7.0 ms, respectively. These time constants returned to 40.5 ± 2.9 and 249.0 ± 12.8 ms at the end of a 30-min wash, which were 85.9% and 77.3% of the control values for {tau}1 and {tau}2. The degree of reversibility in values of inactivation kinetics and peak current after 30 min was similar.

Closed-state inactivation kinetics. In addition to open-state inactivation, Kv4.3 channels can inactivate from the closed state at potentials reported for myocardial cells during acute myocardial ischemia (18). Using previously described protocols (72), we examined the effects of W-7 on the kinetics of closed-state inactivation (Fig. 8). The kinetics of closed-state inactivation were evaluated using subthreshold depolarizing pulses (9, 10, 72). These protocols consisted of four pulses: P1 from –100 to +40 mV (800 ms); P2 to –100 mV (5 s); P3 to variable voltages and durations (from –80 to –40 mV and from 800 ms to 14.4 s); and P4 to +40 mV (800 ms), followed by a return to a holding potential of –100 mV. The magnitude of peak current during P4 represents the degree of inactivation that developed during P3. Closed-state inactivation was monoexponential and accelerated by 85 µM W-7 at –60 to –40 mV (Fig. 8). At –60 mV, W-7 decreased the time constant of inactivation by 59.2%.


Figure 8
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  		Fig. 8. Voltage dependence of closed-state inactivation of IKv4.3. Voltage-clamp protocol consisted of 4 pulses (HP = –100 mV): P1 (+40 mV, 800-ms duration), P2 (–100 mV, 5,000-ms duration), P3 (–80 to –40 mV, 800- to 14,400-ms duration), and P4 (+40 mV, 800-ms duration). Normalized current is plotted as a function of pulse duration at different test potentials during P3 (in mV): –60 (circles), –50 (squares), –40 (diamonds). Filled symbols are control data, and open symbols are W-7 (85 µM) data; 9 cells were used in each experiment.

 
Recovery from open-state inactivation. Our data suggest that the changes in IKv4.3 during exposure to W-7 are due to the simultaneous effect of open-channel block and possible inhibition of Ca2+/CaMKII activity and that KN-93 acts solely through inhibition of Ca2+/CaMKII activity. To further validate this interpretation, we examined recovery from inactivation in the absence and presence of W-7 (Fig. 9) and KN-93, since KN-93 was shown to prolong recovery from inactivation (63). Our data suggested that the association kinetics of W-7 were fast. Hence, for P2, we selected a 10-ms pulse to minimize association of W-7 during P2 in the recovery protocol. Under control conditions, recovery from inactivation was monoexponential, with time constant, {tau}rec = 294 ± 34 ms. Interestingly, 75 µM W-7 changed recovery kinetics, which were best described by two components ({tau}rec1 = 399 ± 44 ms and {tau}rec2 = 8,110 ± 505 ms). The time constant of the first component was increased by 35.7% compared with control values (Fig. 9). The appearance of a second extremely slow component of recovery from inactivation confirmed the presence of open-channel block, representing dissociation of W-7 from the inner channel pore. The y-axis intercept value for the second component is 0.21 ± 0.05, which corresponds to the degree of open-channel block during P1 (Fig. 1). In the presence of 100 µM KN-93, recovery from inactivation remained monoexponential and {tau}rec increased by 22.5% from 338 ± 42 to 414 ± 45 ms (P < 0.03, paired t-test). We suggest that the slowing of the first component of recovery in the presence of W-7 reflects a decrease in CaMKII activity, inasmuch as {tau}rec1 in the presence of W-7 is similar to {tau}rec in the presence of KN-93 (63). KN-92 had no effect on recovery from inactivation (data not shown).


Figure 9
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  		Fig. 9. Recovery of Kv4.3 channel from inactivation. Normalized peak current IKv4.3 is plotted as a function of interpulse interval in the absence and presence of W-7. Standard 2-pulse protocol was used to determine recovery from inactivation. An 800-ms P1 to +50 mV (HP = –90 mV) was followed by a 10-ms P2 to +50 mV. Interval between P1 and P2 was allowed to vary between 0 and 20 s. Recovery kinetics were best fit by a monoexponential function under control conditions ({tau}rec = 294 ± 34 ms). With W-7, recovery from inactivation was best described by a biexponential function. The first component was 399 ± 44 ms, and the second was 8,110 ± 505 ms. W-7 at 75 µM caused the appearance of a second component of recovery. Intercept value with y-axis was 0.21 ± 0.05 (n = 4).

 
To exclude the possibility of association of W-7 with the Kv4.3 channel in the inactive state, we examined the amplitude of the second component of recovery from inactivation as a function of pulse duration. We utilized a standard recovery protocol for determination of recovery kinetics, except for changes in P1 duration. Pulses (P1) of 10, 67, and 2,000 ms to +50 mV were applied in the presence of 35 µM W-7. Our data indicate that the amplitude of the second component of recovery from inactivation was independent of pulse duration. The amplitude of the second component of recovery was 7.93 ± 0.92% at 10 ms, 7.88 ± 1.04% at 67 ms, and 6.78 ± 0.86% at 2,000 ms. These data indicate that there is no significant association of W-7 with the Kv4.3 channel during the inactive state.

Interventions designed to identify selective inhibition of Ca2+/CaMKII kinase activity. If changes in the Kv4.3 channel inactivation kinetics are due to Ca2+-dependent phosphorylation, then a reduction of intracellular Ca2+ concentration ([Ca2+]i) as seen after washout of 500 µM BAPTA-AM should cause these changes. To ascertain whether the effects of W-7 and KN-93 on Kv4.3 channel open-state inactivation kinetics were independent of changes in [Ca2+]i, we evaluated the effects of W-7 and KN-93 on Kv4.3 channels in oocytes after washout of 500 µM BAPTA-AM (see MATERIALS AND METHODS). These experiments showed that {tau}1 decreased by 9.8 ± 3.2%, {tau}2 by 16.3 ± 5.3%, and {tau}3 by 0.3 ± 4.6% after washout of 500 µM BAPTA-AM (Fig. 10, Table 2). To establish whether these changes were mediated by a decrease in Ca2+/CaMKII activity, we utilized a double-mutant Kv4.3 channel with modified CaMKII consensus phosphorylation sites, Kv4.3[S516A, S550A] (see MATERIALS AND METHODS) (63). In contrast to the WT channel, inactivation kinetics were unchanged in the mutated channel after washout of 500 µM BAPTA-AM (Fig. 10, Table 2). These data suggest that BAPTA decreased inactivation time constants through a partial decrease in Ca2+/CaMKII activity.


Figure 10
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  		Fig. 10. Effects of BAPTA-AM, KN-93, and W-7 on open-state inactivation in wild type (WT) and mutant [Mut, modified CaMKII consensus phosphorylation sites (S516A and S550A)] Kv4.3 channels. Current recordings were obtained from 2-electrode voltage clamp on Xenopus oocytes during 2,000-ms depolarizing pulses to +20 mV from HP of –90 mV. A and D: control solution and after BAPTA-AM washout. B and E: after BAPTA-AM washout and subsequent exposure to 35 µM KN-93. C and F: after BAPTA-AM washout and subsequent exposure to 15 µM W-7.

 

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  		Table 2. Effects of BAPTA and KN-93 on inactivation kinetics of WT and mutant Kv4.3 channels

 
The selectivity of KN-93 for CaMKII has been reported to be concentration dependent, which suggests that its effects on WT and Kv4.3[S516A, S550A] channels should also be concentration dependent (19, 63). The concentration of KN-93 that accelerates inactivation kinetics of WT, but not mutated, channels represents the concentration that selectively decreases Ca2+/CaMKII activity. We examined the effects of 35 and 50 µM KN-93 on Kv4.3 inactivation kinetics in oocytes pretreated for 60 min with 500 µM BAPTA-AM (see MATERIALS AND METHODS) (5, 31a, 19, 33, 63). The changes in the time constants of the three components of open-state inactivation during 2,000-ms pulses at 35 and 50 µM were compared with the kinetics measured after washout of BAPTA-AM and are summarized in Figs. 10 and 11 and Table 2. KN-93 had a concentration-dependent effect on Kv4.3 channel inactivation kinetics (35 vs. 50 µM). Only 35 µM KN-93 caused a significant decrease in each of the three components of inactivation without changing any of the components of inactivation of Kv4.3[S516A, S550A] channel (Fig. 11, Table 2). KN-93 at 35 µM decreased {tau}1, {tau}2, and {tau}3 of WT channels by 7.2 ± 1.3%, 8.5 ± 1.5%, and 13.9 ± 3.5%, respectively. Even at 50 µM KN-93, the decrease in Kv4.3 channel inactivation time constants was much greater in WT than in mutant channels (Fig. 11, Table 2). On the basis of the changes in Kv4.3 channel inactivation kinetics, we conclude that 35 µM KN-93 selectively inhibited Ca2+/CaMKII activity, while additional effects at 50 µM were mediated by other enzymes. We examined these effects at test potentials of +20 and +50 mV and found that the data were voltage insensitive.


Figure 11
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  		Fig. 11. Percent change in open-state inactivation kinetics during exposure to KN-93 in WT (shaded bars) and mutant (solid bars) Kv4.3[S516A, S550A] channels. Oocytes were initially exposed to 500 µM BAPTA-AM for 60 min and then washed and incubated in ND-96 solution. Inactivation kinetics for fast, intermediate, and slow components were measured during 2,000-ms pulses to +20 mV (HP = –90 mV) before and after exposure to 35 and 50 µM KN-93. Percent change was calculated for data obtained after washout of BAPTA-AM and during exposure to 35 and 50 µM KN-93.

 
To determine whether the effects of W-7 were also a result of a selective decrease in Ca2+/CaMKII activity, we compared the effects of 15, 25, and 50 µM W-7 on WT and mutant channels after pretreatment with 500 µM BAPTA. Exposure to 15, 25, and 50 µM W-7 resulted in a significant acceleration of all three components of inactivation in WT channels (Figs. 10 and 12, Table 3). W-7 at 50 µM decreased {tau}1 by 39.3 ± 8.8%, {tau}2 by 52.0 ± 5.9%, and {tau}3 by 62.3 ± 5.5% (Fig. 12, Table 3). However, there was no significant difference in inactivation kinetics between WT and mutant channels at any concentration tested. In sum, the W-7-induced changes in inactivation kinetics are inconsistent with selective decrease of Ca2+/CaMKII activity.


Figure 12
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  		Fig. 12. Percent change in open-state inactivation kinetics during exposure to W-7 in WT (shaded bars) and mutant (solid bars) Kv4.3 channels. Oocytes were initially exposed to 500 µM BAPTA-AM for 60 min and then washed and incubated in ND-96 solution. Inactivation kinetics for fast, intermediate, and slow components were measured during 2,000-ms pulses to +20 mV (HP = –90 mV) before and after exposure to 15, 25, and 50 µM W-7. Percent change was calculated for data obtained after washout of BAPTA-AM and during exposure to 15, 25, and 50 µM W-7.

 

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  		Table 3. Effects of W-7 on inactivation kinetics of WT and mutant Kv4.3 channels

 
A selective decrease in Ca2+/CaMKII activity results not only in an acceleration of open-state inactivation kinetics, but also in a slowing of recovery from inactivation (63). As a result, one would predict that this effect on recovery kinetics in the BAPTA-pretreated oocytes would occur in the WT, but not the mutated, Kv4.3 channel. Indeed, the time constant of recovery after washout of BAPTA-AM increased by 23.7 ± 5.7% and 3.6 ± 6.4% in the WT and mutant channels, respectively (Fig. 13, Table 4). We also predict that the effects of W-7 on the first component of recovery kinetics would occur after pretreatment with BAPTA-AM. Hence, we examined the effects of W-7 on the kinetics of inactivation recovery in the WT channel to determine whether these effects were concentration dependent and whether there was a comparable effect in the mutated Kv4.3 channel (Fig. 13, Table 4). Recovery kinetics in the WT channel were selectively slowed at 15 µM W-7. In the mutant channel, W-7 had no or little effect on the first component of recovery kinetics (Fig. 13, Table 4). There were significant differences in the recovery kinetics of WT and mutant channels at all W-7 concentrations tested when the percent changes of the corresponding time constants of recovery were compared (Table 4). These differences between the effects of W-7 on recovery kinetics in WT and mutant channels suggest that the selective effects of W-7 on recovery result from a reduction in Ca2+/CaMKII activity.


Figure 13
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  		Fig. 13. Effects of BAPTA-AM and W-7 on recovery from inactivation in WT and mutant Kv4.3 channels. A–C and E–G: current traces elicited by standard 2-pulse protocol (800-ms P1 to +50 mV, with HP = –90 mV, followed by 10-ms P2 to +50 mV). Interval between P1 and P2 was allowed to vary between 10 and 1,710 ms. D and H: normalized peak currents IKv4.3 plotted as a function of interpulse interval obtained in control solution, after BAPTA-AM washout, and during subsequent exposure to 15 µM W-7.

 

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  		Table 4. Effects of W-7 on {tau}rec1 of WT and mutant Kv4.3 channels

 

   DISCUSSION
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Attention has been drawn to the importance of increased Ca2+/CaMKII activity in the pathophysiology of different types of heart disease, including ventricular arrhythmias (2, 4, 23, 36, 45, 46). W-7, a commonly used inhibitor of Ca2+-CaM, has been reported to suppress ventricular arrhythmias in animal models (4, 26, 36, 45, 46). To gain an understanding of the mechanisms underlying the effects of W-7, we examined the effects of W-7 and KN-93 on the Kv4.3 voltage-gated K+ channel, which plays an important role in cardiac repolarization in atria and ventricles (13, 16, 53).

The goal of our study was to examine the effects of W-7 on the permeation and gating properties of heterologously expressed Kv4.3 channels in Xenopus laevis oocytes. Comparison of the effects of W-7 with those of KN-93 at a concentration that selectively reduces Ca2+/CaMKII activity should help establish whether the inhibition of Ca2+-CaM produces effects similar to those attained by competitive inhibition of Ca2+-CaM binding to CaMKII. Decreased Ca2+/CaMKII activity has been shown to accelerate open-state inactivation and slow recovery from inactivation in HEK-293 cells (19, 63). However, no comparable data are available for such studies performed in Xenopus laevis oocytes. Our data demonstrate voltage-dependent pore block by W-7, but not KN-93. In addition, we have shown that the effects of W-7 did not solely result from inhibition of Ca2+/CaMKII activity.

We have demonstrated that W-7 and KN-93 reduced peak current of heterologously expressed Kv4.3 channels in Xenopus oocytes. Although both compounds reduced the peak current, only W-7 caused voltage- and concentration-dependent block. The power coefficient of 3.5 (Fig. 1) represents pore block and inhibition of Ca2+-CaM (63, 77). The voltage-dependent effects of W-7, but not KN-93, on peak IKv4.3, the hyperpolarizing shift in steady-state inactivation relation, and the use dependence demonstrate that only W-7 fulfills the criteria for open-channel block resulting from binding of W-7 to residues in the cytoplasmic portion of S6, which faces the channel pore (8, 66, 74, 76, 77).

Analysis of recovery from inactivation in the presence of W-7 provides additional evidence for the presence of open-channel block. In the presence of KN-93, recovery was demonstrated to be a monoexponential process, with a slowing of recovery kinetics comparable to that previously described (39). However, recovery from inactivation in the presence of 75 µM W-7 yielded a distinct biexponential time course. The first component is attributable to recovery of unblocked channels and the second component to the slow dissociation of W-7 from the channel pore. The intercept value of the second component, 0.21, corresponds to a 21% decrease in IKv4.3 from open-channel block. Thus the decrease in IKv4.3 in the presence of W-7 is consistent with open-channel block. The unresolved question is whether the very slow recovery kinetics of W-7 reflect unrestricted dissociation of the drug from its binding site or trapping of W-7 in the pore region on the cytoplasmic side of the channel. If the latter is true, trapping can result in retention of the drug in the inner pore region and preclude accurate determination of the dissociation rate of W-7 from its binding site (6, 30).

W-7 and KN-93 decreased the time constants of each of the three components of open-state inactivation. The effects of W-7 and KN-93 on inactivation kinetics were concentration dependent. W-7 was shown to accelerate the kinetics of closed-state inactivation. Recovery from inactivation in the presence of KN-93 was slowed but remained monoexponential, while the first component of recovery from inactivation in the presence of W-7 was slowed to a similar degree. Using WT and mutant [modified CaMKII consensus phosphorylation sites (S516A and S550A)] Kv4.3 channels, we demonstrated that the effects of KN-93 were selective in the WT channel, but only at low concentrations. The nonselective effects at higher concentrations of KN-93 suggest that these effects are mediated independently of the inhibition of CaMKII-mediated phosphorylation of Kv4.3 channels. On the other hand, W-7 did not show a selective effect on open-state inactivation. Despite the nonselectivity of effect on inactivation, the first component of recovery from inactivation (unblocked channels) was slowed selectively by W-7 in WT channels. The nonselective effects of W-7 on open-state inactivation kinetics indicate the presence of multiple Ca2+-CaM-sensitive regulatory pathways.

To understand the basis of the effects of W-7 on inactivation kinetics, it is useful to recall the effects of CaM in cells. CaM, a ubiquitous Ca2+-binding protein, plays an important role in the Ca2+-dependent signaling pathways of eukaryotic cells (37, 48). Among the targets of Ca2+-CaM are Ca2+/CaM-dependent kinases and calcineurin (31a, 34, 49, 71). Ca2+ binding to CaM results in a transition from the closed to the open conformation, thereby creating a hydrophobic pocket on the surface of each domain, which is essential for Ca2+-CaM binding to target enzymes such as Ca2+/CaMKII (32, 41, 47, 48, 51). Ca2+-CaM binds to the regulatory domain of Ca2+/CaMKII and induces a conformational change that releases the catalytic domain from autoinhibition (31a, 44, 64). W-7 binds to Ca2+-CaM, preventing the activation of its target enzymes (28, 54, 56). To further resolve the mechanism of action of W-7, we compared its effects with those produced by KN-93, an inhibitor of Ca2+/CaMKII, with little or no effect on other protein kinases, such as PKA and PKC (68), in WT and mutant Kv4.3 channels. In addition, we also used KN-92, a congener of KN-93 that lacks Ca2+/CaMKII inhibitory activity, as an experimental control (70, 71).

Changes in intracellular Ca2+ levels have also been shown to affect the properties of ion channels through a direct effect on CaM bound to a putative binding site, e.g., the IQ motif, in an ion channel and through activation of Ca2+/CaMKII or other Ca2+-sensitive kinases, resulting in phosphorylation of specific residues (serine/threonine) within the NH2 and COOH termini of the channel (31a, 23, 43). There is no consensus sequence representing a CaM-binding site in the Kv4.3 channel, and no other evidence suggests that Ca2+-CaM binds to this channel. Hence, it is likely that the effects of changes in intracellular Ca2+ on the Kv4.3 channel result from Ca2+/CaMKII-mediated phosphorylation (63). Within the context of these observations and other studies on Ca2+/CaMKII activity (19, 63), we can begin to understand how Ca2+-CaM modulates the properties of the Kv4.3 channel in the presence of W-7.

To assess changes in Ca2+/CaMKII activity, we used changes in open-state inactivation kinetics in a WT vs. a mutant [modified CaMKII consensus phosphorylation sites (S516A and S550A)] Kv4.3 channel as a reporter of the changes in Ca2+/CaMKII activity after washout of BAPTA-AM. We showed that 500 µM, but not 100 µM, BAPTA-AM accelerated inactivation kinetics in the WT, but not the mutant, channel, suggesting that an appreciable reduction in [Ca2+]i was needed to decrease Ca2+/CaMKII activity. We suggest that, at 100 µM BAPTA-AM, [Ca2+]i remained at levels that were insufficient to cause an acceleration of open-state inactivation resulting from phosphorylation of Ca2+/CaMKII consensus sites in the Kv4.3 channel (63). Although our data are consistent with the view that the Kv4.3 channel is phosphorylated under resting conditions (19), the effects of 500 µM BAPTA-AM on gating kinetics could have been mediated also via other Ca2+-sensitive kinases, such as PKC. In this situation, one would expect that 500 µM BAPTA-AM would also have had significant effects on the inactivation kinetics of the mutant channel, which was not the case.

We observed a large change in Kv4.3 channel inactivation kinetics at 100 µM KN-93. However, the concentration employed and the magnitude of decrease were much greater than reported by others (19, 63), raising concerns about the selectivity of KN-93. As there are no data identifying a concentration at which the effects of KN-93 on Ca2+/CaMKII are selective in Xenopus oocytes, we used the approach described above to demonstrate that the effects of KN-93 on open-state inactivation kinetics were selective. We showed that 35 µM KN-93 selectively accelerated open-state inactivation kinetics. Although a higher concentration (50 µM) of KN-93 resulted in a greater acceleration of open-state inactivation kinetics in the WT channel, it also caused a modest effect on open-state inactivation in the mutant Kv4.3 channel, consistent with a modest nonselective effect. Colinas et al. (19) attributed the acceleration of inactivation with KN-93 to a decrease in phosphorylation of this channel, because CaMKII coprecipitated with the Kv4.3 channel in HEK cells, an effect inhibited by KN-93. Additional experiments on inactivation kinetics with use of a Ca2+/CaMKII inhibitory peptide demonstrated selective action on the WT vs. the mutant channel, which is also consistent with a decrease in Ca2+/CaMKII activity (63). Finally, other data suggest a similar relation between a slowing of recovery from inactivation in the Kv4.3 channel and a decrease in Ca2+/CaMKII activity (63).

W-7, on the other hand, interacts with Ca2+-CaM and, as a result, influences multiple Ca2+-CaM-dependent pathways. Our observations illustrate the complexity of its actions. Although we were unable to show that the effects of W-7 on open-state inactivation kinetics were greater in the WT than in the mutant channel, the selective effect on the recovery from inactivation in unblocked channels at 15 µM and the greater effects at higher concentrations suggest that W-7 decreased CaMKII activity. However, the difference in selectivity of W-7 on inactivation and recovery suggests that the Ca2+-CaM-dependent pathways that modulate inactivation and recovery may be different.

Thus our data show that W-7 has five important effects on the Kv4.3 channel. 1) Pore block was voltage dependent, which is consistent with use dependence and a biexponential recovery from inactivation. 2) Inactive state block was not a factor contributing to the acceleration of open-state inactivation. 3) We established a concentration of KN-93 that selectively accelerated open-state inactivation of the WT, but not the mutant, channel in oocytes; however, we could not establish a comparable effect with W-7, suggesting that inhibition of Ca2+-CaM activity in oocytes affects multiple regulatory pathways, including Ca2+/CaMKII. The greater slowing of recovery from inactivation in the WT than in the mutant channels suggests that the selective effects of W-7 on recovery from inactivation result from a reduction in Ca2+/CaMKII activity. 4) The marked acceleration of closed-state inactivation in the presence of W-7 will result in a reduction of peak current elicited from potentials positive to –70 mV. 5) We showed that exposure of the oocytes to 500 µM BAPTA-AM significantly accelerated open-state inactivation only in the WT channel through a decrease in Ca2+/CaMKII activity.

Physiological relevance. Extrapolation of our findings to cardiac myocytes suggests that W-7 and KN-93 would accelerate Ito inactivation kinetics and reduce peak Ito, and these effects would be concentration dependent. Although W-7 and KN-93 accelerated open-state inactivation of the Kv4.3 channel, only W-7 caused open-channel block. Because temperature differences between experiments performed in oocytes and in vivo experiments would have appreciable effects on gating kinetics, our data can only be qualitatively extrapolated to in vivo settings. Since the Kv4.3 channel serves as the molecular basis of Ito in humans and most other mammals, both agents would be predicted to decrease the contribution of Ito to repolarization in the atria and ventricles. Simulations of different ionic currents in ventricular action potentials with use of in silico models have established that a reduction in Ito has its primary effects on the early part of repolarization, namely, the speed of phase 1, the magnitude of the notch, and the voltage level of phase 2 (21, 27, 55, 57). The accuracy of these model predictions for all myocytes in the ventricle is based on the assumption that the channels incorporated in these models are representative of uniform channel density in the myocardium, which we know is not the case, since heterogeneity of channel expression has been widely described (13, 14, 25, 53). The different modeling studies have also shown that the effects of a reduction in Ito or IKv4.3 on simulated action potentials depend on the magnitude of the other repolarizing currents, which vary widely between different animal species (12, 27). In the atria, where heterogeneity of repolarizing currents is even more marked, two sets of simulations have shown that a reduction in Ito can lead to a lengthening of action potential duration (14, 21, 55). Experimental data support these conclusions. For example, Kirchhof et al. (36) showed that W-7 and KN-93 prolonged action potential duration in mouse ventricle, where Ito is dominant, but the effect of W-7 was greater. We hypothesize that the greater effect of W-7 could have been caused by acceleration of inactivation, open-channel block, and use-dependent reduction of Ito. It would then follow that the antiarrhythmic effects of W-7 and KN-93 could differ.

In general, inhibition of Ito leads to an increase in APD, which in turn increases Ca2+ influx into the cell and Ca2+-induced Ca2+ release, thereby affecting excitation-contraction coupling (62). The increase in action potential duration may also terminate reentrant arrhythmias. Inhibition of CaMKII should decrease Ito and L-type Ca2+ current and, thereby, reduce Ca2+ entry into the cell (3). Since Ca2+ overload leads to cardiac arrhythmias, KN-93 and W-7 have the potential to be better antiarrhythmic drugs than other class III antiarrhythmic drugs. In addition to the antiarrhythmic effects, alterations in Ito resulting from a decrease in peak current and/or an acceleration of inactivation can lead to significant depolarizing shifts in notch potential and phase 2 of repolarization (45, 62). The loss of the notch and depolarizing shift in the plateau has been shown to cause desynchronization of Ca2+ sparks, prolongation and reduction in L-type Ca2+ current, and intracellular Ca2+ transients (62).

We showed that W-7 caused a marked acceleration of closed-state inactivation of IKv4.3. If this acceleration occurs in native channels, it would cause Ito to be inactivated and, therefore, unavailable for opening at resting potentials more positive than those normally encountered in atrial and ventricular myocytes. Our observation is relevant to understanding electrophysiological experiments performed in isolated cardiac myocytes, where the use of holding potentials positive to approximately –65 mV fall in a range where a change in the kinetics of closed-state inactivation could result in effects that are attributed to open-state inactivation. Of more profound significance is the rapid efflux of K+ during acute myocardial ischemia, which results in depolarization of the resting membrane potential to a range where closed-state inactivation occurs (18). Failure to routinely evaluate closed-state inactivation in the Kv4.3 channel and Ito under different experimental conditions could result in failure to detect a change in the magnitude and kinetics of closed-state inactivation. Hence, the acceleration of closed-state inactivation would lead to a reduction of Ito that might mistakenly be attributed to other factors such as CaMKII-mediated effects on K+ channel subunit gene expression.


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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-52874 and a grant from the Oishei Foundation.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Arthur M. Edelman for many helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. C. Strauss, Dept. of Physiology and Biophysics, UB, SUNY, School of Medicine and Biomedical Sciences, 124 Sherman Hall, 3435 Main St., Buffalo, NY 14214 (e-mail: hstrauss{at}buffalo.edu)

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.


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 REFERENCES
 

  1. Amberg GC, Baker SA, Koh SD, Hatton WJ, Murray KJ, Horowitz B, Sanders KM. Characterization of the A-type potassium current in murine gastric antrum. J Physiol 544: 417–428, 2002.[Abstract/Free Full Text]
  2. Amberg GC, Koh SD, Imaizumi Y, Ohya S, Sanders KM. A-type potassium currents in smooth muscle. Am J Physiol Cell Physiol 284: C583–C595, 2003.[Abstract/Free Full Text]
  3. Anderson ME. Calmodulin kinase and L-type calcium channels: a recipe for arrhythmias? Trends Cardiovasc Med 14: 152–161, 2004.[CrossRef][Web of Science][Medline]
  4. Anderson ME. Calmodulin kinase signaling in heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias. Pharmacol Ther 106: 39–55, 2005.[CrossRef][Web of Science][Medline]
  5. Anderson ME, Braun AP, Wu Y, Lu T, Wu Y, Schulman H, Sung RJ. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharmacol Exp Ther 287: 996–1006, 1998.[Abstract/Free Full Text]
  6. Armstrong CM. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J Gen Physiol 58: 413–437, 1971.[Abstract/Free Full Text]
  7. Aromolaran AA, Blatter LA. Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells. Am J Physiol Cell Physiol 289: C1426–C1436, 2005.[Abstract/Free Full Text]
  8. Bean BP, Cohen CJ, Tsien RW. Lidocaine block of cardiac sodium channels. J Gen Physiol 81: 613–642, 1983.[Abstract/Free Full Text]
  9. Beck EJ, Bowlby M, An WF, Rhodes KJ, Covarrubias M. Remodelling inactivation gating of Kv4 channels by KChIP1, a small-molecular-weight calcium-binding protein. J Physiol 538: 691–706, 2002.[Abstract/Free Full Text]
  10. Beck EJ, Covarrubias M. Kv4 channels exhibit modulation of closed-state inactivation in inside-out patches. Biophys J 81: 867–883, 2001.[Web of Science][Medline]
  11. Birnbaum SG, Varga AW, Yuan LL, Anderson AE, Sweatt JD, Schrader LA. Structure and function of Kv4-family transient potassium channels. Physiol Rev 84: 803–833, 2004.[Abstract/Free Full Text]
  12. Bondarenko VE, Szigeti GP, Bett GCL, Kim SJ, Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: H1378–H1403, 2004.[Abstract/Free Full Text]
  13. Brahmajothi MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne JM, Strauss HC. Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha-subunits in ferret left ventricular myocytes. J Gen Physiol 113: 581–600, 1999.[Abstract/Free Full Text]
  14. Brahmajothi MV, Morales MJ, Reimer KA, Strauss HC. Regional localization of ERG, the channel protein responsible for the rapid component of the delayed rectifier, K+ current in the ferret heart. Circ Res 81: 128–135, 1997.[Abstract/Free Full Text]
  15. Campbell DL, Rasmusson RL, Comer MB, Strauss HC. The cardiac calcium-independent transient outward potassium current: kinetics, molecular properties, and role in ventricular repolarization. In: Cardiac Electrophysiology. From Cell to Bedside (2nd ed.), edited by Zipes D and Jalife J. Philadelphia, PA: Saunders, 1995, p. 83–96.
  16. Campbell DL, Rasmusson RL, Qu Y, Strauss HC. The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis. J Gen Physiol 101: 571–601, 1993.[Abstract/Free Full Text]
  17. Cascio WE, Johnson TA, Gettes LS. Electrophysiologic changes in ischemic ventricular myocardium. I. Influence of ionic, metabolic, and energetic changes. J Cardiovasc Electrophysiol 11: 1039–1062, 1995.
  18. Colinas O, Gallego M, Setien R, Lopez-Lopez JR, Perez-Garcia MT, Casis O. Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes. Am J Physiol Heart Circ Physiol 291: H1978–H1987, 2006.[Abstract/Free Full Text]
  19. Comer MB, Campbell DL, Rasmusson RL, Lamson DR, Morales MJ, Zhang Y, Strauss HC. Cloning and characterization of an Ito-like potassium channel from ferret ventricle. Am J Physiol Heart Circ Physiol 267: H1383–H1395, 1994.[Abstract/Free Full Text]
  20. Courtemanche M, Ramirez RJ, Nattel S. Ionic targets for drug therapy and atrial fibrillation-induced electrical remodeling: insights from a mathematical model. Cardiovasc Res 42: 477–489, 1999.[Abstract/Free Full Text]
  21. Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, 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]
  22. Dzhura I, Wu Y, Zhang R, Colbran RJ, Hamilton SL, Anderson ME. C terminus L-type Ca2+ channel calmodulin-binding domains are "auto-agonist" ligands in rabbit ventricular myocytes. J Physiol 550: 731–738, 2003.[Abstract/Free Full Text]
  23. Fan GH, Wang LZ, Qiu HC, Ma L, Pei G. Inhibition of calcium/calmodulin-dependent protein kinase II in rat hippocampus attenuates morphine tolerance and dependence. Mol Pharmacol 56: 39–45, 1999.[Abstract/Free Full Text]
  24. Flaim SN, Giles WR, McCulloch AD. Contributions of sustained INa and IKv43 to transmural heterogeneity of early repolarization and arrhythmogenesis in canine left ventricular myocytes. Am J Physiol Heart Circ Physiol 291: H2617–H2629, 2006.[Abstract/Free Full Text]
  25. Gbadebo TD, Trimble RW, Khoo MS, Temple J, Roden DM, Anderson ME. Calmodulin inhibitor W-7 unmasks a novel electrocardiographic parameter that predicts initiation of torsade de pointes. Circulation 105: 770–774, 2002.[Abstract/Free Full Text]
  26. Greenstein JL, Wu R, Po S, Tomaselli GF, Winslow RL. Role of the calcium-independent transient outward current Ito1 in shaping action potential morphology and duration. Circ Res 87: 1026–1033, 2000.[Abstract/Free Full Text]
  27. Hait WN, Lazo JS. Calmodulin: a potential target for cancer chemotherapeutic agents. J Clin Oncol 4: 994–1012, 1986.[Abstract/Free Full Text]
  28. Hille B. Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J Gen Physiol 69: 497–515, 1977.[Abstract/Free Full Text]
  29. Hille B. Ion Channels of Excitable Membranes (3rd ed.). Sunderland, MA: Sinauer, 2001.
  30. Hondeghem LM, Katzung BG. Time- and voltage-dependent interactions of antiarrhythmic drugs with cardiac sodium channels. Biochim Biophys Acta 472: 37–98, 1977.
  31. Hudmon A, Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function. Annu Rev Biochem 71: 473–510, 2002.[CrossRef][Web of Science][Medline]
  32. Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256: 632–638, 1992.[Abstract/Free Full Text]
  33. Ishida A, Kameshita I, Okuno S, Kitani T, Fujisawa H. A novel highly specific and potent inhibitor of calmodulin-dependent protein kinase II. Biochem Biophys Res Commun 212: 806–812, 1995.[CrossRef][Web of Science][Medline]
  34. James P, Vorherr T, Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem Sci 20: 38–42, 1995.[CrossRef][Web of Science][Medline]
  35. Jerng HH, Pfaffinger PJ, Covarrubias M. Molecular physiology and modulation of somatodendritic A-type potassium channels. Mol Cell Neurosci 27: 343–369, 2004.[CrossRef][Web of Science][Medline]
  36. Kirchhof P, Fabritz L, Kilic A, Begrow F, Breithardt G, Kuhn M. Ventricular arrhythmias, increased cardiac calmodulin kinase II expression, and altered repolarization kinetics in ANP receptor deficient mice. J Mol Cell Cardiol 36: 691–700, 2004.[CrossRef][Web of Science][Medline]
  37. Klee CB, Vanaman TC. Calmodulin. Adv Protein Chem 35: 213–232, 1982.[Web of Science][Medline]
  38. Klockner U, Isenberg G. Calmodulin antagonists depress calcium and potassium currents in ventricular and vascular myocytes. Am J Physiol Heart Circ Physiol 253: H1601–H1611, 1987.[Abstract/Free Full Text]
  39. Koh SD, Perrino BA, Hatton WJ, Kenyon JL, Sanders KM. Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II. J Physiol 517: 75–84, 1999.[Abstract/Free Full Text]
  40. Kong ID, Koh SD, Bayguinov O, Sanders KM. Small conductance Ca2+-activated K+ channels are regulated by Ca2+-calmodulin-dependent protein kinase II in murine colonic myocytes. J Physiol 524: 331–337, 2000.[Abstract/Free Full Text]
  41. Kretsinger RH, Rudnick SE, Weissman LJ. Crystal structure of calmodulin. J Inorg Biochem 2–3: 289–302, 1986.[CrossRef][Web of Science]
  42. Ledoux J, Chartier D, Leblanc N. Inhibitors of calmodulin-dependent protein kinase are nonspecific blockers of voltage-dependent K+ channels in vascular myocytes. J Pharmacol Exp Ther 290: 1165–1174, 1999.[Abstract/Free Full Text]
  43. Liang H, DeMaria CD, Erickson MG, Mori MX, Alseikhan BA, Yue DT. Unified mechanisms of Ca2+ regulation across the Ca2+ channel family. Neuron 39: 951–960, 2003.[CrossRef][Web of Science][Medline]
  44. Lou LL, Lloyd SJ, Schulman H. Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc Natl Acad Sci USA 83: 9497–9501, 1986.[Abstract/Free Full Text]
  45. Lu HR, Vlaminckx E, Van de Water A, Gallacher DJ. Calmodulin antagonist W-7 prevents sparfloxacin-induced early afterdepolarizations (EADs) in isolated rabbit Purkinje fibers: importance of beat-to-beat instability of the repolarization. J Cardiovasc Electrophysiol 17: 415–422, 2006.[CrossRef][Web of Science][Medline]
  46. Mazur A, Roden DM, Anderson ME. Systemic administration of calmodulin antagonist W-7 or protein kinase A inhibitor H-8 prevents torsade de pointes in rabbits. Circulation 100: 2437–2442, 1999.[Abstract/Free Full Text]
  47. Meador WE, Means AR, Quiocho FA. Modulation of calmodulin plasticity in molecular recognition on the basis of X-ray structures. Science 262: 1718–1721, 1993.[Abstract/Free Full Text]
  48. Means AR, Tash JS, Chafouleas JG. Physiological implications of the presence, distribution, and regulation of calmodulin in eukaryotic cells. Physiol Rev 62: 1–39, 1982.[Free Full Text]
  49. Means AR, VanBerkum MF, Bagchi I, Lu KP, Rasmussen CD. Regulatory functions of calmodulin. Pharmacol Ther 50: 255–270, 1991.[CrossRef][Web of Science][Medline]
  50. Mellgren G, Bruland T, Døskeland AP, Flatmark T, Vintermyr OK, Døskeland SO. Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes. Endocrinology 138: 4373–4383, 1997.[Abstract/Free Full Text]
  51. Nakayama S, Kretsinger RH. Evolution of the EF-hand family of proteins. Annu Rev Biophys Biomol Struct 23: 473–507, 1994.[Web of Science][Medline]
  52. Nerbonne JM. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525: 285–298, 2000.[Abstract/Free Full Text]
  53. Nerbonne JM, Nichols CG, Schwarz TL. Genetic manipulation of a cardiac K+ channel function in mice. What have we learned and where do we go from here? Circ Res 89: 944–956, 2001.[Abstract/Free Full Text]
  54. Niki I, Niwa T, Yu W, Budzko D, Miki T, Senda T. Ca2+ influx does not trigger glucose-induced traffic of the insulin granules and alteration of their distribution. Exp Biol Med 228: 1218–1226, 2003.[Abstract/Free Full Text]
  55. Nygren A, Fiset C, Firek L, Clark JW, Lindblad DS, Clark RB, Giles WR. Mathematical model of an adult human atrial cell: the role of K+ currents in repolarization. Circ Res 82: 63–81, 1998.[Abstract/Free Full Text]
  56. Osawa M, Swindells MB, Tanikawa J, Tanaka T, Mase T, Furuya T, Ikura M. Solution structure of calmodulin-W-7 complex: the basis of diversity in molecular recognition. J Mol Biol 276: 165–176, 1998.[CrossRef][Web of Science][Medline]
  57. Priebe L, Beuckelmann DJ. Simulation study of cellular electric properties in heart failure. Circ Res 82: 1206–1223, 1998.[Abstract/Free Full Text]
  58. Quamme GA. Chlorpromazine activates chloride currents in Xenopus oocytes. Biochim Biophys Acta 1324: 18–26, 1997.[Medline]
  59. Rakhilin SV, Olson PA, Nishi A, Starkova NN, Fienberg AA, Nairn AC, Surmeier DJ, Greengard P. A network of control mediated by regulator of calcium/calmodulin-dependent signaling. Science 306: 698–701, 2004.[Abstract/Free Full Text]
  60. Rezazadeh S, Claydon TW, Fedida D. KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. J Pharmacol Exp Ther 317: 292–299, 2006.[Abstract/Free Full Text]
  61. Rogalski SL, Cyr C, Chavkin C. Activation of the endothelin receptor inhibits the G protein-coupled inwardly rectifying potassium channel by a phospholipase A2-mediated mechanism. J Neurochem 72: 1409–1416, 1999.[CrossRef][Web of Science][Medline]
  62. Sah R, Ramirez RJ, Backx PH. Modulation of Ca2+ release in cardiac myocytes by changes in repolarization rate: role of phase-1 action potential repolarization in excitation-contraction coupling. Circ Res 90: 165–173, 2002.[Abstract/Free Full Text]
  63. Sergeant GP, Ohya S, Reihill JA, Perrino BA, Amberg GC, Imaizumi Y, Horowitz B, Sanders KM, Koh SD. Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II. Am J Physiol Cell Physiol 288: C304–C313, 2005.[Abstract/Free Full Text]
  64. Shifman JM, Choi MH, Mihalas S, Mayo SL, Kennedy MB. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums. Proc Natl Acad Sci USA 103: 13968–13973, 2006.[Abstract/Free Full Text]
  65. Skeberdis VA, Lan J, Opitz T, Zheng X, Bennett MV, Zukin RS. mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C. Neuropharmacology 40: 856–865, 2001.[CrossRef][Web of Science][Medline]
  66. Starmer CF, Grant AO, Strauss HC. Mechanisms of use-dependent block of sodium channels in excitable membranes by local anesthetics. Biophys J 46: 15–27, 1984.[Web of Science][Medline]
  67. Strauss HC, Morales MJ, Wang S, Brahmajothi MV, Campbell DL. Voltage-dependent K+ channels. In: Heart Physiology and Pathophysiology (4th ed.), edited by Sperelakis N, Kurachi Y, Terzic A, and Cohen MV. San Diego, CA: Academic, 2001, p. 259–280.
  68. Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, Hidaka H. The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun 181: 968–975, 1991.[CrossRef][Web of Science][Medline]
  69. Tessier S, Karczewski P, Krause EG, Pansard Y, Acar C, Lang-Lazdunski M, Mercadier JJ, Hatem SN. Regulation of the transient outward K+ current by Ca2+/calmodulin-dependent protein kinases II in human atrial myocytes. Circ Res 85: 810–819, 1999.[Abstract/Free Full Text]
  70. Tombes RM, Grant S, Westin EH, Krystal G. G1 cell cycle arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an inhibitor of CaMK-II (the multifunctional Ca2+/CaM kinase). Cell Growth Differ 9: 1063–1070, 1995.
  71. Vogel HJ. Calmodulin: a versatile calcium mediator protein. Biochem Cell Biol 72: 357–376, 1994.[Web of Science][Medline]
  72. Wang S, Bondarenko VE, Qu Y, Bett GCL, Morales MJ, Rasmusson RL, Strauss HC. Time- and voltage-dependent components of Kv4.3 inactivation. Biophys J 89: 3026–3041, 2005.[CrossRef][Web of Science][Medline]
  73. Wang S, Bondarenko VE, Qu Y, Morales MJ, Rasmusson RL, Strauss HC. Activation properties of Kv4.3 channels: time, voltage and [K+]o dependence. J Physiol 557: 705–717, 2004.[Abstract/Free Full Text]
  74. Wang S, Morales MJ, Qu YJ, Bett GC, Strauss HC, Rasmusson RL. Kv1.4 channel block by quinidine: evidence for a drug-induced allosteric effect. J Physiol 546: 387–401, 2003.[Abstract/Free Full Text]
  75. Yamniuk AP, Vogel HJ. Calmodulin's flexibility allows for promiscuity in its interactions with target proteins and peptides. Mol Biotechnol 27: 33–57, 2004.[CrossRef][Web of Science][Medline]
  76. Yeola SW, Rich TC, Uebele VN, Tamkun MM, Snyders DJ. Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding. Circ Res 78: 1105–1114, 1996.[Abstract/Free Full Text]
  77. Yeola SW, Snyders DJ. Electrophysiological and pharmacological correspondence between Kv4.2 current and rat cardiac transient outward current. Cardiovasc Res 33: 540–547, 1997.[Abstract/Free Full Text]



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C. Xie, V. E. Bondarenko, M. J. Morales, and H. C. Strauss
Closed-state inactivation in Kv4.3 isoforms is differentially modulated by protein kinase C
Am J Physiol Cell Physiol, November 1, 2009; 297(5): C1236 - C1248.
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