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NEUROPHARMACOLOGY
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
Received October 24, 2005; accepted December 15, 2005.
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Abstract |
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) that are expressed in a wide range of tissues, such as heart [e.g., Kv1, Kv2, Kv4, and human ether a-go-gorelated gene (hERG)], brain (Kv1, Kv2, Kv3, and Kv4), pancreas, and smooth muscle (e.g., Kv1) (Shieh et al., 2000). It is well documented that Kv channels are crucial targets for protein kinase activation and that phosphorylation can affect channel characteristics (Levitan, 1994). Calcium/calmodulin kinase II (CaMK II) is a multifunctional cytoplasmic calcium and calmodulin-dependent protein kinase that phosphorylates and alters the function of a variety of substrates. Given that members of a number of Kv channel subfamilies are expressed in the brain (Shieh et al., 2000) and the abundance of expression of CaMK II in the brain (Braun and Schulman, 1995), it comes as no surprise that CaMK II has been suggested to phosphorylate Kv channels (Roeper et al., 1997). Roeper et al. (1997) showed that the rate of fast N-type inactivation of the Shaker-related Kv1.4 channel is modulated by CaMK II phosphorylation of serine 123 in the N terminus. Similarly, the rate of fast inactivation of Kv4.3 was recently shown to be slowed by CaMK II phosphorylation of serine 550 in the C terminus (Sergeant et al., 2005). In addition to these effects on channel gating, CaMK II also increased surface expression of Kv4.2 and Drosophila ether a go-go (eag) potassium channels (Wang et al., 2002; Sun et al., 2004; Varga et al., 2004). The regulatory effect of CaMK II is not limited to potassium channels, because CaMK II has been shown to alter the activation properties of the sodium channel NaV1.5 (Young and Caldwell, 2005).
The effect of CaMK II on ion channel function has been widely studied through the use of specific inhibitors such as KN-93. KN-93 is a methoxybenzenesulfonamide compound that exerts its effect by competing for the calmodulin binding site of CaMK II with an IC50 of 370 nM (Sumi et al., 1991) and is an extensively used inhibitor of CaMK II. For example, inhibition of CaMK II with KN-93 incubation enhanced N-type inactivation of Kv1.4 and Kv4.3 (Roeper et al., 1997; Sergeant et al., 2005), an observation that is consistent with CaMK II-induced slowing of inactivation. However, Ledoux et al. (1999) have shown that caution must be taken in the interpretation of such experiments because KN-93 affected Kv currents in rabbit portal vein smooth muscles independently of CaMK II action.
In the present study, we have extended these initial observations to show a direct effect of KN-93 on a wide range of cloned Kv channels heterologously expressed in mammalian cells is independent of CaMK II. KN-93 is a potent extracellular open channel blocker of all Kv channels tested and exerts its effect by enhancing C-type inactivation.
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Materials and Methods |
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Cell Culture and Transfection. Experiments were performed on HEK 293 cells either stably (hERG, Kv1.5, Kv1.5 R487V, Kv1.4 
2-147, and Kv4.2) or transiently (Kv1.2, Kv2.1, Kv3.2, and all other Kv1.5 point mutations) expressing cloned channels. HEK 293 cells stably expressing channels were grown in minimum essential medium (MEM), 10% fetal bovine serum, penicillin-streptomycin, and 1 mg ml–1 gentamicin. Transient transfections were performed with HEK 293 cells plated at 20 to 30% confluence on sterile coverslips in 25-mm Petri dishes 1 day before transfection. Two micrograms of ion channel DNA was incubated with 1 µg of enhanced green fluorescent protein DNA (to enable detection of transfected cells) and 3 µl of Lipofectamine 2000 (Invitrogen) in 100 µl of Opti-MEM and added to the cells after changing the media with 900 µl of MEM with 10% fetal bovine serum. All cells were maintained at 37°C in an atmosphere of 5% CO2/air.
Molecular Biology and Channel Mutations. The mammalian expression system pcDNA3.1 (Invitrogen) was used for expression of all constructs in this study. All point mutations in Kv1.5 were performed using the polymerase chain reaction-based QuickChange site-directed mutagenesis kit (Stratagene, San Diego, CA) using primers constructed by Sigma-Genosys (Oakville, ON, Canada). All constructs were sequenced at the University of British Columbia core facilities (Vancouver, BC, Canada) to ensure the fidelity of the polymerase chain reaction reactions.
Electrophysiology Solutions. For recording potassium current, the pipette solution contained 130 mM KCl, 5 mM EGTA, 1 mM MgCl2, 10 mM HEPES, 4 mM Na+2ATP, and 0.1 mM GTP, adjusted to pH 7.2 with KOH. The bath solution contained 5 mM KCl, 135 mM NaCl, 1 mM MgCl2, 2.8 mM sodium acetate, and 10 mM HEPES, adjusted to pH 7.4 with NaOH. High extracellular K+ experiments were performed using bath solution containing 135 mM KCl, 10 mM HEPES, 1 mM MgCl2, and 10 mM dextrose, adjusted to pH 7.4 with KOH. For recording currents through Kv1.5 R487V, the pipette solution contained 130 mM NaCl, 4 mM Na+2ATP, 1 mM MgCl2, 5 mM HEPES, 10 mM EGTA, and 0.1 mM GTP, pH adjusted to 7.2 with NaOH. The bath solution contained 5 mM NaCl, 130 mM N-methyl-L-glucamine, 10 mM HEPES, 10 mM dextrose, and 1 mM MgCl2, pH adjusted to 7.4 with HCl. KN compounds were diluted in bath solution at the appropriate concentration immediately before each experiment and kept in the dark to avoid photo destruction of the drug. For experiments with CIP and internal KN-93, the compounds were diluted in pipette solution at the appropriate concentration.
Electrophysiological Procedures. Glass coverslips to which cells were adhered were removed from the incubator immediately before experiments and placed in a recording chamber mounted on the stage of an inverted phase contrast microscope at room temperature. The bath solution was constantly flowing. Patch electrodes fashioned from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) had a resistance of 1.5 to 2.5 M
when filled with the pipette solution. Whole-cell current recording and data analysis were performed using an Axopatch 200B amplifier, DigiData 1322A digitizer, and pClamp8 software (Axon Instruments, Foster City, CA). Capacity compensation and 80% serial resistance compensation were used in all whole-cell recordings. Data were sampled at 10 kHz and filtered at 2 kHz. No leak subtraction was used, and dashed lines on current records represent the zero current level.
Data Analysis. Potency of each drug was determined by fitting the concentration-response relationships with the Hill equation using GraphPad Prism 3.02 (San Diego, CA):
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The pore structure of Kv1.5 was modeled using the known crystal structure of the related Kv1.2 channel (Protein Data Bank accession no. 2A79) as a template using Swiss-Model's "First Approach Mode" (http://swissmodel.expasy.org/SM_FIRST.html) and "Swiss-Pdb-viewer". Data throughout the text and figures are shown as means ± S.E.M. Statistical significance was determined throughout using Student's t test with P values of less than 0.05 taken to be significant.
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Results |
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) in rabbit portal vein smooth muscle, where the types of Kv channels directly affected by KN-93 were not identified, we examined the effect of KN-93 on a range of Kv channel representatives from a number of different subfamilies. Figure 1A shows typical current traces recorded from Kv1.2, Kv1.5, Kv1.4 2-147, Kv2.1, Kv3.2, Kv4.2, and hERG channels during perfusion of control solution or solution containing 1 µM KN-93. Kv1.4 2-147 was used to remove fast inactivation from the channels and reveal any effect of KN-93. Currents were recorded during voltage pulses to +60 mV for 5 s every 40 s with the exception of Kv4.2 currents, which were recorded during 200-ms pulses every 10 s, and hERG currents, which were recorded during 4-s pulses to –50 mV following a 4-s pulse to +20 mV. In all channels tested, KN-93 enhanced current decay so that the current amplitude at the end of the pulse was significantly reduced (Fig. 1B, filled bars). In some channels, KN-93 also inhibited the peak current (e.g., Kv1.2 and Kv2.1 in Fig. 1A), but this was probably due to the slower activation kinetics of these channels (as shown by the scaled traces) rather than any additional effect of KN-93 on these channels. To demonstrate that the effect of KN-93 on Kv channels was independent of CaMK II, we repeated these experiments using KN-92, the inactive but structurally very similar form of KN-93. The open bars in Fig. 1B show that, like KN-93, KN-92 significantly reduced the sustained current amplitudes at the end of the 5-s depolarizing pulses of all channels tested. These data show that KN-93 inhibits a wide range of Kv channels in a manner that is independent of CaMK II.
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KN-93 Directly Blocks the Kv1.5 Channel. Because Kv1.5 is thought to be the major constituent of the delayed rectifier current in the rabbit portal vein smooth muscle cells (Overturf et al., 1994), and KN-93 has been shown to act as a direct blocker of Kv currents in these cells (Ledoux et al., 1999), we used Kv1.5 to examine the nature of the KN-93 inhibition of Kv currents. Data in Fig. 2A show typical traces recorded from Kv1.5 channels in the presence of a number of different concentrations of KN-93. The current amplitude at the end of the 5-s pulse at each concentration was used to construct the concentration-response curve shown in Fig. 2B. A nonlinear least-squares fit of the Hill equation to the concentration-response data yielded an IC50 value of 307 ± 12 nM and a Hill coefficient, n, of 1.3 ± 0.3 (Fig. 2B).
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In Fig. 3, the effect of KN-93 on the voltage dependence of Kv1.5 channel activation was examined. Figure 3A shows currents recorded during 300-ms pulses from –60 mV to +60 mV (in 10-mV increments) from a holding potential of –80 mV in the absence and presence of 1 µM KN-93. Conductance-voltage curves (Fig. 3B) were generated by calculating the chord conductance from the current amplitude at the end of each pulse. The data were fitted to a single Boltzmann function (see eq. 2 under Materials and Methods). The deviation from the Boltzmann fit seen at depolarized potentials most likely represents an artifact that arises from the calculation of conductance from the current at the end of the pulse. It was not possible to calculate conductance from tail currents in these experiments because KN-93 reduced tail currents to such an extent that it was not possible to obtain accurate measurements. Figure 3B shows that KN-93 appeared to shift the voltage dependence of channel opening to more hyperpolarizing potentials, as reported for the sustained K+ currents of rabbit portal vein smooth muscle cells (Ledoux et al., 1999); however, the data in Fig. 3B did not reach statistical significance. The V1/2 of activation was –7.7 ± 1.0 mV during control conditions and –12.8 ± 3.0 mV during perfusion of 1 µM KN-93. KN-93 resulted in a significant decrease in the Boltzmann's constant k from 8.6 ± 0.7 mV during control conditions to 4.8 ± 0.3 mV. Note that in Fig. 3A on the first pulse following channel opening at –20 mV there is no apparent block because of KN-93. From data such as those recorded in Fig. 3A, we measured the degree of channel block in the presence of different concentrations of KN-93 over a range of potentials where the probability of channel opening was close to 1 (Vm 
10 mV) to construct concentration-response curves at each potential (Fig. 3C). The inset of Fig. 3C shows the dependence of the IC50 on membrane potential and highlights that KN-93 is only a weak voltage-dependent blocker of Kv1.5.
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KN-93 Is an Extracellular Open Channel Blocker of Kv1.5 Channels. To examine the state dependence of accessibility of KN-93, a control current trace was recorded (Fig. 4A, left), and then 1 µM KN-93 was added to the bath while channels were held in the closed state (–80 mV) (Fig. 4A). The peak current obtained on the first opening of the channels following the 3-min incubation was not different from that of control; the average peak current on the first pulse following a 3-min rest was 96 ± 2% that of the control value (n = 6, paired t test, not significant). There was, however, a pronounced effect on the peak current during the second depolarizing pulse, suggesting that KN-93 could not access its binding site in the closed state of the channel. We further examined the open state-dependent binding of KN-93 by inspecting the tail currents (Fig. 4B). Currents were recorded during 200-ms pulses to allow KN-93 binding before a hyperpolarizing pulse to –80 mV to observe tail currents. These experiments were performed in symmetrical K+ conditions to increase tail current amplitude and therefore aid analysis. Tail currents obtained from the same cell in control conditions and in the presence of 1 µM KN-93 are shown in Fig. 4B. The tail currents were fitted to a double exponential function, which shows that KN-93 significantly slowed the slow component of the tail current (mean time constant, 
2, slowed from 52 ± 8 to 77 ± 5 ms, n = 5; paired t test, p < 0.05), causing the tail currents to crossover, a phenomenon that suggests that KN-93 is an open channel blocker that must unbind before the channel can close.
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To identify which side of the membrane KN-93 exerts its effect, we dialyzed the cells with intracellular solution containing 1 µM KN-93 for 5 min after the formation of the whole-cell configuration. Constant drug-free bath perfusion was maintained throughout. Cells were held at –80 mV and pulsed to +60 mV every 40 s for 5 min to document the effect of intracellularly applied KN-93. Figure 4, C and D, shows typical traces and a diary plot from such an experiment. It is clear that at the end of the 5-min period with KN-93 applied intracellularly, sustained currents were not different from that obtained immediately after formation of whole-cell (Fig. 4C). To the same cell, we then applied KN-93 extracellularly by bath perfusion. Figure 4, C and D, documents that addition of KN-93 to the extracellular solution caused a rapid reduction of sustained currents. These data suggest that KN-93 induces its effect from the extracellular side and is ineffective as a blocker from the intracellular solution.
Because nearly all known blockers of hERG involve drug binding at a site located in the central cavity of the pore (Mitcheson et al., 2000), we were interested to know whether KN-93 was an extracellular blocker of hERG as it is in Kv1.5. Figure 4, E and F, shows that hERG currents were not affected following a 5-min period with KN-93 applied intracellularly (n = 3, paired t test, not significant). In contrast, external KN-93 dramatically reduced hERG currents. These data suggest that KN-93 does not act by binding at the "classical" internal drug binding site of the hERG channel.
KN-93 Delays Recovery from Inactivation. Data in Fig. 1 show that Kv1.5 channels activate rapidly upon depolarization and then undergo slow inactivation over the course of a number of seconds, which results in the decay of the current while depolarization is sustained. Slow inactivation is thought to be caused by a local conformational change in the outer pore (P-type inactivation), followed by an energetically and structurally more complete stabilization of the inactivated conformation (C-type inactivation) (De Biasi et al., 1993; Loots and Isacoff, 1998; Kurata and Fedida, 2005). We measured the rate of recovery of Kv1.5 channels from inactivation in the absence (Fig. 5A) and presence of 1 µM KN-93 (Fig. 5B) by applying a +60-mV conditioning pulse for 5 s followed by brief (10 ms) test pulses to +60 mV applied at increasing intervals. The peak current amplitude obtained during each test pulse was normalized to that obtained during the conditioning pulse and plotted against the interpulse interval (Fig. 5C). The data points were fitted to a double exponential function to represent recovery from P-type inactivation (fast) and the more stable C-type inactivation (slow). The data show that KN-93 slowed the fast phase of recovery from 255 ± 28 to 546 ± 61 ms (n = 5; paired t test, p < 0.05) and increased its contribution afast from 0.13 ± 0.04 to 0.32 ± 0.09 of the total inactivation (n = 5; paired t test, p < 0.05). KN-93 had no effect on the slow component of recovery from inactivation (n = 5; paired t test, p = N.S.).
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; Wang et al., 2000)]. R487V mutant channels showed little inactivation during the pulse, and the effect of 1 µM KN-93 was significantly reduced (Fig. 6A). Figure 6B shows a model of the primary sequence of the Kv1.5 outer pore, based on that of the recently crystallized structure of the Kv1.2 channel (Long et al., 2005), and reveals that the outer vestibule of the Kv1.5 pore is formed by the extracellular loops between the fifth and sixth transmembrane helices (S5-P-S6), and R487 is positioned close to the external mouth of the pore. In contrast to the R487V mutation, mutation of threonine 462 to cysteine (T462C) in the outer pore of the channel (Fig. 6B) resulted in an increased rate of inactivation (Fig. 6C). T462C currents inactivated so rapidly that currents were recorded during much shorter pulses; during a 500-ms depolarizing pulse to +60 mV, inactivation was comparable with that in the wild-type channel following a 5-s depolarization (Fig. 1B). Enhancement of inactivation by the T462C mutant was accompanied by increased block upon exposure to KN-93 (Fig. 6C). Concentration-response curves generated using T462C current amplitudes at the end of the 500-ms depolarizing pulses in (Fig. 6D) show that the IC50 of block was significantly reduced to 69 ± 18 nM (n = 4; P < 0.05, paired t test, compared with wild-type). To test the correlation between KN-93 potency and outer pore-mediated inactivation, we mutated each residue with the pore lining region of the outer vestibule (residues highlighted in Fig. 6B) and assessed the effect of KN-93. Of the eight mutations made, four were nonfunctional. However, H463C, S465C, S466C, and P468C showed robust currents, and these showed similar inactivation kinetics to the wild-type channel. The bar graph in Fig. 6E shows the effect of all of the mutations tested on the fractional current, normalized to control, remaining at the end of the pulse following KN-93 block. In contrast to the R487V and T462C mutations, which alter inactivation, the pore mutations that did not alter inactivation (H463C, S465C, S466C, and P468C) showed a level of block that was not significantly different from control.
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Discussion |
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KN-93 Stabilizes the Inactivated State. Here, we have shown that KN-93 induces a rapid decay of current and a reduction of peak current that is dependent upon the ability of the channel to inactivate. Reduction of the rate of inactivation, as in the Kv1.5 R487V mutation, markedly attenuated the effect of KN-93 (Fig. 6A). This suggests that KN-93 binding to open and/or inactivated channels promotes the transition of channels to the inactivated state and stabilizes them there. Consistent with this, recovery of channels from inactivation was slowed in the presence of KN-93 (Fig. 5). Because the data in Fig. 6 suggest that KN-93 binds with a lower affinity to channels that inactivate slowly (e.g., R487V), this suggests that the concentration-response curve of R487V channels is right-shifted, and we therefore expected that higher concentrations of KN-93 would induce greater block of R487V. Interestingly, we observed enhanced inactivation of Kv1.4 even in the absence of the N terminus and therefore N-type inactivation (Fig. 1A), suggesting that at least part of the decay of current observed in Kv1.4 by Roeper et al. (1997) is due to a direct effect of KN-93 on slow inactivation of the channel. Enhancement of inactivation by the mutation Kv1.5 T462C in the outer pore enhanced the potency of KN-93 action (Fig. 6, C and D). This increased block was unlikely to be due to allosteric effects on the outer pore structure because mutation of neighboring residues had no effect on either the rate of inactivation or the potency of KN-93 binding (Fig. 6E). Given that KN-93 acts at the extracellular side of membrane and interacts with the open channel (Fig. 4) and that the drug slows the recovery from the P-type inactivated state (Fig. 5), which involves reconfiguration of the outer pore, these results are consistent with the conclusion that, following open channel block, KN-93 promotes and stabilizes outer pore-dependent inactivation.
As a precedent for such an action, it is well documented that drug binding to the hERG channel requires channel opening (Trudeau et al., 1995; Zhou et al., 1998), and there is a developing body of evidence suggesting that the block for most drugs occur via the inactivated state of the channel (Ficker et al., 1998; Mitcheson et al., 2000). Molecular determinants of high-affinity hERG block by a wide range of agents have been attributed to two aromatic residues in the S6 domain (Mitcheson et al., 2000). It has been suggested that through inactivation, these two aromatic residues undergo a rotation and become exposed for drug block (Chen et al., 2002). Because inactivation of Kv channels involves a physical conformational change in the outer pore (Liu et al., 1996), a similar mechanism may provide a favorable binding site for KN-93 in these channels.
In conclusion, the CaMK II inhibitor KN-93 and its inactive form KN-92 inhibit a wide range of Kv channels. The present study shows that KN-93 is an external open channel blocker that shows little voltage dependence and exerts its action by enhancement of inactivation. Because KN-93 is a potent blocker of many Kv channels, it must be used with caution. However, because KN-93 had no effect on Kv1.5 when applied intracellularly, CaMK II-independent effects of KN-93 on Kv channels can probably be circumvented by its intracellular application, although this should be confirmed for each channel system in which it is used.
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Acknowledgements |
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Footnotes |
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S.R. and T.W.C. contributed equally to this work.
ABBREVIATIONS: Kv, voltage-gated potassium channels; hERG, human ether a-go-go related gene; eag, ether a go-go; KN-93, 2-[N(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; KN-92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate; CaMK II, Ca2+/calmodulin-dependent protein kinase II; CIP, CaMK II inhibitory peptide fragment; HEK, human embryonic kidney; MEM, minimal essential medium.
Address correspondence to: Dr. David Fedida, Department of Cellular and Physiological Sciences, 2350 Health Sciences Mall, Vancouver BC V6T 1Z3, Canada. E-mail: fedida{at}interchange.ubc.ca
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