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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2015 Apr 24;172(13):3370–3382. doi: 10.1111/bph.13127

Effect of tyrphostin AG879 on Kv4.2 and Kv4.3 potassium channels

Haibo Yu 1,2,✉, Beiyan Zou 2,*, Xiaoliang Wang 1, Min Li 2,†
PMCID: PMC4500372  PMID: 25752739

Abstract

Background and Purpose

A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv4.2/Kv channel-interacting protein 2 (KChIP2) channels.

Experimental Approach

To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv4.2/KChIP2 channels using a whole-cell patch-clamp technique.

Key Results

Tyrphostin AG879 selectively and dose-dependently inhibited Kv4.2 and Kv4.3 channels. In Kv4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons.

Conclusion and Implications

AG879 was identified as a selective and potent inhibitor the Kv4.2 channel. AG879 inhibited Kv4.2 channels by preferentially interacting with the open state and further accelerating their inactivation.

Tables of Links

Targets
Ion channelsa2013b Catalytic receptorsb2013b
Kv1.1 Kv2.1 ErbB2 receptor (HER2)
Kv1.3 Kv4.2 TrkA
Kv1.4 Kv4.3 VEGFR-2 (FLK-1)
Kv1.5
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Ligands
Genistein
Tyrphostin AG879
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,bAlexander et al., 2013a,b).

Introduction

A-type potassium channels (IA) are important proteins with a variety of functional roles in modulating neuronal membrane excitability (Hoffman et al., 1997; Birnbaum et al., 2004; Kim et al., 2005; Yuan et al., 2005). Several cloned Kv channels, including Kv4.2, Kv4.3 and Kv1.4, exhibit transient, rapidly activating A-type currents when expressed in heterologous systems. Recent experiments using knock-out (KO) mice with a targeted disruption of the Kv4.2 gene (Kv4.2−/−) have revealed that Kv4.2 is the major subunit contributing to IA in hippocampal and cortical pyramidal neurons, as well as in dorsal horn neurons of the spinal cord (Chen et al., 2006; Hu et al., 2006; Norris and Nerbonne, 2010). Kv4.2 KO mice exhibit pronounced defects in performing hippocampus-dependent learning and memory tasks (Lugo et al., 2012; Truchet et al., 2012). The localization of Kv4.2 channels at synapses and their ability to regulate neuron firing support a role for these channels in modulating synaptic activity in the hippocampus and influencing hippocampus-dependent tasks (Chen et al., 2006). These studies have provided compelling evidence that the expression and activity of Kv4.2 channels are critical for neurological functions.

Recent studies have shown that the expression of Kv4.2 channels is markedly increased in FMR1 (coding for fragile X mental retardation protein) KO mice that lack fragile X mental retardation protein, a Fragile X syndrome (FXS) model (Lee et al., 2011). Furthermore, the defective long-term potentiation (LTP) induced in hippocampus slices from FMR1 KO mice can be restored by reducing Kv4 channel activity. Therefore, pharmacological inhibitors of Kv4.2 may be considered for potential therapeutic applications. However, only a few pharmacological gating modifiers of Kv4.2 have been reported (Tseng et al., 1996; Sanguinetti et al., 1997; Diochot et al., 1999; Ebbinghaus et al., 2004). There is still a need to identify more Kv4.2 inhibitors, which may be used to analyse mental retardation in which Kv4.2 channels are involved and could potentially serve as lead compounds for the development of new treatments. We screened a 1280 compound library on Kv4.2 potassium channels. Tyrphostin AG879, a protein tyrosine kinase (PTK) inhibitor, appeared to be the most potent compound. Here we mainly studied the effects of AG879 on Kv4.2 channels co-expressed with Kv channel-interacting protein 2 (KChIP2), an accessory subunit. The possible mechanism of the inhibitory effect of AG879 is also discussed.

Methods

Cell culture and transient transfection

HEK-293 and CHO-K1 cells were routinely cultured in either DMEM or 50/50 DMEM/F-12 (Mediatech, Manassas, VA, USA), supplemented with 10% FBS (Gemini Bio-products, West Sacramento, CA, USA) and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA). Cells were maintained and passaged when reaching 80% confluence.

For the thallium-based assay, HEK-293 cells were transfected with the Kv4.2-pcDNA3.1 plasmid with MaxCyte STX® Scalable Transfection System (MaxCyte, Gaithersburg, MD, USA) to achieve a consistent expression level. For the patch-clamp recording, CHO-K1 cells were transfected with Kv4.2 or Kv4.2/KChIP2 plasmids using LipofectamineTM LTX with Plus™ Reagent according to the manufacture's instruction (Invitrogen). Plasmid containing EGFP was co-transfected to identify transfection-positive cells as a marker. After transfection for 24 h, the cells were split and replated onto coverslips coated with poly-L-lysine (Sigma, St. Louis, MO, USA) for electrophysiological recording.

Hippocampus neuron culture

The neuronal culture method was adopted and modified from a protocol described previously (Ahlemeyer and Baumgart-Vogt, 2005). The study was approved by the Institutional Animal Care and Use Committee at the Chinese Academy of Medical Sciences (CAMS) and was performed in accordance with the National Institute of Health Guide for the care and use of laboratory animals. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). Pregnant Sprague-Dawley rats were killed by an overdose of isoflurane at embryonic day 18. For the experiment, a total of 10 fetal rats were used for hippocampus neuron dissection. The uterus was dissected out and placed in a sterile Petri dish. The fetuses were removed from the uterus and hippocampus tissues were dissected out from their brains. The tissue was digested with 0.25% Trypsin and resuspended into single cells. The single-cell suspension was plated on poly-D-lysine–coated coverslips. The neurons were maintained in culture medium consisting of neurobasal medium with B-27 supplement (Invitrogen), penicillin, streptomycin and 2 mM L-glutamine. Cultured neurons on day 10–14 were used for electrophysiological recording.

Thallium-based fluorescence assay

The library of pharmacologically active compounds (LOPAC) (Sigma-Aldrich) was chosen to screen for Kv4.2 inhibitors. HEK-293 cells expressing Kv4.2 were used. The activity of Kv4.2 potassium channels was monitored by the influx of a surrogate ion for potassium, thallium (Tl+). Thallium influx was detected through the use of a thallium-sensitive fluorescent dye, FluxORTM (Invitrogen). Cells were seeded at 6500 cells per well into BD Biocoat 384-well-plates (poly-D-Lysine coated) and incubated overnight at 37°C and 5% CO2. On the day of assay, medium was removed; cells were incubated with 1× FluxOR solution for 90 min at room temperature (RT) in the dark; the 1× FluxOR solution was replaced by assay buffer (HBSS containing 5.8 mM potassium; Catalogue # 14065, Invitrogen); test compounds 10 μM were then added to cells and incubated for 20 min; cell plates were loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader (Hamamatsu Photonics, Hamamatsu City, Japan); after the establishment of a fluorescence baseline by 1 Hz scanning for 10 s, the Kv4.2 channels were activated with a stimulus buffer containing 2.5 mM K2SO4 and 12 mM Tl2SO4 giving a final potassium concentration of 10.8 mM and final Tl+ concentration of 24 mM; fluorescence measurement was continued at 1 Hz for another 110 s. The fluorescence ratio, F(max−min)/F0 (F/F0), was calculated for each well using the entire 120 s detection window and then normalized to the positive and negative control wells. Compared with negative controls, if a compound caused the signal to decrease by more than three times the standard deviation (SD) of the fluorescence ratio, it was classified as an inhibitor.

Electrophysiological recordings

Traditional whole-cell voltage-clamp recordings were performed at RT to record the Kv4.2 and Kv4.2/KChIP2 currents of the transiently transfected CHO-K1 cells. Transfected cells were dissociated and seeded onto glass coverslips at a low density on the day of recording. Recording pipettes were pulled with borosilicate glass (World Precision Instruments, Inc., Sarasota, FL, USA) to 2–5 MΩ when filled with a pipette solution containing 145 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES and 5 mM MgATP at pH 7.2 and placed in a bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose and 10 mM HEPES at pH 7.4 adjusted with NaOH. Isolated cells were voltage-clamped in whole-cell mode with an AxoPatch 200B amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA), and currents were recorded at 10 kHz. Cells were continuously perfused with bath solution through a gravity-driven perfusion system (ALA Scientific, Farmingdale, NY, USA). Stock solutions of test drugs were made with DMSO. Immediately before each experiment, drugs were diluted in appropriate external solutions to the desired concentrations and applied by perfusion. Recordings were performed at RT (22–25°C).

Chemicals

The library of pharmacologically active compounds (LOPAC), genistein and sodium orthovanadate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tyrphostin AG879 was from Selleck (Chemicals (Houston, TX, USA); the compound stock was prepared in DMSO at 30 mM.

Data analysis

The data from the thallium-based flux assay were analysed by the manufacturer's software package (Hamamatsu Photonics). Patch-clamp data were processed in Clampfit 9.2 (Molecular Devices,Sunnyvale, CA, USA) and then analysed in Excel and Origin 6.0 (OriginLab Corporation, Northampton, MA, USA). Dose–response curves were fitted by the Hill equation in Origin software: Y = 1/[1 + (IC50/C)P], where Y is the fractional block at drug concentration C; IC50 the drug concentration producing half of the maximum block; and P the Hill coefficient. The steady-state activation and inactivation curves were fitted with the Boltzmann equation G = 1/[1 + exp(V − V1/2)/S]. V1/2 is the voltage needed to open half of the total number of channels and S is the slope factor. The voltage-dependence of the fractional block was fitted to the Woodhull equation (Woodhull, 1973): f = [D]/[[D] | Kd(0)e−zdFV/RT], where Kd represents the binding affinity at the reference voltage (0 mV); δ is the fractional electrical distance (the fraction of the transmembrane electrical field sensed by a single charge at the receptor site); z is the charge valence of the compound; F is the Faraday constant; V is the test potential; R is the universal gas constant; T is absolute temperature.

Statistics

Data were summarized as the mean ± SEM. Student's t-test was used for statistical analysis. P < 0.05 was considered statistically significant.

Results

Identification of tyrphostin AG879 as a selective Kv4.2 inhibitor

A High throughput screen was done using HEK-293 cells that were transiently transfected with rat Kv4.2 plasmid using the Maxcyte transfection system. Here HEK-293 cells were well-optimized for the transfection system. LOPAC was screened at 10 μM in duplicate using the optimized conditions (see Methods). Among the screened plates in duplicate, the Z′ factor was 0.52 ± 0.05 (n = 8), a robust range for a cell-based high-throughput screen campaign. Supporting Information Figure S1 shows the comparison of two repeats with R2 = 0.95, indicative of an excellent correlation between the different experiments when the same compounds were tested. A screening of 1280 known bioactive compounds yielded 11 compounds that inhibited Kv4.2-mediated Tl+ influx when a criterion of five times the SD value or more decrease in activity in comparison with the mean of buffer controls was used, with a cut-off at 10 μM, and six of these compounds were confirmed in the Tl+ assay. Then we examined their effects on Kv4.2 channels using electrophysiological recordings.

Among all of the compounds tested, tyrphostin AG879, a tyrosine kinase inhibitor of the nerve growth factor receptor, ErbB2 (Snyders et al., 1992; He et al., 2004; Zhou and Brattain, 2005), was confirmed and appeared to be the most potent Kv4.2 inhibitor. Its chemical structure and inhibitory effect are shown in Figure 1A and B. For the whole-cell patch-clamp experiments, CHO-K1 cells were used for transient transfection. As shown in Supporting Information Figure S2, the Kv4.2 channel alone expressed in CHO-K1 cells displayed a medium expression level and fast inactivation kinetics. As reported previously (An et al., 2000), Kv channel-interacting protein 2 (KChIP2), one of the KChIPs, is associated and co-localized with Kv4.2 in the CNS resulting in increased surface expression, slower inactivation and accelerated recovery from inactivation. To mimic the native channel complex and improve the expression level of Kv4.2 channels, we co-trasfected cDNAs of Kv4.2 and KChIP2 to study the effect of Kv4.2 inhibitors. Consistent with earlier reports, co-expression of Kv4.2 with KChIP2 increased the amplitude of the Kv4.2-mediated current by more than 15-fold when peak currents were measured. And the inactivation phase was also slowed (Supporting Information Figure S2C).

Figure 1.

Figure 1

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Identification of tyrphostin AG879 as a Kv4.2 channel inhibitor. (A) Chemical structure of AG879. (B) Effect of AG879 on Kv4.2 channels in CHO-K1 cells. AG879 10 μM reduced the Kv4.2-mediated peak current by 31.27 ± 2.4%. The Kv4.2 channel currents were recorded by a depolarization to +40 mV for 300 ms from a holding potential −80 mV. (C) Inhibition by AG879 3, 10 and 20 μM of Kv4.2/KChIP2 co-expressed channels. (D) Concentration-dependent inhibition by AG879 of Kv4.2/KChIP2 channel shown by measuring peak and integral currents. Concentration–response curves for AG879 were fitted to the Hill equation. (E) Reversible inhibition of Kv4.2 by AG879. Maximal inhibition occurred ∼5 min after drug application. (F) Concentration-dependent inhibition by AG879 of Kv4.3 channels.

As shown in Figure 1B and C, AG879 10 μM induced similar levels of inhibition when the peak currents for both Kv4.2 alone and Kv4.2/KChIP2-coexpressed channels were measured. Thus, the Kv4.2/KChIP2 channel complex was used to explore the effect of AG879. When cells were depolarized to +40 mV once every 5 s from a holding potential −80 mV, AG879 not only caused a reduction in the peak current of Kv4.2/KChIP2 (Figure 1D) but also preferentially accelerated the inactivation rate. As a measurement of the peak current underestimates the steady-state compound effect on the Kv4.2 channels, we measured the integrated area of Kv4.2 current during the entire time of depolarization. IC50 values for AG879 to block the peak and the integrated Kv4.2/KChIP2 currents were 17.87 ± 1.75 μM, with Hill coefficient 0.72 ± 0.06, and 4.68 ± 0.61 μM, with Hill coefficient 0.86 ± 0.10, respectively. The inhibitory effect of AG879 on the Kv4.2/KChIP2 current was reversible (Figure 1E). AG879 also induced a similar level of inhibition on Kv4.3channels, a close family member of the Kv4.2 potassium channel, with IC50 value 14.94 ± 1.69 μM for peak current and 4.14 ± 0.16 μM for integrated current respectively (Figure 1F). However, AG879 10 μM did not have a significant effect on the other voltage-gated potassium channels Kv1.1, Kv1.3, Kv1.4, Kv1.5 and Kv2.1 (Supporting Information Figure S3). In addition, we investigated the mechanism of action of tyrphostin AG879 on Kv4.2/KChIP2 channels.

AG879 facilitated the inactivation of Kv4.2 channels

As shown in Figure 2A, the peak values of the control and AG879-blocked currents were set equal to 1; a very pronounced feature induced by AG879 was the increase in the inactivation rate of Kv4.2/KChIP2 channels during depolarization, which suggests an open-channel block mechanism (Hatano et al., 2003). As described previously (Caballero et al., 2003; Hatano et al., 2003), the inactivation time course was fitted to a double exponential equation:

graphic file with name bph0172-3370-m1.jpg

where Afast and τfast and Aslow and τslow are the initial amplitudes and time constants of the fast and slow components of inactivation, respectively; and A0 is a time-independent component. The fast component of the inactivation current was predominant at +40 mV in control cells, with a relative contribution of 87% (determined as Afast/(Afast + Aslow)). Therefore, the inactivation rate of Kv4.2-mediated currents was mainly determined by τfast under control conditions. The fast inactivation time constant at +40 mV was significantly decreased from a control value of 45.8 ± 2.70 ms (n = 10) to 13.57 ± 1.07 ms after AG879 20 μM treatment (*P << 0.01) (Figure 2B). The contribution of the fast component was significantly increased by AG879 to 99%. However, the slow inactivation time constant was not altered significantly (Figure 2C). Further experiments demonstrated that AG879 also accelerated the inactivation rate of Kv4.2 channels in the absence of KChIP2. Therefore, the increased inactivation rate of Kv4.2 channels by AG879 was independent of the KChIP2 subunit (Supporting Information Figure S4).

Figure 2.

Figure 2

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Effect of AG879 on the inactivation time course. (A) Kv4.2 currents were activated by depolarization pulses from −80 to +40 mV. The current traces in the absence and presence of AG879 were superimposed and the dashed line represents the current trace obtained by adjusting their peak amplitudes. (B) and (C) The effect of AG879 on the fast and slow inactivation time course. The inactivation time course of Kv4.2 currents were well fitted with a double-exponential function (n = 5). (D) Rate of open-channel block (1/τblock) as a function of the drug concentration.

Based on the rate of inactivation, we further calculated the rate of open-channel block. As reported in the methods (Slawsky and Castle, 1994; Hatano et al., 2003), the time constant for decay of Kv4.2 (1/τdecay) in the presence of a drug should reflect the sum of the time constants of channel inactivation (1/τinactivation) and of open-channel block(1/τblock) as follows: 1/τdecay = 1/τblock + 1/τinactivation. The time constant of fast inactivation was used to represent the τinactivation considering its predominant role in the inactivation of the Kv4.2 channel. The rate of open-channel block (1/τblock) was plotted against AG879 concentration (Figure 2D), and the data were well fitted by a line with the least squares linear fit: 1/τblock = κ+1[D] + κ−1, where the association rate constant (κ+1) is 2.43 ± 1.17 μM−1·s−1, and the dissociation rate constant κ−1 = 2.24 ± 0.19 s−1. The Kd (κ−1/κ+1) was estimated to be 0.92 μM, which was in reasonable agreement with the IC50 value of 4.68 μM derived from the concentration–response curve shown in Figure 1. These results also imply that AG879 inhibited the Kv4.2/KChIP2 channels through an open-channel block mechanism.

Use and frequency-dependent block of Kv4.2 channels

When the cells were exposed to AG879 20 μM for 5∼7 min, the inhibition of peak current amplitude reached a plateau with the depolarization at 0.2 Hz (Figure 3A). The inhibition for the first pulse was identical to that of 20th pulse (0.50 ± 0.01 and 0.51 ± 0.01, P < 0.05), and no use-dependence was observed. The frequency-dependence of the AG879 block was further examined at 1, 3 and 5 Hz respectively. In the absence of AG879, 20 depolarizing pulses for 200 ms in duration from −80 to +40 mV were applied at 1, 3 and 5 Hz; the peak amplitude from the first to the 20th pulse was not changed significantly at 1 Hz. However, it was significantly decreased at 3 and 5 Hz, suggesting a frequency-dependent increase in inactivation. The relative amplitude ratios for the inhibition caused by AG879 20 μM at the first pulse were apparent with 0.51 ± 0.01, 0.51 ± 0.01 and 0.55 ± 0.02 for 1, 3 and 5 Hz respectively. The inhibitory effect showed no progressive change at 1 Hz, tended to increase at 3 Hz (P < 0.05, blocking effect between first and 20th pulses), and significantly increased at 5 Hz (P < 0.01). The relative amplitudes of the inhibition at the 20th pulse were 0.51 ± 0.02, 0.32 ± 0.03 and 0.23 ± 0.03, indicating a strong use-dependent inhibition of Kv4.2 by AG879 (Figure 3B).

Figure 3.

Figure 3

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Use-dependent block by AG879 of Kv4.2 currents. (A) Kv4.2 currents were recorded with 0.2 Hz of 20 depolarization pulses from −80 to +40 mV and peak current change is shown in (A) before and after 7 min of incubation with AG879 20 μM, membrane potential held at −80 mV. (B) The peak amplitude of Kv4.2 at each pulse in the presence of AG879 was normalized to the corresponding one in the absence for 1, 3 and 5 Hz with holding potential at −80 mV. And then the ratios were plotted against the pulse number (n = 4–7). (C) Peak current changes for the different frequencies, 1, 3 and 5 Hz, after cells were loaded with AG879 for 7 min and then washed out, with membrane potential held at −80 mV. (D) Peak current changes for the different frequencies, 1, 3 and 5 Hz, after cells were loaded with AG879 for 7 min and then washed out, with membrane potential held at −100 mV.

After the cell was exposed to AG879 20 μM for 7 min with the holding potential at −80 mV, the compound was washed out before the channel was activated (1, 3 and 5 Hz). Under these conditions, the use-dependent block could still be observed for 3 and 5 Hz, but not 1 Hz (Figure 3C). Theoretically, no use-dependent block should be observed in the no-channel-opened state. One interpretation is that there were a certain amount of Kv4.2 channels inactivated at the holding potential −80 mV, thus AG879 could access the inactivated channels to block the channel activity. Therefore, the holding potential was set to a more hyperpolarized voltage, −100 mV, to drive all the channels to a closed state. Under these conditions, no use- dependent block was observed for the different frequencies of depolarization (1, 3 and 5 Hz) after treatment with AG879 for 7 min and then washed out (Figure 3D). In contrast, when the cells were incubated with AG879 for a longer duration and then activated, the use-dependent inhibitory effect was restored. These experiments indicate that the inhibitory effect of AG879 on Kv4.2 channels was state-dependent.

Slowed recovery from inactivation of Kv4.2 channels by AG879

A double-pulse protocol was used to characterize the recovery of Kv4.2 channels from inactivation in the absence and presence of AG879 20 μM (as shown in Figure 4A). Peak current amplitude at the test pulse was normalized to that elicited by the conditioning pulses in the same cells. The normalized current amplitude was plotted against the inter-pulse durations. The time course for recovery from inactivation was well fitted by a single exponential function with a time constant of 53.0 ± 3.4 ms under control conditions and 129.6 ± 3.35 ms (n = 5, P < 0.01) in the presence of AG879 (Figure 4B and 4C). AG879 significantly slowed the recovery from inactivation of the Kv4.2 channel.

Figure 4.

Figure 4

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Effect of AG879 on the recovery from inactivation of Kv4.2 channels. (A) A double-pulse protocol was used to characterize the recovery of Kv4.2 channels from inactivation in the absence and presence of AG879. A pre-pulse of 500 ms depolarization to +40 mV from holding potential −80 mV was followed by a test pulse with 500 ms depolarization to +40 mV by increasing the inter-pulse duration from 10 to 2000 ms at −80 mV. The protocol is shown in the insert. (B) Peak current amplitude at the test pulse was normalized to that recorded from the conditioning pulses in the same cells. (C) Normalized data were plotted against the inter-pulse duration and fitted by a single exponential function. Data are expressed as mean ± SEM (n = 6).

Voltage-dependent block of Kv4.2 channels by AG879

Current–voltage relationships were examined in the CHO-K1 cells expressing Kv4.2/KChIP2 in the absence or presence of AG879. Kv4.2-mediated currents were generated by depolarizing the cells from holding potential −80 mV to a series of voltage steps ranging from −70 to +70 mV for 300 ms in 10 mV increments and activated at voltage potentials more than −30 mV (Figure 5A).The current–voltage relationships for peak currents are shown in Figure 5B. AG879 significantly reduced the amplitude of the Kv4.2-mediated peak current in the voltage range from 0 to +70 mV (*, P << 0.01).

Figure 5.

Figure 5

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Effect of AG879 on current–voltage relationships of Kv4.2 channels in CHO-K1 cells. (A) Representative Kv4.2 channel current traces in the absence and presence of AG879 20 μM. Each CHO-K1 cell expressing Kv4.2 channels was depolarized for 300 ms from a holding potential of −80 mV in 10 mV increments, at 5 s intervals. (B) Current–voltage curves for peak current amplitude in the absence and presence of AG879 20 μM (n = 5). In each cell, the current amplitude measured at the peak current was normalized to that at +70 mV in the absence of AG879. Data are expressed as mean ± SEM (*P << 0.01).

To determine the voltage-dependent activation of Kv4.2 channels, the normalized tail currents were fitted to a Boltzmann function. AG879 induced a slight hyperpolarizing shift of the voltage-dependence, in which the half maximal activation voltage (V1/2) was leftward shifted for 3.5 mV from control −0.40 ± 1.21 mV (mean ± SEM) to −3.92 ± 1.24 mV (Figure 6A and B). These results indicate that AG879 does not markedly change the voltage-dependence of the steady-state activation of Kv4.2 channels. To quantify the inhibitory effect of AG879, the relative current amplitudes for peak currents were plotted against test potentials. As shown in Figure 6C, the inhibitory effect induced by AG879 was increased steeply in the voltage range from −30 to +0 mV, when the Kv4.2 channels were in the unsaturated open status according to the activation curve (as shown in the dashed line in Figure 6C).The voltage-dependence of the block was determined at the potentials in the range of full channel opening (between +10 and +70 mV). The Woodhull model was used to generate fractional electrical distance δ = 0.10 ± 0.01 (Woodhull, 1973), suggesting a mild voltage-dependent inhibition.

Figure 6.

Figure 6

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Effect of AG879 on the steady-state activation and inactivation of Kv4.2 channels. (A)The representative tail current traces of Kv4.2 channels in the absence and presence of AG879. Depolarized voltage steps from −70 to +80 mV were applied from a holding potential of −80 mV for 10 ms in 10 mV increments, followed by a hyperpolarizing step to −50 mV for 500 ms to record tail currents. (B) The activation curves in the absence and presence of AG879. The curves were well fitted to the Boltzmann equation. Data are expressed as mean ± SEM (n = 8). (C) Fraction block by 20 μM AG879 of the peak currents was plotted against the membrane potentials. The current amplitude in the presence of AG879 20 μM was normalized to that at each voltage in the absence of AG879 (control cells n = 5). (D) Inactivation curves in the absence and presence of AG879. The steady-state curves were generated with a two-pulse protocol, in which 1 s conditioning pulses from a holding potential of −80 mV to voltages between −110 and +10 mV in 10 mV increments were followed by a test pulse to +40 mV for 500 ms. (E) Fraction block was plotted as a function of the voltage from the preceding pulse.

Steady-state inactivation was examined using a double-pulse protocol as shown in the inset of Figure 6D. The half inactivation voltage was −45.68 ± 0.29 mV under control conditions with a slope of 5.09 ± 0.32. In the presence of AG879, the V1/2 was shifted in a hyperpolarizing direction of ∼6 mV (V1/2 = −51.56 ± 0.80 mV and slope 5.30 ± 0.36). The blocking effect was plotted against the pre-pulse potentials as shown in Figure 6E; it remained at a relative stable level at potentials between −110 mV (23.30 ± 11.96%) and −60 mV (32.13 ± 10.47%, n = 5, P < 0.05). At more positive voltages, the blocking effect was enhanced as the number of inactivated channels increased, reaching a maximum at the pre-pulse voltage −40 mV (62.57 ± 8.7%, P < 0.01 vs. blocking effect at −110 mV).

Inhibition of A-type potassium currents by AG879 in hippocampus neurons

In the cultured hippocampal neurons the transient A-type current component (IA) was separated from the total outward currents by a pre-pulse inactivation and subtraction protocol. Briefly, starting from a holding voltage of −80 mV, currents were activated at +40 mV, either following a 500 ms pre-pulse to −110 mV to obtain the total neuronal outward K current, or following a 500 ms pre-pulse to −30 mV to obtain the non- or slowly-inactivating delayed rectifier current ID. The ID component was then subtracted offline from the total neuronal outward K current in a point-by-point fashion to obtain IA (Figure 7A). As shown in Figure 7A and 7B, AG879 20 μM significantly inhibited A-type potassium currents by around 55% in the cultured hippocampus neurons, comparable with the inhibition by AG879 of the current mediated by recombinant Kv4.2/KChIP2 channels.

Figure 7.

Figure 7

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Effect of AG879 on A-type potassium currents in cultured hippocampus neurons. (A) Isolated A-type current from a cultured hippocampal neuron in the absence (upper trace) and presence of 20 μM AG879 (lower trace).(B) Bar chart plot for the inhibition of the peak current amplitude of Kv4.2/KChIP2 and A-type currents in the presence of AG879 (n = 5).

Discussion and conclusions

In the present study, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a selective inhibitor of Kv4 channels. The main findings of the present study can be summarized as follows. AG879 selectively inhibited both Kv4.2/KChIP2 and Kv4.3 channels dose-dependently without a significant effect on the other voltage-gated potassium channels. The fast inactivation of Kv4.2 channels was significantly accelerated by AG879 in a KChIP2-independent manner. AG879 inhibited Kv4.2 currents in a use-dependent manner and also slowed their recovery from inactivation. Furthermore, AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv4.2 channels. AG879 also exhibited apparent inhibition of the A-type potassium currents in cultured hippocampus neurons.

KChIPs co-localize with brain Kv4 α-subunits and are integral components of the native Kv4 channel complex (An et al., 2000). To mimic the native channel complex, we co-transfected the cDNAs of Kv4.2 and KChIP2 channels to study the effect of AG879. For the activation kinetics of the Kv4.2 channel, the peak current reflects the open state of the channel and the rest of the current reflects the steady state of equilibrium between open and inactivated states. As a measurement of the peak current would underestimate steady-state compound effect on the Kv4.2 channels, we also measured the integrated area of Kv4.2 currents for the entire time of depolarization. AG879 blocked Kv4.2/KChIP2-mediated currents in a dose-dependent manner. The IC50 values were 17.87 and 4.68 μM for the reduction of the peak current and integrated current respectively. In addition, AG879 also displayed a similar affinity for blockade of Kv4.3 channels. It is not too surprising that AG879 blocked both Kv4.2 and Kv4.3 channels because there is a high percentage of sequence identity between Kv4.2 and Kv4.3 channels (99% in pore region and 92% in voltage-sensor domain S1–S4). However, AG879 did not inhibit the other Kv channels tested (Kv1.1, Kv1.3, Kv1.4, Kv1.5 and Kv2.1). In addition, KChIP2 not only increased the current density, but also slowed the inactivation rate when co-expressed with the Kv4.2 channels; AG879 accelerated the inactivation rate of Kv4.2/KChIP2 channels. To determine whether AG879 attenuates the KChIP2-mediated slowing of inactivation of the Kv4.2 channels, we tested the effect of AG879 on the Kv4.2 inactivation rate in the absence of KChIP2 expression. The results demonstrated that the AG879-induced effects are independent of the KChIP2 subunit.

AG879 induced about a 20% block of the current in the resting state of Kv4.2 channels. However, its inhibitory effect was greatly enhanced when the channels were stimulated/activated, so that it accelerated inactivation or current reduction, which indicates that AG879 might interact with either the open state or the inactivated state. The characteristics of the AG879-induced blockade of Kv4.2 channels were also compared with those of other drugs that have been shown to affect either Kv4 channels or Ito channels in heart tissue (Slawsky and Castle, 1994; Wang et al., 1995; Caballero et al., 2003; Hatano et al., 2003). Results with drugs such as flecanide, quinidine, nicardipine and propafenone that inhibit either cardiac Ito or Kv4 channels have been interpreted as indicative of them acting by an open-channel block mechanism. An important common characteristic of these agents and AG879 is the prominent acceleration of the inactivation rate. However, there are also differences in some aspects by which these drugs affect the channels. For example, the inhibition produced by quinidine on Kv4.2 and Ito channels is dependent on the test potential; however, the inhibition by AG879, as well as that induced by nicardipine and flecainide, was only slightly dependent on the test potential. In addition, unlike propafenone, AG879, flecainide, quinidine and nicardipine all generated use-dependent block at higher frequencies of stimulation. These were also associated with a slower recovery from inactivation for the time course of Kv4.2 and Ito channels.

Kv4.2 channels can be inactivated from the open state at strongly depolarized voltages or directly from closed states at more negative potentials (Bahring et al., 2001). The peak current amplitude was significantly decreased from the first pulse to the 20th pulse while the stimulation frequencies were increased from 1 to 3 and 5 Hz, suggesting that the fraction of inactivated channels was increased. AG879-induced inhibition was also increased at higher frequencies of stimulation. In addition, we also observed that AG879 reduced the Kv4.2-mediated current use-dependently even though the channel was in a closed/inactivated state (membrane potential −80 mV). In addition, AG879 caused a hyperpolarizing-shift of the inactivation curve and its inhibitory effect became more pronounced as the fraction of inactivated channels increased. These results suggest it interacts with an inactivated-state or it accelerates the inactivation. More direct experiments, such as excised patch recordings and site-directed mutagenesis to knock-down or cancel the inactivation property of the channel, are needed to unravel the precise mechanisms of the compound.

Tyrphostin AG879 has long been widely used as a protein tyrosine kinase inhibitor and is known to have beneficial anti-tumour properties, which include inhibition of NGF-induced PLC-γ 1 phosphorylation and PI3K activation (Ohmichi et al., 1993; Rende et al., 2000; 2006), inhibition of ErbB2 receptors and the VEGF receptor 2 (FLK-1) (He et al., 2001; 2004; Zhou and Brattain, 2005; Lee et al., 2008) and indirect inhibition of the MAPK cascade (Gil et al., 2003; Larsson, 2004). Although AG879 potently inhibits, with an IC50 of 1 μM, the expression of human epidermal growth factor receptor 2/ErbB2 receptors, the compound starts to cause apoptosis or reduce the proliferation of cells at concentrations more than 5∼20 μM in TrkA-highly expressed tumour cell lines (Rende et al., 2006). In our study, tyrphostin AG879 was identified as a selective inhibitor of Kv4.2 channels with an IC50 value similar to that of the above-mentioned concentrations. It has been reported that ion channels can be modulated by tyrosine kinase inhibitors (Son et al., 2013). To differentiate any tyrosine kinase-dependent actions of AG879 from those induced by direct inhibition of Kv4.2 channels, a structurally dissimilar broad-spectrum tyrosine kinase inhibitor, genistein 30 μM, was pre-applied before the addition of AG879. AG879 20 μM could still inhibit the peak current mediated by Kv4.2 channels by around 70% after the tyrosine kinase activity had been completely inhibited by genistein (Supporting Information Figure S5). If AG879 inhibited the Kv4.2-mediated currents by decreasing tyrosine kinase activity, pre-application of a broad-spectrum tyrosine kinase inhibitor, such as genistein, would reduce or prevent the further inhibition of the current mediated by Kv4.2 channels induced by AG879. However, our results do not support this. Furthermore, if the AG879-induced inhibitory effect was dependent on tyrosine kinase, it would be weakened or cancelled when a protein phosphatase inhibitor was co-applied, as reported previously (Li et al., 2006; Gierten et al., 2008; Zhang et al., 2012). However, an effective concentration of orthovanadate, an inhibitor of protein phosphates (Swarup et al., 1982), did not reverse the inhibitory effect of AG879 (Supporting Information Figure S5). All these results indicate that the tyrosine-phosphorylation pathway is not involved in the process of AG879-induced channel inhibition and that AG879's inhibition of the Kv4.2 channel is unlikely to be mediated by an effect on tyrosine kinase. However, although our results revealed a PTK-independent modulation by AG879 of Kv4.2/KChIP2, we cannot completely rule out the possibility that this compound might inhibit the channel via a tyrosine-phosphorylation sensitive pathway, which is secondary to the direct interaction.

A-type currents play an important role in the shaping of an action potential and regulate the firing frequency of neurons in the CNS (Rudy, 1988). Kv4.2 is the major contributing subunit for IA in the CNS and plays an important role in modulating synaptic activity, influencing hippocampus-dependent tasks and neuronal excitability. AG879 caused significant inhibition of IA currents in cultured hippocampus neurons. Inhibition of neuronal A-type currents would affect the refractory period and the action potential duration, and further alter the firing frequency of neurons. Furthermore, Kv4.2 inhibitors can restore the deficits in LTP induced in the FMR1 KO mice model (Lee et al., 2011). Thus pharmacological inhibition of Kv4.2 currents by AG879 or similar compounds may have potential as a treatment for individuals with Fragile X syndrome.

In summary, the results of the present study suggest that AG879 selectively and directly inhibits Kv4.2/Kv4.3 channels by preferentially interacting with or stabilizing the open/inactivated states. Furthermore, this inhibitory effect of AG879 is not mediated through inhibition of PTK activity. It should be noted that Kv4.2 potassium channels are also expressed in heart tissue (Barry et al., 1995) and the corresponding cardiac safety issues might be an issue if AG879 is used in vivo. Therefore, we recommend caution in the use of the drug in physiological experiments designed to determine the role of PKs in the modulation of ion channels. However, AG879 selectively inhibited Kv4.2 and Kv4.3 channels with no apparent effect on the other Kv channels tested. AG879 may have potential as a tool to study the role of neuronal A-type currents, especially Kv4.2 channels, in Fragile X syndrome in the CNS.

Acknowledgments

This work was supported by the start-up fund from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (to H. Y.) and by National Institutes of Health and Molecular Libraries Probe Production Centers Network (MLPCN) Grants U54MH084691 (to M. L.), and Johns Hopkins University is a member of the MLPCN and houses the Johns Hopkins Ion Channel Center.

Glossary

KChIP2

Kv channel-interacting protein 2

KO

knock-out

PTK

protein tyrosine kinase

Author contributions

H. Y. designed the experiments, analysed data and wrote the paper. B. Z. performed the experiments and analysed data. X. W. contributed new reagents/analytical tools. M. L. designed the experiments and revised the paper.

Conflict of interest

None.

Supporting Information

Figure S1 A pilot screening for Kv4.2 channel modulators in the LOPAC library using a thallium-influx assay.

Figure S2 Improved Kv4.2 potassium channel expression by accessory subunit KChIP2.

Figure S3 Effect of AG879 on Kv channels.

Figure S4 Effect of AG879 on the inactivation time course of Kv4.2 channels.

Figure S5 AG879 inhibited Kv4.2/KChIP2 channels in a TK insensitive manner.

bph0172-3370-sd1.pdf (431.3KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 A pilot screening for Kv4.2 channel modulators in the LOPAC library using a thallium-influx assay.

Figure S2 Improved Kv4.2 potassium channel expression by accessory subunit KChIP2.

Figure S3 Effect of AG879 on Kv channels.

Figure S4 Effect of AG879 on the inactivation time course of Kv4.2 channels.

Figure S5 AG879 inhibited Kv4.2/KChIP2 channels in a TK insensitive manner.

bph0172-3370-sd1.pdf (431.3KB, pdf)

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