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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Neuroscience, Baylor College of Medicine, Houston, Texas
Submitted 29 April 2005 ; accepted in final form 26 August 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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voltage-gated potassium channel; kinase; gating
The Kv4.2 channel gene encodes a channel-forming 
-subunit that exhibits rapid activation and inactivation when exogenously expressed and is probably the major contributor to A-type K+ current found in many types of neurons (5). Biochemically, Kv4.2 subunits are directly phosphorylated by PKA, PKC, CaMKII, and MAPK-ERK (1, 3, 21). Phosphorylation of Kv4.2 subunits affects certain aspects of channel behavior, including surface expression and voltage-dependent kinetics (12, 21, 25). The phosphorylation of Kv4.2 by ERK is especially interesting because ERK appears to mediate downstream effects of both PKA and PKC in kinase modulation of both hippocampal A-type K+ channels and Kv4.2 subunits (25).
The study of the ERK signaling pathway, however, is limited by the lack of specific pharmacological agents directly targeting ERK. Nevertheless, manipulation of ERK activity can be achieved by using organic compounds to inhibit MEK, a kinase immediately upstream from ERK. U0126 and PD-98059 are two MEK inhibitors proved to be specific among kinases (7, 10) and are thus valuable pharmacological tools for studying the ERK signaling pathway. Specifically, it was reported previously that 10 µM U0126 treatment completely blocked intracellular ERK phosphorylation in cultured COS cells (10). Using these pharmacological tools, it has been demonstrated in various preparations and cell types that the ERK signaling pathway plays important roles in modulating K+ channels (13, 14, 25), as well as the induction and expression of synaptic plasticity (8, 9, 17, 22, 23, 24). The concentration of U0126 used in these studies involving brain slices has typically been 20 µM.
Accumulating evidence, however, also suggests that some protein kinase inhibitors interact directly with voltage-gated ion channels in ways separate from kinase inhibition. For example, the tyrosine kinase inhibitor AG-1478 has been shown to block Kv1.5-mediated K+ currents, possibly acting as an open channel blocker (6). Similarly, calphostin directly inhibits muscarinic K+ channels (15), whereas staurosporine can directly inhibit L-type Ca2+ channels (11). It is therefore important to investigate possible nonspecific effects associated with a kinase inhibitor.
In this study, we investigated MAPK-ERK regulation of Kv4.2 subunits by using the MEK inhibitor U0126. We found that U0126 significantly accelerated the inactivation of Kv4.2-mediated A-type K+ currents. The effect had rapid onset, was fully reversible, and did not depend on the modulation of protein phosphorylation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Electrophysiological recordings.
A Zeiss Axioskop fitted with a x40 magnification water-immersion objective and differential interference contrast was used to view cultured cells. Transfected cells expressing EGFP were identified under the fluorescence microscope. Whole cell voltage-clamp and outside-out patch-clamp experiments were performed in CHO cells using an Axopatch 200 amplifier. All experiments were performed at room temperature. The bath solutioncontained (in mM) 134 NaCl, 2 KCl, 5.6 NaOH, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, with pH 7.4. The pipette solution contained (in mM) 140 KCl, 1 MgCl2, 1 EGTA, 0.133 CaCl2 (free Ca2+ concentration 
10 nM), and HEPES 10, pH 7.4. The K+ current reversal potential measured under this condition was approximately –85 mV. The liquid junctional potential between the pipette solution and the bath solution was measured as –7 mV and was not corrected. Pipettes had resistances between 2 and 5 M
. Whole cell capacitance and series resistances were compensated to >80%.
Data acquisition and analysis. Pulse generation and data acquisition were controlled with custom software written in Igor Pro (Wavemetrics). Current records were analog filtered at 2 kHz and digitized at 10 kHz. Linear leak and capacitive currents were digitally subtracted using null traces or scaled traces with smaller voltage steps. Steady-state activation and inactivation curves were constructed and fitted as described previously (12). Current inactivation and recovery from inactivation kinetics were determined by single or double exponential curve fitting. Statistical significance was set at P < 0.05 and was determined using two-sample t-tests. Error bars represent SE.
Drugs. U0126 (Promega, Madison, WI), U0125, and PD-98059 (Calbiochem, CA) were dissolved in DMSO to 10 mM, kept frozen until use, and then diluted to the appropriate concentrations.
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RESULTS |
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) that became slower as a function of membrane voltage depolarization under control conditions (Fig. 1C and Table 1). In the presence of U0126, however, the decay phase of the K+ currents became accelerated and were best fit with two exponentials. The accelerating effect of U0126 on channel inactivation was also voltage dependent; however, in this case, both the fast and slow values became faster with depolarization (Fig. 1, C and D, and Table 1).
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1–2, 10, and 20 µM), both slow (100.6 ± 1.5 ms, 86.2 ± 1.9 ms, and 58.8 ± 2.2 ms) and fast (8.1 ± 1.9 ms, 4.7 ± 0.9 ms, and 4 ± 0.7 ms) accelerated as a function of U0126 concentration at the holding potential (Vh) of –80 mV and command voltage (Vc) of +100 mV (Fig. 1E). To exclude the possibility that the coexpressed KChIP3 mediated this effect, we conducted similar experiments on CHO cells expressing Kv4.2 subunits alone. Although currents mediated by Kv4.2 alone were much smaller in amplitude and faster in inactivation kinetics (2), U0126 accelerated inactivation in a manner similar to that of currents mediated by Kv4.2 with KChIP3. For example, inactivation of currents mediated by Kv4.2 subunits alone can be fitted with a single exponential with = 20.7 ± 3.0 ms, while the presence of 20 µM U0126 accelerated to 2.7 ± 1.0 ms (Vc = 60–80 ms; n = 4). We tested whether modulation with another commonly used MEK inhibitor could mimic these effects on current decay. As previously reported, PD-98059 at 25 µM shows effects on inhibiting MEK activity similar to those of 20 µM U0126. We found that 25 µM PD-98059 had no significant effect on the inactivation of Kv4.2/KChIP3-encoded K+ currents (control, 86.7 ± 5.3 ms, n = 7, vs. PD-98059, 73.9 ± 5.8 ms, n = 4; P = 0.15).
U0126 exerted its effect on Kv4.2 channel inactivation independently of protein kinase inhibition. The quick onset and rapid washout of the effect of U0126 on channel inactivation and the lack of effect of 25 µM PD-98059 suggested that U0126 may have exerted this effect independently of protein kinase inhibition. We tested this hypothesis in several ways.
We first tested whether the effect persisted in the absence of GTP/ATP, substrates that are necessary for protein phosphorylation. We pulled outside-out patches from CHO cells expressing Kv4.2 and KChIP3 without including ATP/GTP in the pipette solution. The decay phase of currents recorded under this condition was fitted with a single exponential with of 64.1 ± 1.5 ms (Vc = +80 mV; n = 5). Consistently, bath application of 10 µM U0126 significantly accelerated the decay kinetics, which were best fit with a double exponential (slow = 54.8 ± 4.7 ms, fast = 6.0 ± 1.6 ms, Vc = +80 mV; n = 5).
We then tested the U0126 derivative U0125 that structurally differs from U0126 by only two amine groups, while its potency for MEK inhibition is 10-fold less than U0126 (10). Application of U0125 also produced a dose-dependent acceleration of channel inactivation (Fig. 2A). To compare the accelerating effect on channel inactivation produced by 20 µM U0126 and U0125, we measured channel half-inactivation time, or the time needed for current to decay to one-half of the peak amplitude. As shown in Fig. 2B, half-inactivation times in the presence of 20 µM U0126 and U0125 were 5.5 ± 1.0 ms (n = 7) and 8.1 ± 2.5 ms (n = 4), respectively, at Vc of +100 mV. Both were significantly different from that of control (60.4 ± 5.4 ms, n = 7; P < 0.005).
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: wild type, 81.3 ± 2.6 ms, n = 11; ERK mutant 79.1 ± 4.7 ms, n = 7, Vh = –80 mV, Vc = +80 mV). Despite the removal of all known ERK phosphorylation sites on the channel, application of 10 µM U0126 still resulted in a faster rate of current inactivation (Fig. 2, C and D), suggesting that U0126 was acting through a phosphorylation-independent mechanism (ERK mutant with 10 µM U0126; slow = 81.8 ± 6.5 ms, fast = 4.4 ± 2.1 ms; n = 4).
Site of action of U0126.
The results obtained from the above experiments do not support a mechanism whereby U0126 accelerated channel inactivation through inhibition of protein phosphorylation. To test further whether U0126 was acting through an intracellular signaling pathway, 20 µM U0126 was included in the pipette solution that recorded Kv4.2-mediated K+ current in CHO cells expressing Kv4.2 and KChIP. After breakin and establishment of a whole cell configuration, the magnitude and kinetics of Kv4.2-mediated A-type currents were monitored and found to be unaltered compared with control experiments (percentage change of inactivation = 7.6 ± 2.5%, n = 4, measured within 10–15 min). Subsequent external application of 20 µM U0126, however, promptly changed the amplitude and kinetics of Kv4.2 currents (n = 3). The results from exponential fitting of the current decay phases before and after U0126 in these experiments are summarized in Table 1. These results suggest that U0126 exerts its effect on Kv4.2 subunits at a site external to the channel. We have observed a similar phenomenon in cultured hippocampal neurons (Yuan LL and Johnston D, unpublished data) in which application of 20 µM U0126 in pipette solution appeared not to change the amplitude or kinetics of native K+ currents in these neurons (n = 4).
U0126 exerted similar effects on other Kv subunits.
Because U0126 appeared to interact with Kv4.2 subunits directly from outside the plasma membrane, we tested whether this interaction was specific for Kv4.2 subunits by expressing Kv4.3 in CHO cells (without KChIP3). We found that in the absence of KChIP3, Kv4.3 subunits in CHO cells produced A-type K+ currents with comparable amplitude and kinetics similar to those of Kv4.2/KChIP3. Application of 10 µM U0126 to cells expressing Kv4.3 subunits accelerated the channel inactivation from 57.9 ± 0.4 ms to 10.4 ± 0.4 ms (Vc = +80 mV), which is similar to that observed with Kv4.2 subunits (Fig. 3A).
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-subunits, the currents mediated by Kv1.1 represented typical noninactivating K+ currents. U0126 changed the current kinetics dramatically in a concentration-dependent manner. For example, 20 µM U0126 significantly decreased the peak current amplitude and, more strikingly, converted these noninactivating currents into fast-inactivating or transient ones (Fig. 3B). Inactivation kinetics of those transient currents can be fit with single exponentials with on the order of several milliseconds ( = 3.2 ± 0.2 ms, Vc = 80 mV; n = 3).
Mechanisms of U0126 blocking.
The increased rate of current inactivation produced by the application of U0126 could result from channel open state blocking, modulation of gating mechanisms, or both. The voltage dependence of acceleration of current decay by U0126 that we observed (Fig. 1, C and D, and Table 1) suggested that this ERK inhibitor might work as an open channel blocker. To explore these different possibilities further, we chose to measure the low-voltage inactivation kinetic properties of Kv4.2-encoded channel kinetics in the absence and presence of U0126. Most studies on Kv4.2 channels (4) suggest that at voltage near the one-half steady-state inactivation voltage (
) the closed-state inactivation process dominates channels inactivation. Consequently, modulation of closed-state inactivation by U0126 should shift and change the kinetics of entry into closed-state inactivation at low voltages. We first verified that U0126 did not produce any significant effects on channel steady-state activation properties. As expected, steady-state activation of Kv4.2-mediated currents were not significantly changed by 10 µM U0126 ( before and after drug addition were 13.5 ± 4.1 mV and 17.4 ± 4.4 mV, respectively; P > 0.05). of steady-state inactivation curve, however, was shifted in a hyperpolarizing direction by 7.2 mV (Fig. 4A) from –49.0 ± 2.1 mV in control to –56.2 ± 3.1 mV in U0126 (n = 5; P < 0.005, paired t-test). To assess kinetics of entry into closed-state inactivation, we used a protocol by which Kv4.2 channels were inactivated by a prepulse to –50 mV (Fig. 4A, dashed line), followed by a test pulse to +40 mV (Fig. 4B). The difference between currents measured using this protocol (protocol 1) and the protocol without a prepulse (protocol 2) (Fig. 4B) represents the portion of Kv4.2 channels that went directly into closed-state inactivation. As shown in Fig. 4, C and D, the ratio of current 1 to current 2 was reduced from 53.2 ± 9.2% to 32.0 ± 9.7% (P < 0.05) by the application of U0126. In other words, the percentage of channels directly entering into closed-state inactivation was higher in the presence of U0126 than that in control experiments. These results suggest that U0126, an ERK inhibitor, sped up the entry of Kv4.2 channels from the closed state directly into closed-state inactivation without opening.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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First, the effect took place rapidly, within 1 min of U0126 application, and reversed within a few minutes of washout. Although one cannot reach a conclusion solely on the basis of this observation without further testing for any involvement of protein kinases, the quick onset and offset of the drug effect seem to favor U0126’s accelerating the inactivation process by interacting directly with channel subunits. Second, U0125, a 10-fold less potent analog of U0126 in MEK inhibition, showed an effect on inactivation similar to that of U0126 at the same concentrations. Third, U0126 showed similar effects on outside-out patches without ATP/GTP supplied. Fourth, we observed that greater inhibition occurred at higher voltage steps, suggesting that U0126 binding of channel subunits modulates a voltage-dependent process. This process is most likely closed-state inactivation. Fifth, we found membrane polarity to the effects of U0126. If U0126 had to cross the membrane to exert its effect on kinase inhibition, then inclusion of U0126 in recording pipette solution should have mimicked the effect of the bath application; however, this was not the case.
Finally, if the channel-inhibiting effects of U0126 were dependent on protein kinase phosphorylation, we would have expected that U0126 would not have less effect on a triple mutant of Kv4.2 2 in which three identified phosphorylation sites were mutated to alanine residues. Instead, U0126 had effects on this triple mutant that were similar to the effects it had on wild-type Kv4.2 channels. The effects of U0126 on current amplitude and channel inactivation kinetics also were not specific to Kv4.2-encoded K+ channels. The specificity of U0126’s direct interaction with K+ channels was tested on two representative subunits, Kv4.3 and Kv1.1. Kv4.3 is a Kv4 subfamily member that possesses potential MAPK phosphorylation consensus similar to that of Kv4.2 subunits and also underlies transient K+ currents. In contrast, Kv1.1 shows less sequence conservation with Kv4.2, has no potential MAPK phosphorylation consensus, and encodes noninactivating K+ currents when expressed without appropriate -subunits. Both Kv4.2 and Kv1.1 behaved similarly to Kv4.2 subunits in the presence of U0126, which suggests that the effects of U0126 reported herein were not specific to Kv4.2 subunits and not dependent on the ERK phosphorylation sites.
Apart from the unexpected effects of U0126 on Kv4.2 reported herein, an important question is whether ERK phosphorylation alters Kv4.2 channel kinetics. This hypothesis has been tested recently using a different approach (Schrader L, Nadin B, Birnbaum S, Bui D, Anderson A, and Sweatt J, unpublished observations). Schrader and colleagues have mutated the three phospho-sites of Kv4.2 subunits to aspartate to mimic phosphorylation. They found that only the T607D mutation mimicked the expected kinetic changes elicited by ERK/MAPK activation, which is a rightward shift of the activation curve and an overall reduction in current amplitude compared with the wild type. This effect is dependent on the ancillary KChIP subunits. Contrary to expectations, the S616D mutation resulted in an opposite effect: a leftward shift of the activation curve. These results suggest that ERK modulation of Kv4.2 channel kinetics is complex and that the overall effect of modulation may well depend on ancillary subunits.
Finally, it is interesting to note that although the effects of U0126 in suppressing Kv current amplitude and accelerating inactivation are dramatic, we previously performed similar experiments on the native dendritic A-type K+ currents of hippocampal CA1 pyramidal neurons without noting such effects (23). Application of 20 µM U0126 to the native channel had no effect, regardless of whether it was applied to the outside of the neurons or included in the whole cell patch pipette, despite the fact that these native channels are likely from Kv4.2 subunits (or possibly Kv4.3 subunits) that are clearly sensitive to U0126 (16, 19, 20). One significant difference from native channels is that they inactivate much faster than the channels we recorded in the present study. Thus it is possible that some native modulatory protein or other cofactor missing from our heterologous expression system was acting on the site modulated by U0126.
In conclusion, we observed dramatic and unexpected effects of U0126 on Kv channels. Our results favor U0126’s exerting these effects on Kv channels not through kinase-dependent mechanisms but instead through direct action on gating mechanisms (18). The data reported herein suggest that caution should be exercised when interpreting experimental results using U0126.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of D. Johnston and X. Chen: Center for Learning and Memory, University of Texas at Austin, 1 University Station, C7000, Austin, TX 78712.
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FOOTNOTES |
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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 |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2. An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, and Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature 403: 553–556, 2000.[CrossRef][Medline]
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16. Maletic-Savatic M, Lenn NJ, and Trimmer JS. Differential spatiotemporal expression of K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro. J Neurosci 15: 3840–3851, 1995.[Abstract]
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23. Watanabe S, Hoffman DA, Migliore M, and Johnston D. Dendritic K+ channels contribute to spike-timing dependent long-term potentiation in hippocampal pyramidal neurons. Proc Natl Acad Sci USA 99: 8366–8371, 2002.
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). In the presence of U0126, current inactivation was best fit with a double exponential function. Both fast 
) and slow 
) decreased as a function of voltage. The voltage dependence of the fast 
