MinK-Related Peptide 1 Associates With Kv4.2 and Modulates Its Gating Function
Potential Role as β Subunit of Cardiac Transient Outward Channel?
Abstract—Inherited mutations and a polymorphism in minK-related peptide 1 (MiRP1) have been linked to congenital or acquired long-QT syndrome, pointing to the importance of MiRP1 in maintaining the cardiac electrical stability. We tested whether MiRP1 could affect the function of Kv4.x (x=2 and 3), the major pore-forming (α) subunits of transient outward (Ito) channels in the heart. We used the Xenopus oocyte expression system to examine the effects of MiRP1 on Kv4.x channel gating kinetics and current amplitude and correlated these effects with MiRP1 expression level. MiRP1 slowed the rates of Kv4.2 activation and inactivation and shifted the voltage dependence of channel gating in the positive direction. These effects had a similar “dose” dependence: they plateaued at a cRNA ratio (MiRP1:Kv4.2) of 13:1, with half-maximum effects at estimated cRNA ratios of 2 to 4. On the other hand, MiRP1 had no significant effects on Kv4.2 current amplitude in the same range of expression level. When expressed at a comparable low level, MiRP1 had similar (although smaller) effects on Kv4.3 but could not modulate Kv1.4 (another α subunit of Ito channels in the heart). Kv4.2 could be coimmunoprecipitated with epitope-tagged MiRP1, indicating that the 2 could form a stable complex. Our data suggest that MiRP1 may serve as a regulatory (β) subunit of Ito channels in the heart. This is supported by the observation that MiRP1 induced an “overshoot” of Kv4.2 current amplitude during channel recovery from inactivation, similar to the overshoot of Ito described for human epicardial myocytes.
- Kv channel β subunits
- long QT
- rapid component of delayed rectifier channels
- transient outward channels
- Xenopus oocytes
In 1999, minK-related peptide 1 (MiRP1) was cloned by Abbott et al1 as one of three MiRPs. minK and MiRP1 through MiRP3 are encoded by KCNE genes (KCNE1 through KCNE4, respectively). They function as regulatory (β) subunits by associating with major pore-forming (α) subunits of voltage-gated K (Kv) channels and regulating their function. Inherited mutations or a common polymorphism in the MiRP1 amino acid sequence has been linked to congenital long QT syndrome (LQT6) or acquired long QT syndrome,1 2 3 suggesting that MiRP1 plays a role in maintaining the cardiac electrical stability. Initial functional expression experiments suggested that MiRP1 can associate with HERG, the α subunit of the rapid component of the delayed rectifier channel, and modulate the channel function.1 More recently, it has been shown that MiRP1 can also associate with KvLQT1, the α subunit of the slow component of delayed rectifier channel, and modulate its function.4 These reports suggest that the KCNE β subunits may engage in “promiscuous” interactions with α subunits from different gene families. Indeed, MiRP2 can affect the function of not only KvLQT1 (KCNQ1) but also KCNQ4, HERG, and Kv3.4.5 6
We report in the present study that MiRP1 can form a stable complex with Kv4.2, the major α subunit of the transient outward channel (Ito) in cardiac myocytes,7 and induce profound effects on the Kv4.2 gating kinetics. Therefore, MiRP1 can potentially serve as a β subunit for Ito channels in cardiac myocytes. Furthermore, our data suggest that at a low expression level, MiRP1 preferentially modulates the function of Kv4.2 (and Kv4.3) over other α subunits tested (HERG and Kv1.4). Part of the data have appeared in abstract form.8
Materials and Methods
cRNA Cloning, Mutagenesis, Transcription, and Quantification
We used the human isoform of MiRP1 (a generous gift of Dr S.A.N. Goldstein, Yale University, New Haven, Conn),1 minK, and HERG and the rat isoforms of Kv4.2, Kv4.3, and Kv1.4. We cloned the rat isoforms of MiRP1 and a Kv channel–interacting protein (KChIP2) by reverse transcription–polymerase chain reaction from rat ventricular RNA. The amino acid sequences are the same as those reported in the literature.1 9 Epitope tagging of human MiRP1 was performed by using the polymerase chain reaction–based overlap extension method.10 cRNAs were transcribed by using a commercial kit (mMessage mMachine), followed by DNase 1 digestion. All cRNA samples were run on a denaturing RNA gel and stained with ethidium bromide. The cRNA band sizes and intensities were quantified by use of densitometry (ChemiImager model 4400, Alpha Innotech Corp). The cRNA solutions were then diluted to reach the desired final concentrations for injections.
Oocyte Preparation and Injection
Oocyte isolation and cRNA injections were as described previously.11 On the day of oocyte isolation (day 1), each oocyte was injected with 5 ng cRNA of Kv4.2 (or another α subunit). On day 3, some oocytes were further injected with varying amounts of cRNA (1.5 to 79 ng) of MiRP1 (or another β subunit). The molar ratios of cRNAs injected were calculated by dividing the nanogram amounts of cRNAs injected by the cRNA sizes.
On days 4 and 5, channel function was studied by using the 2-microelectrode voltage-clamp method, as described previously.12 Oocytes were continuously superfused with a low-chloride ND96 solution at room temperature. Voltage-clamp protocol generation and data acquisition were controlled by pClamp 5.5 (Axon Instruments). Data analysis was performed with pClamp 6 or 8, Excel (Microsoft), and PeakFit (Jandel Scientific). Specific voltage-clamp protocols and methods of data analysis are described in the figure legends. Where appropriate, data are presented as mean±SEM. Statistical analysis was performed with rank-based 1-way ANOVA followed by the Dunn test (SigmaStat 2.0, SPSS).
In Vitro Translation and Immunoprecipitation
In vitro translation of c-myc–tagged human MiRP1 and Kv4.2 was performed by use of a commercial kit (Rabbit Reticulocyte Lysate System, Promega) with canine pancreatic microsomes in the presence of [35S]methionine. The 2 proteins were translated separately and then mixed. Immunoprecipitation of the translation products was carried out by using Immunopure Immobilized Protein G (Pierce) and an anti–c-myc monoclonal antibody (Oncogene) according to the manufacturer’s instructions. The immunoprecipitates were fractionated on 10% SDS gel. After drying, the gel was exposed to a PhosphorImager (Molecular Dynamics) screen for an appropriate amount of time, followed by PhosphorImager quantification.
In all experiments, oocytes were injected with the same amount (5 ng) of Kv4.2 cRNA and, 2 days later, varying amounts of human MiRP1 cRNA. The expression levels of MiRP1 are noted as MiRP1:Kv4.2 (cRNA molar ratio).
MiRP1 Affects Kv4.2 Gating Kinetics
Figure 1A⇓ illustrates 2 distinct effects of MiRP1 on Kv4.2: slowing of activation and slowing of inactivation. The changes in the activation kinetics were quantified by “time-to-peak” current (TTP) at +60 mV. Data are summarized in Figure 1C⇓. MiRP1 prolonged TTP of Kv4.2 in a “dose”-dependent manner (from 5.1±0.1 ms in Kv4.2 alone to a maximum of 26.0±3.0 ms at an MiRP1:Kv4.2 cRNA molar ratio of 66:1), with the half-maximum effect occurring at an estimated cRNA ratio of 4. The inactivation of Kv4.2 during depolarization to +60 mV followed an apparent double-exponential function. To simplify data presentation and comparison, we quantified the data with the half-time of current decay, which is the time for current to decay to 50% of its peak value during a 1-second pulse to +60 mV. Figure 1D⇓ shows that MiRP1 prolonged the decay half-time from 13.2±0.8 ms (Kv4.2 alone) to a maximum of 44.4±3.9 ms, with a dose dependence similar to that of its effect on TTP.
Figure 1B⇑ shows that the Kv4.2 current amplitude measured at +60 mV appeared to be increased modestly in the presence of MiRP1 at MiRP1:Kv4.2 cRNA molar ratios up to 13:1. However, the differences were not statistically significant. At a supramaximal level of MiRP1 expression (MiRP1:Kv4.2 66:1), there was a pronounced reduction in Kv4.2 current amplitude (to 54±6% of Kv4.2 alone).
MiRP1 also induced a dose-dependent positive shift in the voltage dependence of Kv4.2 activation (Figure 2⇓) and inactivation (Figure 3⇓). The dose dependence resembled those shown for the changes in gating kinetics in Figures 1C⇑ and 1D⇑. On the other hand, MiRP1 did not alter the shape of the instantaneous current-voltage (I-V) relationship (Figure 2A⇓), indicating that it does not affect the ion permeation process of the Kv4.2 channel.
MiRP1 Retards 4AP Unbinding From Kv4.2 Channel
The above data suggest that MiRP1 can interact with Kv4.2 and hinder both the activation and the inactivation processes. The activation process of voltage-gated K+ channels involves conformational changes in the inner mouth region that result in the opening of the pore, allowing K+ ion fluxes through the channel.13 14 15 The molecular mechanism of Kv4.2 inactivation is not entirely clear at present but likely also involves conformational changes in the inner mouth region.16 Therefore, it is possible that at depolarized membrane voltages, the conformational changes in the inner mouth region of Kv4.2 are hindered by MiRP1. To test this hypothesis, we examined whether MiRP1 could affect the interaction between 4-aminopyridine (4AP) and Kv4.2. 4AP binds to the inner mouth region of Kv4.2 in the closed (resting) state at negative voltages.17 Unbinding occurs at depolarized voltages when channels open and is further enhanced by channel inactivation.18 According to our hypothesis, we predicted that 4AP unbinding from Kv4.2 should be hindered by MiRP1. Figure 4⇓ shows the results of these experiments. The voltage-clamp protocol and rationale are described in the legend. The time course of 4AP (10 mmol/L) unbinding from Kv4.2 at +60 mV could be described by a single exponential function both in the absence and in the presence of MiRP1. Consistent with our prediction, the τ value of 4AP unbinding was prolonged from 59±9 to 530±77 ms by MiRP1 (MiRP1:Kv4.2=13:1).
MiRP1 Can Form a Stable Complex With Kv4.2
The above oocyte experiments indicate that MiRP1 could associate with Kv4.2 and modulate its function. Furthermore, because the MiRP1 cRNA was injected 2 days after Kv4.2 cRNA injection (at which time the Kv4.2 channel function had been well expressed), the association between MiRP1 and Kv4.2 did not require that the 2 be translated together. To further confirm these, we attached an epitope (c-myc peptide sequence EQKLISEEDL) to MiRP1, so that we could use an antibody to test whether MiRP1 and Kv4.2 could form a stable complex and be immunoprecipitated together. We inserted the c-myc sequence between amino acids 19 and 20 in the extracellular domain of MiRP1 (diagram in Figure 5A⇓), close to the consensus N-glycosylation site (N26). The rationale was that if the interaction between MiRP1 and Kv4.2 occurs intracellularly, inserting the c-myc sequence in the extracellular domain should not interfere with the function of MiRP1. Indeed, Figure 5A⇓ shows that the c-myc–tagged MiRP1 (c-myc-MiRP1) induced a prominent positive shift in the voltage dependence of Kv4.2 activation, similar to the effect of the unmodified MiRP1. Furthermore, c-myc-MiRP1 also slowed Kv4.2 activation and inactivation and delayed 4AP (10 mmol/L) unbinding from Kv4.2 at depolarized voltages.
The results of a representative immunoprecipitation experiment are shown in Figure 5B⇑. c-myc-MiRP1 and Kv4.2 were in vitro–translated in separate reactions in the presence of [35S]methionine. The translation products were then mixed and subjected to immunoprecipitation by an anti–c-myc monoclonal antibody. The immunoprecipitates were run on an SDS gel along with the Kv4.2 translation product. The autoradiogram clearly shows that Kv4.2 was copurified with c-myc-MiRP1 by the antibody (compare the left and right lanes of Figure 5B⇑). Similar results were obtained in 3 experiments. The middle lane of Figure 5B⇑ shows that coimmunoprecipitation of Kv4.2 with c-myc-MiRP1 was prevented by including a detergent (Triton X-100, 1%) in the immunoprecipitation reaction that could destabilize protein-protein interactions.19
MiRP1:Kv4.2 Interaction Is Preferred Over Other α:β Pairs
The above data, in conjunction with the known ability of MiRP1 to associate with HERG and modulate its function,1 suggest that MiRP1 may be able to engage in promiscuous interactions with α subunits of different gene families. To explore whether there is a preference in the interactions of MiRP1 with different α subunits, we tested the effects of a low expression level of MiRP1 on various α subunits (Kv4.2, Kv4.3, Kv1.4, and HERG). In all cases, MiRP1 cRNA was injected 2 days after α-subunit cRNA injections, and the cRNA molar ratios (MiRP1:α subunit) were kept at 2:1 except for HERG (MiRP1:HERG=6.6:1).
Figure 6A⇓ shows that MiRP1 caused a positive shift in the inactivation curve of Kv4.3, similar to its effect on Kv4.2, although to a smaller degree. On the other hand, MiRP1 did not have detectable effects on Kv1.4. We also compared the effects of minK on Kv4.2 with those of MiRP1. At a low expression level (minK:Kv4.2 cRNA molar ratio 2:1), minK did not affect the voltage dependence of Kv4.2 inactivation or its gating kinetics. Figure 6B⇓ shows that MiRP1 had little or no effects on HERG current amplitude or gating kinetics (although the same level of MiRP1 expression induced significant changes in Kv4.2 gating kinetics).
MiRP1 Induced an Overshoot of Kv4.2 Peak Current Amplitude During Recovery From Inactivation
The voltage-clamp protocol and data analysis for studying the time course of Kv4.2 recovery from inactivation are described in the Figure 7⇓ legend. Figure 7A⇓ shows that at a holding voltage (Vh) of −80 mV and a recovery interval of 2 seconds, Kv4.2 expressed alone completely recovered from inactivation (V1 and V2 current traces superimposed). However, when MiRP1 was coexpressed, the peak current amplitude during V2 became significantly larger than that during V1 (“overshoot”). Figure 7B⇓ depicts the complete time courses of changes in the V2 current amplitude relative to that during V1 in oocytes expressing Kv4.2 alone or coexpressing Kv4.2 with MiRP1 (MiRP1:Kv4.2=2:1). The Kv4.2 current recovered from inactivation in a monoexponential function. On the other hand, in the presence of MiRP1, the V2 current amplitude overshot that during V1, with a peak at V1−V2 intervals of 1 to 3 seconds. MiRP1 also induced an overshoot of Kv4.3 peak current amplitude during recovery from inactivation (data not shown).
A similar overshoot phenomenon has been described for the Ito channel in human ventricular myocytes of the epicardial origin.20 The major α subunit of Ito channels in these myocytes is believed to be Kv4.3.21 Interestingly, the Ito channel in human ventricular myocytes from the endocardial region (whose major α subunit is believed to be Kv1.4)21 does not display such an overshoot phenomenon.20 This distinction between epicardial and endocardial Ito can be explained by our data: Kv4.x (x=2 or 3) can be modulated by MiRP1, whereas Kv1.4 cannot. To further compare the MiRP1-induced overshoot in Kv4.2 with the Ito overshoot in human epicardial myocytes, we examined the degree of overshoot measured at different Vhs. In human epicardial myocytes, the degree of Ito overshoot is enhanced by making the Vh more negative, from −80 to −100 mV.20 Figure 7C⇑ compares the peak levels of MiRP1-induced Kv4.2 overshoot measured at Vh values of −80, −100, and −120 mV. As the Vh became more negative, the overshoot peaked earlier (shorter V1−V2 intervals) because the underlying rate of Kv4.2 recovery from inactivation became faster (τ=743±52, 286±12, and 166±6 ms at −80, −100, and −120 mV). The peak level of overshoot at Vh of −120 mV was significantly larger than that at Vh of −80 mV.
The major α subunit of Ito channels in rat ventricle is Kv4.2. However, rat Ito does not show an overshoot during restitution. To investigate whether this is due to differences between rat and human MiRP1 isoforms (18% differences in amino acid sequence), we compared the effects of rat MiRP1 on Kv4.2 restitution with those of human MiRP1. Figure 7D⇑ shows that rat MiRP1 induced little or no overshoot during Kv4.2 restitution.
Our major findings can be summarized as follows: (1) MiRP1 coexpressed with Kv4.2 in oocytes slowed Kv4.2 activation and inactivation and shifted the voltage dependence of gating in the positive direction. (2) MiRP1 also greatly slowed the rate of 4AP unbinding from Kv4.2. (3) Inserting an epitope tag (c-myc) in the extracellular domain of MiRP1 did not affect its function. A c-myc antibody could immunoprecipitate c-myc–tagged MiRP1 along with Kv4.2 when the 2 were translated in separate reactions and then mixed. (4) MiRP1 induced an overshoot of Kv4.2 peak current amplitude during the course of recovery from inactivation. (5) At a comparable low level of expression, MiRP1 had similar effects on Kv4.3 but did not affect Kv1.4 or HERG.
Dose Dependence of Effects of MiRP1 on Kv4.2
Our data showed that MiRP1 modulated the Kv4.2 gating function in a dose-dependent manner. All the gating effects plateaued at an MiRP1:Kv4.2 cRNA molar ratio of 13:1 and were not increased further by elevating the ratio to 66:1. Therefore, the maximal effects of MiRP1 on Kv4.2 gating under our experimental conditions could be defined, although these data do not provide any information about the stoichiometry of MiRP1:Kv4.2.22 23
The similarity in the dose dependence of the effects of MiRP1 on Kv4.2 gating suggests common or closely related mechanisms. On the other hand, MiRP1 did not have statistically significant effects on the Kv4.2 current amplitude at cRNA ratios up to 13:1. Further elevating the cRNA ratio to 66:1 produced a prominent reduction in the Kv4.2 current amplitude, indicating that a different mechanism might be involved. Note that we injected the MiRP1 cRNA 2 days after the injection of Kv4.2 cRNA. In preliminary experiments, coinjecting MiRP1 and Kv4.2 cRNAs at the same time produced a marked suppression of Kv4.2 current amplitude even at a MiRP1:Kv4.2 ratio close to 2:1. It is possible that the much smaller MiRP1 cRNA (0.55 kb) could interfere with the biosynthesis processes of Kv4.2 cRNA (2.3 kb) in oocytes.
Many β subunits known so far can increase the current amplitudes through channels formed by α subunits with which they associate: minK on KvLQT1,24 Kv β subunits on Kv1,25 KChIPs on Kv4,9 and the K+ channel–associated protein (KChAP) on Kv2.1.26 MiRP1 seems to be an exceptional case in this aspect. It has been shown that MiRP1 coexpressed with HERG in oocytes1 or in mammalian cells27 can significantly reduce the HERG current amplitude, although there is no consensus as to the mechanism.1 27 Our data highlight the importance of testing the effects of MiRP1 on different channel properties (eg, gating function versus current amplitude) at different expression levels and of using strategies (eg, separate cRNA injections) to minimize the potential interference of α-subunit biosynthesis by the small MiRP1 cRNA.
Mechanism of Effects of MiRP1 on Kv4.2: A Common Theme for Interactions Between KCNE β Subunits and α Subunits?
The best studied example of α-β interactions in Kv channels is minK/KvLQT1.24 28 29 Our data show that the interaction between MiRP1 and Kv4.2 is similar to that between minK and KvLQT1 in the following aspects. First, MiRP1 slowed both the activation and the inactivation processes of Kv4.2, similar to the effects of minK on KvLQT1 gating.24 29 Second, because both the activation and the inactivation processes of Kv4.2 involve conformational changes in the inner mouth region of the pore15 16 and because the extracellular domain of MiRP1 did not seem to play a major role in its effects on Kv4.2 gating (inserting an epitope tag here did not affect its effects), it is possible that the cytoplasmic domain of MiRP1 interacts with the inner mouth region of Kv4.2. This is similar to the proposed domains of interactions between minK and KvLQT1.28 Third, MiRP1 could associate with Kv4.2 and modulate its function even when the 2 were translated separately. This is similar to minK association with KvLQT128 but appears different from Kv β association with Kv1 α subunits (which occurs in the endoplasmic reticulum when both proteins are being translated in proximity).25
Does MiRP1 Association With Kv4.x Recapitulate the Native Ito Function?
It is well established that Kv4.x (x=2 and/or 3) are the major α subunits of Ito channels in the heart.7 However, there are still distinct differences in the behavior between Kv4.x expressed in mammalian cells or Xenopus oocytes and native Ito channels in cardiac myocytes, suggesting that an additional factor(s) or subunit(s) is required to recapitulate the native Ito channel function. For example, under comparable recording conditions (in terms of temperature and the ionic composition of bath solution), the voltage dependence of activation and inactivation of native Ito channels occurs in a more positive voltage range than that of Kv4.x in heterologous expression systems.30 MiRP1 can shift the voltage dependence of Kv4.x gating in the positive direction, thus making Kv4.x more similar to the native Ito. Furthermore, the Ito in human ventricular myocytes of epicardial origin displays a peculiar overshoot phenomenon during recovery from inactivation (V2 current was larger than V1 current by ≈30%).20 This is not seen when Kv4.x is expressed alone. MiRP1 coexpression with Kv4.x could reproduce this phenomenon: the current during V2 overshot the current during V1 by ≈20%. The degree of MiRP1-induced Kv4.2 overshoot was increased by shifting the Vh from −80 to −120 mV, similar to the voltage dependence of Ito overshoot in human epicardial myocytes.20 However, the human Ito overshoot peaks at a V1−V2 interval of 100 to 200 ms, whereas the MiRP1-induced Kv4.2 overshoot in oocytes peaked at 1000 to 2000 ms under similar conditions (Vh −80 mV and room temperature). It is possible that other subunit(s), such as KChIP, may be also involved in determining the native Ito kinetics.9 Indeed, Figure 8⇓ supports this notion.
Implications of Our Findings
The present data suggest that MiRP1 has the potential of serving as a regulatory (β) subunit of native Ito channels in cardiac myocytes. However, to test this hypothesis, one needs a reliable MiRP1 antibody that can detect the expression and location of MiRP1 protein in cardiac myocytes. On the other hand, MiRP1 is expressed at a much higher level in the brain and may affect the native Ito channels there. We showed that when tested in oocytes at a low expression level, MiRP1 appears to preferentially modulate the gating function of Kv4.x (more for Kv4.2 than for Kv4.3) over HERG or Kv1.4. It is not clear whether this preference in MiRP1 actions is due to the oocyte expression environment. Future experiments should test whether this phenomenon can be reproduced in mammalian cells. In cardiac myocytes, the importance of interactions between different α and β subunits may depend on the expression levels and other factors (eg, subcellular distribution, subunit trafficking, and clustering). It will be important to sort out these selection processes and understand whether and how these processes are affected by mutations of channel (α or β) subunits and by disease conditions of the heart. This information will enhance our understanding of the molecular basis for the heterogeneity of Kv channel function in normal and in diseased hearts.
This study was supported by HL-46451 from the National Heart, Lung, and Blood Institute, National Institutes of Health, and by a Grant-in-Aid award from the American Heart Association/Mid-Atlantic Affiliate.
Original received January 2, 2001; revision received March 26, 2001; accepted March 29, 2001.
- © 2001 American Heart Association, Inc.
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