Circulation Research. 2001
Published online before print May 10, 2001,
doi: 10.1161/hh1001.090839
A more recent version of this article appeared on May 25, 2001
(Circulation Research. 2001;0:hh1001.090839.)
© 2001 American Heart Association, Inc.
MinK-Related Peptide 1 Associates With Kv4.2 and Modulates Its Gating Function
Potential Role as ß Subunit of Cardiac Transient Outward Channel?
Mei Zhang,
Min Jiang
Gea-Ny Tseng
From the Department of Physiology, Virginia Commonwealth University,
Richmond, Va.
Correspondence to Gea-Ny Tseng, PhD, Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298. E-mail gtseng{at}hsc.vcu.edu
Abstract
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 (
) 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 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 (ß)
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.
Key Words: Kv channel ß 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 (ß) subunits by associating with major
pore-forming () 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 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 subunit of the slow component of delayed rectifier channel, and
modulate its function.4 These
reports suggest that the KCNE
ß subunits may engage in "promiscuous" interactions with
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 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 ß 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 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 subunit). On day 3, some oocytes were
further injected with varying amounts of cRNA (1.5 to 79 ng) of MiRP1
(or another ß subunit). The molar ratios of cRNAs injected were
calculated by dividing the nanogram amounts of cRNAs injected by the
cRNA sizes.
Voltage-Clamp Experiments
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.
Results
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.
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Figure 1. Effects of MiRP1 on Kv4.2 current amplitude and kinetics of activation and inactivation. A, Superimposed current traces were recorded at +60 mV from oocytes expressing Kv4.2 alone or with MiRP1 (miRP1:Kv4.2 cRNA molar ratios marked). The display gain was adjusted so that peak current amplitudes match each other. B, MiRP1 had no statistically significant effects on Kv4.2 peak current amplitudes (measured at +60 mV) except when expressed at an extremely high level (MiRP1:Kv4.2=66:1). Data were from 2 batches of oocytes. For each batch, current amplitudes in oocytes coexpressing Kv4.2+MiRP1 (16 to 18 in each group) were normalized by the mean value from oocytes expressing Kv4.2 alone (*P<0.001 for Kv4.2+MiRP1 vs Kv4.2 alone). C, MiRP1 prolonged TTP of Kv4.2 current (measured at +60 mV). The superimposed curve was calculated from the following equation: parameter (TTP)=Amin+Amax/(1+K/x), where Amin is TTP in the absence of MiRP1 (4.9 ms), Amax is the maximal TTP prolongation induced by MiRP1 (22.4 ms), and K is the MiRP1:Kv4.2 cRNA ratio that induced half-maximum effect (4.4). D, MiRP1 prolonged the half-time of Kv4.2 decay (at +60 mV). The superimposed curve was calculated as parameter (decay half-time)=Amin+Amax/(1+K/x), where Amin is 13.1 ms, Amax is 31.3 ms, and K is 2.4.
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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.
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Figure 2. A, MiRP1 had no effects on the instantaneous I-V relationship of Kv4.2. Voltage-clamp protocol (inset) was as follows: from Vh of -80 mV, short pulses to +90 mV for 4 ms (Kv4.2 alone) to 24 ms (with MiRP1) were used to activate currents to peak without appreciate decay. These were followed by repolarization steps (Vrs) from +80 to -50 mV in 10-mV steps. Current amplitudes 3 ms into Vr were measured, leak-subtracted, and plotted against Vr. To average data from different oocytes, current amplitudes in each cell measured at various Vr levels were normalized to the level at Vr +80 mV. B, MiRP1 caused a positive shift in the peak I-V relationship of Kv4.2. Voltage clamp protocol (inset) was as follows: from Vh of -80 mV, depolarization steps (Vts) for 250 ms from -50 to +80 mV were applied once every 15 seconds. Peak currents were measured, leak-subtracted, and normalized to that at Vt +80 mV in the same oocytes (for data averaging). C, MiRP1 caused a positive shift in the voltage dependence of Kv4.2 activation. The activation curve was constructed in the following manner: for each oocyte, the peak current amplitudes at various Vt levels were divided by the instantaneous current amplitudes measured at the same voltages (both without normalization). This gave an estimate of the fractions of channels activated at various Vt levels. The relationship between y-axis (fraction activated) and x-axis (Vt) was fit with a simple Boltzmann function: fraction activated=1/{1+exp[(Vt-V0.5)/k]}, which estimates the half-maximum activation voltage (V0.5) and slope factor (k). D, Dose dependence of MiRP1-induced positive shift in V0.5 of activation (n=17 to 27 each, from 6 batches of oocytes) is shown. The superimposed curve was calculated from Amin+Amax/(1+K/x), where Amin is -7.3 mV, Amax is 30.3 mV, and k is 1.8.
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Figure 3. A, Effects of MiRP1 on the voltage dependence of Kv4.2 inactivation. Voltage-clamp protocol (inset in panel A) was as follows: from Vh of -80 mV, conditioning pulses (Vcs) were applied for 2 seconds to -90 to -20 mV in 5-mV increments. Each Vc was followed by a test pulse (Vt) to +60 mV. Peak current amplitudes during Vt pulses after various Vc levels were normalized to that after Vc to -90 mV. This gave an estimate of fractions of channels available for activation after various Vc levels. The relationship between fraction available and Vc was fit with 1/{1+exp[(Vt-V0.5)/k]}. B, Dose dependence of MiRP1-induced positive shifts in V0.5 of Kv4.2 inactivation (n=23 to 35 each, from 5 batches of oocytes). The superimposed curve was calculated from Amin+Amax/(1+K/x), where Amin is -59.4 mV, Amax is 13.8 mV, and K is 2.3.
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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 
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.
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Figure 5. A, Adding a c-myc epitope tag to the extracellular domain of MiRP1 (between amino acids 19 and 20) did not interfere with its function. Top, Diagram of c-myc insertion site in MiRP1 (TMD denotes transmembrane domain, and N and C denote the amino and carboxyl ends, respectively). Bottom, Effects of c-myc–tagged MiRP1 on the voltage dependence of Kv4.2 activation (cRNA molar ratio 13:1). Inset shows superimposed current traces recorded at +60 mV of Kv4.2 alone or of Kv4.2+c-myc-MiRP1. B, Coimmunoprecipitation of Kv4.2 with c-myc-MiRP1. The 2 proteins were made separately by in vitro translation in the presence of [35S]methionine, mixed, and subjected to immunoprecipitation by a c-myc antibody in the presence (+) or absence (-) of Triton X-100 (1%). The immunoprecipitates were run on an SDS gel along with Kv4.2 translation product. Shown is the autoradiogram of the gel, with the locations of size markers on the left and the locations of Kv4.2 (black arrow) and c-myc-MiRP1 (white arrow) on the right.
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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 :ß 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 subunits of
different gene families. To explore whether there is a preference in
the interactions of MiRP1 with different subunits, we tested the
effects of a low expression level of MiRP1 on various subunits
(Kv4.2, Kv4.3, Kv1.4, and HERG). In all cases, MiRP1 cRNA was injected
2 days after -subunit cRNA injections, and the cRNA molar ratios
(MiRP1: 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).
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Figure 7. Time course of Kv4.2 recovery from inactivation in the absence and presence of MiRP1. Voltage-clamp protocol (inset in panel A) was as follows: from Vh of -80, -100, or -120 mV, double pulses (V1 and V2) each to +40 mV for 500 ms were applied once every 15 seconds. The V1-V2 (recovery) interval ranged from 5 to 12 000 ms. Degree of recovery was measured by normalizing the peak current amplitude during V2 to that during V1 (V2/V1). A, Representative V1 and V2 current traces recorded from Kv4.2 expressed alone or with MiRP1 (MiRP1:Kv4.2=66:1). Vh was -80 mV, and V1-V2 interval was 2000 ms. In the presence of MiRP1, the peak current during V2 overshot the peak current during V1. B, Complete time courses of changes in V2/V1 against V1-V2 interval (plotted on a logarithmic scale). Shown are mean±SEM from 3 oocytes expressing Kv4.2 alone and 3 oocytes coexpressing Kv4.2 and MiRP1. Vh was -80 mV. C, Degrees of MiRP1-induced overshoot of V2 current amplitude at different Vh levels. Shown are peak levels of overshoot (represented by V2/V1 >1) at Vh of -80 mV (measured at V1-V2=3 seconds), -100 mV (V1-V2=1 second), and -120 mV (V1-V2=0.8 second). For comparison, the V2/V1 values from oocytes expressing Kv4.2 alone are also shown. D, At a comparable cRNA molar ratio (MiRP1:Kv4.2=7:1), the rat isoform (rMiRP1) induced much less Kv4.2 overshoot than did the human isoform (hMiRP1). Vh was -80 mV, and V1-V2 interval was 2 seconds (n=5 to 9 for each group). *P<0.001, Kv4.2+hMiRP1 vs Kv4.2 alone.
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A similar overshoot phenomenon has been described for the
Ito
channel in human ventricular myocytes of the epicardial
origin.20 The major
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 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 (=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 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.
Discussion
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 ß subunits known so far can increase the current
amplitudes through channels formed by subunits with which they
associate: minK on KvLQT1,24
Kv ß 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
-subunit biosynthesis by the small MiRP1 cRNA.
Mechanism of Effects of MiRP1 on Kv4.2:
A Common Theme for Interactions Between
KCNE ß Subunits and
Subunits?
The best studied example of -ß 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 ß association with Kv1 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 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.
View larger version (22K):
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Figure 8. Effects of KChIP2 and/or MiRP1 on Kv4.2 restitution. The cRNA molar ratios of KChIP2:Kv4.2 and MiRP1:Kv4.2 in all cases were 3.3:1. The voltage-clamp protocol and data analysis were the same as those described for Figure 7. KChIP2 accelerated Kv4.2 restitution. In the presence of KChIP2, the MiRP1-induced overshoot of Kv4.2 peak amplitude began at a V1-V2 interval of 250 ms, instead of 800 ms as in the absence of KChIP2 (denoted by vertical lines).
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|
Implications of Our Findings
The present data suggest that MiRP1 has the
potential of serving as a regulatory (ß) 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 and ß
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
( or ß) 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.
Acknowledgments
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.
Footnotes
Original received January 2, 2001; revision received March 26, 2001; accepted March 29, 2001.
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277(29):
26436 - 26443.
[Abstract]
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Y. F. Melman, A. Krumerman, and T. V. McDonald
A Single Transmembrane Site in the KCNE-encoded Proteins Controls the Specificity of KvLQT1 Channel Gating
J. Biol. Chem.,
July 5, 2002;
277(28):
25187 - 25194.
[Abstract]
[Full Text]
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M. Grunnet, T. Jespersen, H. B. Rasmussen, T. Ljungstrom, N. K Jorgensen, S.-P. Olesen, and D. A Klaerke
KCNE4 is an inhibitory subunit to the KCNQ1 channel
J. Physiol.,
July 1, 2002;
542(1):
119 - 130.
[Abstract]
[Full Text]
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M. Weerapura, S. Nattel, D. Chartier, R. Caballero, and T. E Hebert
A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link?
J. Physiol.,
April 1, 2002;
540(1):
15 - 27.
[Abstract]
[Full Text]
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S. P Patel, D. L Campbell, M. J Morales, and H. C Strauss
Heterogeneous expression of KChIP2 isoforms in the ferret heart
J. Physiol.,
March 15, 2002;
539(3):
649 - 656.
[Abstract]
[Full Text]
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G. W. ABBOTT and S. A. N. GOLDSTEIN
Disease-associated mutations in KCNE potassium channel subunits (MiRPs) reveal promiscuous disruption of multiple currents and conservation of mechanism
FASEB J,
March 1, 2002;
16(3):
390 - 400.
[Abstract]
[Full Text]
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C. Proenza, D. Angoli, E. Agranovich, V. Macri, and E. A. Accili
Pacemaker Channels Produce an Instantaneous Current
J. Biol. Chem.,
February 8, 2002;
277(7):
5101 - 5109.
[Abstract]
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M. S Nadal, Y. Amarillo, E. V.-S. de Miera, and B. Rudy
Evidence for the presence of a novel Kv4-mediated A-type K+ channel-modifying factor
J. Physiol.,
December 15, 2001;
537(3):
801 - 809.
[Abstract]
[Full Text]
[PDF]
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J. M. Nerbonne, C. G. Nichols, T. L. Schwarz, and D. Escande
Genetic Manipulation of Cardiac K+ Channel Function in Mice: What Have We Learned, and Where Do We Go From Here?
Circ. Res.,
November 23, 2001;
89(11):
944 - 956.
[Abstract]
[Full Text]
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G. W. Abbott and S. A. N. Goldstein
Potassium Channel Subunits: The MiRP Family
Mol. Interv.,
June 1, 2001;
1(2):
95 - 107.
[Abstract]
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G. W. Abbott, S. A. N. Goldstein, and F. Sesti
Do All Voltage-Gated Potassium Channels Use MiRPs?
Circ. Res.,
May 25, 2001;
88(10):
981 - 983.
[Full Text]
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T. Y. Nakamura, D. J. Pountney, A. Ozaita, S. Nandi, S. Ueda, B. Rudy, and W. A. Coetzee
A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents
PNAS,
October 23, 2001;
98(22):
12808 - 12813.
[Abstract]
[Full Text]
[PDF]
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M. Weerapura, S. Nattel, D. Chartier, R. Caballero, and T. E Hebert
A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link?
J. Physiol.,
April 1, 2002;
540(1):
15 - 27.
[Abstract]
[Full Text]
[PDF]
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M. S Nadal, Y. Amarillo, E. V.-S. de Miera, and B. Rudy
Evidence for the presence of a novel Kv4-mediated A-type K+ channel-modifying factor
J. Physiol.,
December 15, 2001;
537(3):
801 - 809.
[Abstract]
[Full Text]
[PDF]
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