From the § Division of Cardiology, Departments of
Medicine and Pharmacology, ¶ Graduate Program of Neuroscience, and
Graduate Program of Pharmacology, Weill Medical College of
Cornell University, New York, New York 10021
Received for publication, December 16, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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The physiological properties
of most ion channels are defined experimentally by functional
expression of their pore-forming Voltage-gated potassium
(Kv)1 channels
form an aqueous pore through the plasma membrane by the assembly of
four Previous studies using either Xenopus oocytes or mammalian
cells as expression systems have produced conflicting reports and often
no consensus as to the functional consequences of Molecular Biology--
xMinK, xMiRP2, xMIRP4.1, and xMiRP4.2
were cloned from cDNA isolated by RT-PCR from stages V and VI
X. laevis oocyte mRNA using primer sequences as follows:
xMinK, 5'-ATGCCAGGGTTAAACACCACTGCC-3' (forward) and
5'-CTAGTTGCTGGG AGAAGAGGGGATATA-3'(reverse); XMIRP2, 5'-CAGTTTGATTGGAGAGTGGGATTC-3' (forward) and 5'-TAGACCCCTGGGGCCTCGTC-3' (reverse); xMIRP4.1, 5'-ATGAATTGTAGTAATACTTCTC GT-3' (forward) and
5'-CTAAAGTAAGTTTTCACTTTCAATACT-3' (reverse); xMIRP4.2,
5'-CGGGAATCAGACTTCTGTCC-3' (forward) and
5'-GCTTTACAGGATACACAGTAGC-3' (reverse). Genes were identified by
a BLAST search of EST data bases with mammalian KCNE gene
sequences using the tblastn algorithm. All genes showed a canonical
initiation ATG site with G at
For RNAi, 500 pg of double-stranded siRNA 21-mer oligos (Dharmacon)
corresponding to bases 104-124 of xMiRP2 (top strand
5'-AAGGGAACCACACGGACGCCA-3') was injected into oocytes immediately
after the injection of Electrophysiology--
Whole-cell currents in oocytes were
recorded at room temperature by TEVC using an OC-725C Amplifier (Warner
Instruments, Hamden, CT), an IBM computer, and pCLAMP8 software
(Axon Instruments, Foster City, CA). Data analysis was performed using
CLAMPFIT (Axon Instruments). Bath solution was (in mM) 4 KCl, 96 NaCl, 0.7 MgCl2, 1 CaCl2, and 10 HEPES
(pH 7.4). Whole-cell currents were recorded at room temperature in CHO
cells using a Multiclamp 700A Amplifier (Axon Instruments), an IBM
computer, and pCLAMP8 software. Cells were studied on an inverted
microscope equipped with epifluorescence optics for green fluorescent
protein detection. The bath solution was composed of (in
mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 5 HEPES, 2.5 CaCl2, and 10 D-glucose (pH 7.4). Pipettes were
of 3-5 Megaohms resistance when filled with intracellular solution
containing (in mM) 10 NaCl, 117 KCl, 2 MgCl2,
11 HEPES, 11 EGTA, and CaCl2 (pH 7.2).
Biochemistry--
Oocytes were lysed with: (in mM)
150 NaCl, 0.2 phenylmethylsulfonyl fluoride, 20 NaF, 10 Na3VO4, 50 Tris (pH 7.4), and the following
detergents: 1% Nonidet P-40, 1% CHAPS, 1% Triton X-100, and 0.5%
SDS. Samples were clarified by two separate centrifugations at
10,000 × g for 20 min before the addition of standard
SDS-PAGE loading buffer and separation by SDS-PAGE on 15% or 4-20%
gradient gels. After transfer to polyvinylidene difluoride membrane,
Western blots were performed with an in-house primary antibody raised by injection into rabbits of a mammalian expression vector expressing full-length hMiRP2. Detection was via goat anti-rabbit horseradish peroxidase-coupled secondary antibodies (Bio-Rad) for fluorography. Band intensities for assessment of RNAi efficiency at the protein level
were quantified as for cDNA bands.
A Xenopus MiRP Family Is Constitutively Expressed in
Oocytes--
Xenopus EST data bases were searched with
mammalian KCNE gene sequences, leading to the identification
of four Xenopus clones designated here as xMinK, xMiRP2,
xMiRP4.1, and xMiRP4.2 on the basis of their closest mammalian KCNE
relatives. The four Xenopus genes were subsequently cloned
from cDNA isolated by RT-PCR from stages V and VI
Xenopus oocyte mRNA (Fig.
1). Homologues of mammalian MiRP1 and
MiRP3 were not detected in Xenopus EST data bases. Mammalian MiRP2 co-assembles with several diverse Kv channel Injected HERG and KCNQ1 Are Modulated by Overexpressed xMiRP2 in
Oocytes--
Injection of HERG cRNA into oocytes resulted in the
expression of rapidly inactivating delayed rectifier K+
currents that passed large tail currents during a Endogenous Oocyte xMiRP2 Is Suppressed by the Injection of siRNA
Oligos--
To evaluate whether endogenous levels of xKCNE
subunits are sufficient to modulate cloned mammalian Kv
channel Endogenous Oocyte xMiRP2 Inhibits Heterologously Expressed HERG
Currents--
The effects of the suppression of endogenous xMiRP2
expression on injected HERG currents were assessed next. HERG is an
especially suitable indicator of endogenous xMiRP2 contribution to
heterologously expressed K+ currents, because the unique
gating attributes of HERG channels (i.e. rapid inactivation
at positive voltages and rapid recovery from inactivation at negative
voltages (31)) allow the assessment of small HERG tail currents at Variability in Effects of hMiRP1 on HERG Suggests Rescue from
Inhibition by Endogenous xMiRP2--
In contrast to human and
Xenopus MiRP2, which completely suppress HERG currents
expressed in oocytes (9) (presumably by inhibiting functional HERG
channel formation), hMiRP1 partially reduces HERG currents by the
formation of lower conductance fast deactivating functional
channel complexes (6). The variability of hMiRP1 effects on HERG
currents depending on expression system or HERG expression level has
been reported previously. Although hMiRP1 mutations have been
linked to inherited and acquired arrhythmia in three studies, and the
effects of hMiRP1 on HERG have been noted by several groups, the
reported effects differ (6, 11, 13-18). We hypothesized that some of
the variability of the effects of hMiRP1 on HERG results from the
displacement of xMiRP2 and that addition of hMiRP1 cRNA would rescue
HERG currents at lower HERG levels (at which HERG is normally inhibited
by endogenous xMiRP2) by forming functional hMiRP1/HERG complexes in
favor of nonfunctional xMiRP2/HERG complexes.
We first recapitulated previous reports of hMiRP1 modulation of HERG in
both Xenopus oocytes and CHO cells. Using 10 ng of HERG
cRNA/oocyte to achieve a high HERG current density, we compared current
attributes with and without the injection of hMiRP1 cRNA. Co-injection
of either 10 or 20 ng of hMiRP1 cRNA/oocyte resulted in similar ~60%
reductions in HERG tail currents (Fig.
5A, left panel).
hMiRP1 also reduced HERG currents in CHO cells, producing an ~40%
reduction in tail current density compared with HERG co-transfected with blank plasmid (Fig. 5A, right panel). Next,
similar experiments were conducted using lower concentrations of HERG
cRNA in oocyte expression studies. In contrast to the high current
density HERG experiments, co-injection of hMiRP1 cRNA (2 ng/oocyte)
with lower levels of HERG cRNA (0.5 ng/oocyte) actually up-regulated
HERG Previously, the finding that the KCNQ1 The issue of MiRP/ Oocytes remain an expression system with numerous advantages for the
assessment of ion channel function. Here we have described the first
reported use (to our knowledge) of RNAi post-transcriptional gene
silencing in the oocyte system. The use of RNAi to control endogenous
modifying subunits in oocytes, mammalian cell expression systems (which
may also contain endogenous MiRPs), and native studies will improve our
understanding of the heteromultimeric molecular basis of human
Kv currents and also allow us to accurately define the
properties of "pure" subunits in Xenopus
laevis oocytes. Here, we cloned a family of
Xenopus KCNE genes that encode MinK-related
peptide K+ channel
subunits (xMiRPs) and demonstrated
their constitutive expression in oocytes. Electrophysiological analysis
of xMiRP2 revealed that when overexpressed this gene modulates
human cardiac K+ channel
subunits HERG (human
ether-a-go-go-related gene) and KCNQ1 by suppressing HERG
currents and removing the voltage dependence of KCNQ1 activation. The
ability of endogenous levels of xMiRP2 to contribute to the biophysical
attributes of overexpressed mammalian K+ channels in
oocyte studies was assessed next. Injection of an xMiRP2
sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had
no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of
endogenous xMiRP2 silencing were tested using electrophysiological
analysis of heterologously expressed HERG channels. The RNA
interference-mediated reduction of endogenous xMiRP2 expression
increased macroscopic HERG current as much as 10-fold depending on HERG
cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG
interaction were also affected by endogenous xMiRP2. At high HERG
channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the
base-line properties of K+ channels like HERG in oocyte
expression studies, which could explain expression level- and
expression system-dependent variation in K+
channel function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, each with six transmembrane helices and a pore loop
(1). The favored method for evaluating potassium channel function is by microinjection of channel cRNA into Xenopus
laevis oocytes to facilitate two-electrode voltage clamp
(TEVC) or patch clamp studies. However, observed differences in the
behavior of potassium channel genes when expressed in oocytes compared
with mammalian cells suggest that endogenous factors in either or both
systems dictate channel properties to some extent (2-5). Studies of
cardiac Kv channels HERG and KCNQ1 involve a further level
of complexity because they co-assemble with transmembrane
subunits,
designated KCNE subunits, or MinK-related peptides (MiRPs),
to generate some native currents (6-10). The mammalian KCNE
superfamily was cloned by a BLAST search of EST data bases using
the sequence for MinK encoded by KCNE1 (6, 11, 12).
Co-assembly of HERG with MiRP1 encoded by KCNE2 forms a
channel with the properties of the IKr cardiac
repolarization current (6, 11, 13-18), and KCNQ1 co-assembles with
MinK to form cardiac IKs (8, 10). The necessity
of
and
subunits alike in human physiology is highlighted by the
linkage of mutations in both to cardiac arrhythmia (6, 15, 17, 19-24).
MiRP1, for example, is thought to play a central and diverse role in
human cardiac physiology on the basis of cardiac expression (6, 25,
26), association with cardiac arrhythmia (6, 15, 17), and ability to
modulate not only HERG but also KCNQ1 (26, 27), Kv4.2 (cardiac
Ito) (16), and even the distantly related
HCN pacemaker channels (28). Further, because
subunits alter the
sensitivity and functional effects of both therapeutic and unwanted
drug interactions and are linked to drug-induced long QT
syndrome, the assignment of both
and
subunits to native
currents is essential for drug screening and rational drug design (6,
15).
-
subunit interactions, particularly in the case of HERG, which interacts with
human MinK, MiRP1, and MiRP2 (6, 9, 29). In addition, many
investigators report that within a single study the properties of
heterologously expressed MiRP/
subunit currents vary with the
expression system or with the concentration of MiRP cRNA, even at
levels where
-subunit saturation would be expected (6, 16, 17). This
finding suggests that these interactions depend on factors intrinsic to
the expression system used. Clearly, resolving the true
heteromultimeric identity of native ion channels and currents recorded
in heterologous expression studies is important for our understanding
of channel structure-function and physiology. Here we cloned a family
of xMiRPs endogenously expressed in Xenopus oocytes and
demonstrated that xMiRP2 interacts with overexpressed HERG to shape the
functional attributes of HERG during oocyte expression studies.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3, A at +3, and no intervening
methionines 3' of the nearest 5' stop codon. For functional expression,
xMiRP2 was ligated into pGA1 and linearized with SacI for
transcription of cRNA for injection into oocytes. For studies in
oocytes, cRNA transcripts for xMiRP2, human MiRP1 (hMiRP1) (in pGA1),
hKCNQ1 (in a pBluescript-based vector), and HERG (in pSP64, Promega)
were produced as described previously (6). To avoid dilution errors,
all oocytes were injected first with the appropriate
subunit cRNA
and then immediately afterward with modifying
subunits or short
interfering RNA (siRNA). For studies in CHO cells, cDNAs in pCINeo
(Promega) were co-transfected with green fluorescent protein (in
pBOB) using Superfect transfection reagent (Qiagen), and
currents were recorded 24-36 h later.
subunit cRNA. siRNA oligo for xMiRP2 was
co-transfected into CHO cells with the appropriate channel cDNA.
For the assessment of RNAi gene silencing at the mRNA level, an
equal amount of oocytes was injected either with xMiRP2 cRNA or
scrambled control siRNA, and RNA was extracted using Trizol
(Invitrogen) on the same day that functional experiments were carried
out and on the same batch of oocytes. RNA concentration was measured by
spectrophotometry (SmartSpec3000; Bio-Rad), and RT-PCR was performed
with Xenopus
-actin primers (forward,
5'-AAGGAGACAGTCTGTGTGCGTCCA-3'; reverse, 5'-CAACATGATTTCTGCAAGAGCTCC-3') to normalize mRNA concentration. The optical density of samples run on a 1% agarose gel and stained with ethidium bromide was measured using a Fluor-S MultiImager (Bio-Rad). Samples normalized to
-actin cDNA concentration were subsequently amplified using xMiRP2-specific primers, and optical density was quantified.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, removing the voltage dependence of KCNQ1 (9), suppressing
HERG current without altering gating kinetics (9, 11), and converting Kv3.4 to a subthreshold-activating delayed rectifier (7). Here we
investigated the effects of the interaction between xMiRP2 and the
cardiac-delayed rectifiers HERG and KCNQ1. Effects of human MiRP2 on
Kv3.4 are pronounced in mammalian cells but not in Xenopus
oocytes (7). This interaction was not covered here but is currently
under study.
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Fig. 1.
An X. laevis MiRP
family is expressed in oocytes. A, sequence alignment of
Xenopus KCNE gene products and their human homologues shows
predicted transmembrane domains (underlined), 100%
conserved (*), and partially conserved residues (. :) using ClustalW
1.81 with default settings. B, unrooted dendrogram of
evolutionary relationships between Xenopus and mammalian
MiRP amino acid sequences using ClustalW 1.81 with default settings.
C, PCR of Xenopus MinK, MiRP2, MiRP4.1, and
MiRP4.2 cDNA after RT-PCR from oocyte mRNA. Arrow
indicates 500-bp marker position after electrophoresis on ethidium
bromide-stained 1% agarose gels.
30-mV tail pulse
because of rapid recovery from inactivation and slow deactivation. Co-injection of xMiRP2 cRNA with HERG in oocytes resulted in the complete suppression of HERG current (Fig.
2, A and B). The
results demonstrate that xMiRP2 functions like hMiRP2 to strongly
inhibit HERG current when it is overexpressed in Xenopus
oocytes. Overexpression of xMiRP2 with KCNQ1 in oocytes resulted in an
increase in the time- and voltage-independent component of KCNQ1
current as observed previously for human MiRP2 (Fig. 2, C
and D). Xenopus MiRP2 can thus modulate human
K+ channels when overexpressed in oocytes.
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Fig. 2.
Overexpressed xMiRP2 modulates HERG and KCNQ1
in oocytes. A, exemplar raw current traces generated from
TEVC of oocytes injected with 5 ng of HERG or co-injected with 3 ng of
xMiRP2 and 5 ng of HERG cRNA. Oocytes were held at 80 mV and stepped
to voltages between
120 and +60 mV, with a
30-mV tail pulse
(protocol inset). Dashed lines indicate zero
current level. B, mean tail current-voltage relationships
from traces as in panel A for HERG (solid
squares) or xMiRP2/HERG (open squares) channels in
oocytes, n = 8. Error bars indicate S.E.
C, exemplar raw current traces generated from TEVC of
oocytes injected with 10 ng of hKCNQ1 or co-injected with 3 ng of
xMiRP2 and 10 ng of hKCNQ1 cRNA. Dashed lines indicate zero
current level. Oocytes were held at
80 mV and stepped to voltages
between
120 and +60 mV, with a
30-mV tail pulse (protocol
inset). D, mean current-voltage relationships
from traces as in panel C, for hKCNQ1 (solid
squares) or xMiRP2/hKCNQ1 (open squares) channels in
oocytes (n = 9-12). Error bars (S.E.) are
smaller than point size.
subunits expressed in oocytes, we tested the applicability
in oocytes of the RNAi sequence-specific post-transcriptional silencing
technique that has been shown to suppress gene expression in a variety
of other cell types (30). A double-stranded xMiRP2-specific, siRNA oligo was injected into Xenopus oocytes, and the effects
were compared against those of a scrambled control siRNA oligo. To ensure that xMiRP2 siRNA was causing a loss of xMiRP2 mRNA, RNA was
isolated from oocytes injected with siRNA for xMiRP2, scrambled siRNA,
or cRNA for xMiRP2. RT-PCR to produce cDNA was followed by
normalization of samples to Xenopus
-actin cDNA
concentration (Fig. 3A,
top panel). Next, normalized cDNA samples were probed for xMiRP2 cDNA by PCR using sequence-specific primers (Fig.
3A, middle panel). Compared with batches of
oocytes injected with scrambled control siRNA, the band intensity of
amplified xMiRP2 cDNA from batches of oocytes injected with xMiRP2
siRNA was reduced 8-fold as assessed by densitometry (Fig.
3A, bottom panel). Injection of 3 ng of xMiRP2
cRNA, in contrast, increased the band intensity of amplified xMiRP2
cRNA 30-fold (n = two batches of 8-12 oocytes). To
assess the effects of xMiRP2 siRNA at the protein level, antibodies raised to full-length human KCNE3 and 80% similar to xMiRP2 in the
transmembrane and C-terminal domains were used to probe injected oocyte
lysates (Fig. 3B, upper panel). Probing the
oocyte lysates with anti-MiRP2 antibody gave a band at 15-18 kDa, in
the range expected for xMiRP2. Although xMiRP2 has one or more
predicted N-glycosylation sites, depending on the prediction
method used, only one band was visible here. We concluded therefore
that the large majority of xMiRP2 protein present in the oocyte at
baseline is in one particular glycosylation state, probably the mature form, as observed previously for mouse MiRP2 in the murine C2C12 skeletal muscle cell line (7). Similar levels of xMiRP2 protein were
detected in water-injected oocytes and oocytes injected with scrambled
control siRNA, whereas oocytes injected with xMiRP2 siRNA showed a
5-fold reduction of endogenous xMiRP2 protein as assessed by
densitometry (n = two batches of oocytes) (Fig.
3B, lower panel). Thus, RNAi can be used in
Xenopus oocytes to specifically suppress endogenous genes,
in this case xMiRP2.
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Fig. 3.
Sequence-specific RNAi efficiently suppresses
endogenous xMiRP2 in oocytes. A, semiquantitative PCR of
cDNA generated by RT-PCR of mRNA isolated from
Xenopus oocytes. Upper panel, Xenopus
mRNA normalized to endogenous -actin gene. Left to
right, oocytes injected with 500 pg of scrambled control
siRNA, 500 pg of xMiRP2 siRNA, and 3 ng of positive control xMiRP2
cRNA. Bands were generated by PCR using primers specific for
Xenopus
-actin and indicate equal amounts of
mRNA-generated cDNA loaded in each lane. Center
panel, PCR from cDNA generated from Xenopus oocyte
mRNA by RT-PCR and normalized to Xenopus
-actin (see
above). Left to right, oocytes injected with 500 pg of scrambled control siRNA (con siRNA), 500 pg of xMiRP2
siRNA (xM2 siRNA), or 3 ng of positive control xMiRP2 cRNA
(xM2 cRNA). Bands were generated by PCR using primers
specific for Xenopus MiRP2 and indicate specific
post-transcriptional gene silencing of xMiRP2 by xMiRP2 siRNA.
Lower panel, densitometry of xMiRP2 bands indicating an
8-fold reduction of xMiRP2 cDNA band intensity using xMiRP2 siRNA
and a 30-fold increase using MiRP2 cRNA. B, upper
panel, Western blot using KCNE3-specific antibodies from lysates
of Xenopus oocytes injected with (left to
right) water, 500 pg of xMiRP2 siRNA (xM2 siRNA),
of 500 pg of scrambled control siRNA (con siRNA). Protein
loaded per lane was normalized to total protein concentration (20 µg/lane) using the Bradford assay. Lines indicate 18 kDa
(upper) and 6.4 kDa (lower). Lower
panel, densitometry of xMiRP2 bands indicating a 5-fold reduction
of xMiRP2 protein concentration using xMiRP2 siRNA and no significant
reduction with scrambled control siRNA.
30
mV, a voltage at which contamination from other oocyte currents and
leaks is negligible. Varying amounts of HERG cRNA were injected into
oocytes in the absence or presence of xMiRP2-specific siRNA to silence
endogenous xMiRP2, and HERG tail currents were measured by TEVC after 3 days. At levels of HERG cRNA that normally expressed virtually zero
HERG tail current (as much as 0.7 ng), co-injection with xMiRP2 siRNA
rescued HERG currents to 500 nA as anticipated when an endogenous
protein that suppresses HERG is silenced (Fig.
4, A and B).
Indeed, xMiRP2 silencing increased HERG currents at all concentrations
tested below 5 ng of HERG (Fig. 4C). This effect was
specific because scrambled control siRNA had no effect (Fig.
4D). To eliminate the possibility that xMiRP2 siRNA oligos
increased HERG current because of an artifact via direct interaction
with HERG protein rather than by post-transcriptional xMiRP2 gene
silencing, we switched expression systems and assessed the effects of
xMiRP2 siRNA on HERG currents in CHO cells using a whole-cell voltage clamp. xMiRP2 siRNA should have no effect on HERG current density in
CHO cells, because even if CHO cells express endogenous hamster MiRP2,
xMiRP2 siRNA is sequence-specific for Xenopus MiRP2.
Transfection of cells with xMiRP2, siRNA, and HERG cDNA produced a
mean current density not significantly different from cells transfected
with HERG cDNA alone. This result argues against our previous
oocyte results being an artifact caused by direct interaction of xMiRP2 siRNA with HERG protein (Fig. 4E).
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Fig. 4.
Endogenous xMiRP2 inhibits heterologous HERG
current in oocytes. A, exemplar current families recorded
between 120 and +60 mV from oocytes injected with 0.7 ng of HERG cRNA
or co-injected with 0.7 ng of HERG cRNA and 500 pg of xMiRP2 siRNA
(xM2 siRNA). Dashed lines indicate zero current
level. Oocytes were held at
80 mV and then stepped to voltages
between
120 and +60 mV with a
30-mV tail pulse (protocol
inset). B, shown is the mean tail current/voltage
relationship from oocytes injected with 0.7 ng of HERG cRNA
(solid squares) or co-injected with 0.7 ng of HERG cRNA and
500 pg of xMiRP2 siRNA (open squares), protocol as in
A, n = 14-17. Error bars
indicate S.E. C, HERG cRNA dose response. Peak tail currents
from oocytes injected with various concentrations of HERG cRNA with or
without 500 pg of xMiRP2 siRNA using the protocol in A
(inset), n = 10-17 oocytes/point. Points
were joined arbitrarily with straight lines. D,
mean peak tail currents recorded using the protocol in A
from oocytes injected with 0.5 or 1 ng of HERG cRNA with or without 500 pg scrambled control siRNA (con siRNA), n = 7-11 per condition. No significant differences were observed between
current densities recorded on the same day (p > 0.05, unpaired Student's t test). Error bars indicate
S.E. E, mean peak tail current densities recorded using a
protocol similar to that shown in A (inset) from
CHO cells transfected with HERG cDNA or co-transfected with HERG
cDNA and xMiRP2 siRNA; n = 8-10. The current was
normalized to cell capacitance. No significant differences were
observed between current densities (p > 0.05, unpaired
Student's t test). Error bars indicate
S.E.
30-mV tail currents ~3-fold after 2 and 3 days of expression (Fig. 5B). Similar effects were observed with xMiRP2 siRNA
or a combination of hMiRP1 cRNA and xMiRP2 siRNA in the same batch of
oocytes (Fig. 5B). The results suggest that, paradoxically, because hMiRP1 is able to couple with HERG and prevent suppression by
endogenous levels of xMiRP2, at low HERG levels hMiRP1 increases HERG
currents despite the formation of channels with lower unitary conductance.
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Fig. 5.
Endogenous xMiRP2 mediates
density-dependent variation in modulation of HERG by human
MiRP1. A, left panel, mean tail current densities
recorded at 120 mV using TEVC of oocytes after injection with 10 ng
of HERG or 10 ng of HERG cRNA co-injected with 10 or 20 ng of hMiRP1
cRNA as indicated, n = 5-9. Error bars
indicate S.E.; asterisk for HERG-alone currents indicates a
significant difference from other currents (p < 0.05, unpaired Student's t test). Right panel, mean
tail current densities recorded at
30 mV using whole-cell patch clamp
of CHO cells co-transfected with 3 µg of HERG cDNA and either 2 µg of hMiRP1 cDNA or blank plasmid, with current normalized to
cell capacitance; n = 8-12. Error bars
indicate S.E., asterisk for HERG-alone currents indicates
significant difference from other currents (p < 0.05, unpaired Student's t test) B, mean tail
current densities recorded at
30 mV using TEVC of oocytes injected
with 0.5 ng of HERG cRNA alone or with either 2 ng of hMiRP1 or 500 pg
of xMiRP2 siRNA, or both; n = 5 oocytes (day 2) and 10 oocytes (day 3)/condition. Asterisks for HERG-alone currents
indicate significant differences from other currents on same day
(p < 0.01, unpaired Student's t
test).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit is expressed
endogenously in X. laevis oocytes and
up-regulated by the heterologous expression of mammalian MinK led to
the discovery that human cardiac IKs is formed
by MinK/KCNQ1 complexes in vivo (10). Here we demonstrate
that Xenopus oocytes also express relatives of mammalian MiRPs and that these endogenous xMiRPs can interact with injected mammalian Kv channel
subunits. HERG, like KCNQ1, is a
human cardiac Kv channel widely studied because of the role
it plays in cardiac repolarization and the propensity of HERG to
interact nonspecifically with a wide range of therapeutic agents, a
propensity that contributes to acquired cardiac arrhythmia (21,
32). The functional properties of HERG are modified by mammalian MinK, MiRP1, and MiRP2 (6, 9, 11, 14, 18, 29). We have demonstrated that
endogenous levels of xMiRP2 are sufficient to modify overexpressed HERG
in oocytes. HERG currents recorded after the injection of HERG cRNA
into oocytes are thus the sum product of "pure" HERG channels,
xMiRP2-suppressed HERG, and/or channels formed by HERG with other xMiRP
subunits. This fact can dramatically affect the interpretation of
previous and future studies on HERG gating, pharmacology, and
trafficking. We have yet to elucidate the mechanism by which xMiRP2
inhibits HERG, but this complete suppression suggests the formation of
a non-functional channel, perhaps one that fails even to reach the
plasma membrane. A question then remains to be answered. What happens
to MiRP2-sequestered HERG, and can it be rescued by stimulating
regulatory pathways or with use of drugs that bind to HERG and that
rescue HERG channels containing trafficking mutations associated with
long QT syndrome (33, 34)? If so, the association of endogenous xMiRP2
with HERG may impact previous and future oocyte-based studies of HERG regulation and pharmacological rescue. The hMiRP1 versus
xMiRP2 data presented here suggest that endogenous xMiRP2 forms the
molecular basis for some of the previously reported variability in
hMiRP1/HERG oocyte studies. The hMiRP1 versus xMiRP2
findings also highlight the importance of understanding the molecular
background of expression systems used in
-
subunit interaction
studies. We anticipate that this phenomenon (i.e. the
functional effects of an introduced mammalian
subunit resulting
partially from the displacement of an endogenous expression system
subunit) will prove to be a recurring theme once the full range of
Kv channel interactions with Xenopus and
mammalian MiRPs is explored in oocytes and in other cell types.
-subunit interaction is particularly contentious
when cardiac Kv channels are considered. Both HERG and KCNQ1, the
subunits that generate the two principal repolarization currents, IKr and IKs,
respectively, in human ventricular myocardium, are modulated by MinK,
MiRP1, and MiRP2 in at least one expression system (35). KCNQ1 is also
strongly suppressed by MiRP3 (KCNE4) (36) and forms with MiRP4 a slowly
activating channel that resembles MinK/KCNQ1 channels but with a much
more positive voltage dependence of activation (37). Little doubt
exists that MinK/KCNQ1 complexes form human cardiac
IKs. Compared with currents formed by
overexpression of KCNQ1 alone, MinK/KCNQ1 channels exhibit a 4-fold
higher conductance and a greatly slowed activation rate, generating a
current that strongly resembles IKs in terms of
biophysical attributes, regulation, and pharmacology. Further,
mutations in both subunits that diminish channel function are
associated with long QT syndrome in humans (19, 23, 24, 38-40). More
debate exists concerning the precise molecular correlate of human
IKr. Although the introduction of HERG
subunits into oocytes or immortal mammalian cell lines creates a
potassium current with many of the attributes of cardiac IKr (21, 32), coexpression with human MiRP1
alters the single channel properties of HERG to produce a current more
closely resembling previous in vivo recordings of
IKr (6). Mutations in hMiRP1 diminish flux
through hMiRP1/HERG channels and are associated with inherited long QT
syndrome (6, 17). Further, some hMiRP1 mutations increase sensitivity
to the blockade of hMiRP1/HERG channels in vitro by drugs
known to block IKr (e.g.
clarithromycin). Importantly, in each case the same drug
(e.g. sulfamethoxazole) had precipitated drug-induced
torsades de pointes and a prolonged QTc interval,
specifically in patients from whom each mutation was isolated (6, 15).
However, largely as a result of the reported wide variability in
effects of MiRP1/HERG coexpression, the assignment of this combination
of subunits as the molecular basis of human IKr
is still questioned. Here we have noted one cause of the reported
variability in hMiRP1/HERG behavior: the dependence of hMiRP1
effects on HERG expression level because of interference from
endogenous MiRPs such as xMiRP2. At low levels of HERG cRNA, HERG
current is completely suppressed by endogenous xMiRP2. The fact that we
were able to subsequently reconstitute HERG current by either silencing
the expression of xMiRP2 or introducing an ostensibly preferred partner
illustrates just how promiscuous the association between an
subunit
and the MiRPs can be. Can we infer from this that HERG will favor in
every cell type an interaction with MiRP1 over other
subunits?
Enough evidence does not exist at this time to support such a claim.
However, our findings in conjunction with those from other studies
highlight the important idea that native currents such as
IKr are probably formed from a spectrum of
molecular components with temporal and spatial dependence, and thus the
success of direct native-cloned current comparisons is governed not
only by the choice of expression system but also by the inclusion of
possible
subunit partners and by the precise choice of native
tissue source. Increasingly, for example, evidence suggests that
IKr is regulated by MinK in some cardiac
myocytes: MinK up-regulates HERG in some mammalian cell lines,
knockdown of MinK suppresses HERG in murine myocytes, and
immunoprecipitation of MinK from horse heart pulls down HERG, and KCNQ1
(25, 29, 41, 42).
subunit complexes. This knowledge will
facilitate the linkage of channel
and
subunit genes to inherited and acquired disease, the rational design of channel-directed therapeutic drugs, and screening for avoidance of unwanted channel-drug interactions. Consideration of endogenous modifying subunits in heterologous expression systems is likely to be most crucial when undertaking single channel recordings, because the lower
subunit expression levels often required for these studies (when compared with
macroscopic studies) will generate a higher molar ratio of endogenous
subunits to exogenous
subunits. The impact of endogenous xMiRPs
on macroscopic studies should not be overlooked, however, because our
results here show that endogenous xMiRP2 affects the behavior of HERG,
for example, at tail current densities in the range widely used by
investigators of whole-cell HERG biophysics, pharmacology, and
intersubunit interactions (6, 11, 43, 44).
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ACKNOWLEDGEMENT |
---|
We are grateful to D. Christini for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Greenberg Atrial Fibrillation Fund and the American Heart Association (to G. W. A. and Z. A. M.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF545500, AF545501, and AF545502, for X. laevis MinK, MiRP2, and MiRP4.1, respectively.
These authors contributed equally to this work.
** To whom correspondence should be addressed: Weill Medical College of Cornell University, 520 E. 70th St., Starr 463, New York, NY 10021. Tel.: 212-746-6275; Fax: 212-746-7984; E-mail: gwa2001@med.cornell.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212751200
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ABBREVIATIONS |
---|
The abbreviations used are: Kv channel, voltage-gated potassium channel; MiRP, MinK-related peptide; hMiRP, human MiRP; CHO, Chinese hamster ovary; RNAi, RNA interference; siRNA, short interfering RNA; TEVC, two-electrode voltage clamp; IKs, slow activating potassium current; IKr, rapidly activating potassium current; Ito, transient outward current; RT, reverse transcriptase; EST, expressed sequence tag; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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