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1.2 interactions and their
effects on cardiac Kv channel expression
1 The Rammelkamp Center for Education and Research, MetroHealth Campus, and Departments of 2 Physiology and Biophysics and 3 Biochemistry, Case Western Reserve University, Cleveland, Ohio 44109
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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KChAP and voltage-dependent K+ (Kv)
-subunits are two different types of cytoplasmic proteins that
interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and
Kv4.3 channels. It also binds to Kv1.x channels but, with the exception
of Kv1.3, does not increase Kv1.x currents. Kv-subunits are
assembled with Kv1.x channels; they exhibit "chaperone-like"
behavior and change gating properties. In addition, KChAP and
Kv-subunits interact with each other. Here we examine the
consequences of this interaction on Kv currents in Xenopus
oocytes injected with different combinations of cRNAs, including
Kv1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found
that KChAP attenuated the depression of Kv1.5 currents produced by
Kv1.2, and Kv1.2 eliminated the increase of Kv2.1 and Kv4.3
currents produced by KChAP. Both KChAP and Kv1.2 are expressed in
cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward
currents and Kv4.3 and Kv1.4 generate transient outward currents.
Because they interact, either KChAP or Kv1.2 may alter both
sustained and transient cardiac Kv currents. The interaction of these
two different classes of modulatory proteins may constitute a novel
mechanism for regulating cardiac K+ currents.
chaperone; modulation; potassium channels; voltage-gated potassium 1.4 channel; voltage-gated potassium 1.5 channel; voltage-gated potassium 2.1 channel; voltage-gated potassium 4.3 channel
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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KCHAP (11,
24) and Kv-subunits (5, 7, 27) are two types of
cytoplasmic proteins known to interact with and alter processing of
voltage-dependent K+ (Kv) channels. Kv-subunits interact
with members of the Kv1.x subfamily (10, 13, 14, 18), with
Kv2.2 (11) and Kv4.x (16) channels, whereas
KChAP in addition to Kv1.x interacts with Kv2.1 and Kv4.3 channels
(12, 25). Kv-subunits and KChAP differ in how they
affect Kv currents, however. First, Kv-subunits coassemble with
Kv
-subunits to form stable Kv/ heterooligomeric channels
(16, 20, 21), whereas KChAP interacts with Kv-subunits only transiently (12, 25). Second, Kv-subunits enhance
the trafficking of Kv channels to the plasma membrane without affecting whole cell Kv-subunit protein levels (17, 21), whereas
KChAP increases Kv-subunit protein levels (12, 25).
Third, Kv-subunits change Kv1.x channel gating by virtue of their
tight association with mature channels (1, 10, 18),
whereas KChAP has no effect on gating (12, 25). Thus KChAP
manifests true chaperone behavior while Kv-subunits exhibit
pseudochaperone behavior that more closely resembles that of other
coassembled, accessory K+ channel subunits such as minK
with KvLQT1 (4, 19, 23) or the -subunit with the
Ca2+-activated maxi-K+ channel
(22).
Not only do KChAP and Kv-subunits interact with Kv channels, they
also interact with each other. In fact, KChAP was discovered in yeast
two-hybrid experiments using Kv1.2 as bait (25). This interaction led us to propose that interactions between KChAP and
Kv-subunits might significantly alter membrane currents in cells
expressing Kv1.x, Kv2.1, or Kv4.3, such as cardiomyocytes. Kv4.x and
Kv1.4 contribute to the transient outward current
(ITo) in heart, and the differential expression
of these channels between endocardium and epicardium is thought to be
critical for the timing and directionality of ventricular
repolarization. In a previous paper, we showed that KChAP interacted
with Kv2.1 and Kv4.3 in rat heart (12). Here we report
that Kv1.2 is expressed in rat and human heart and that complexes
between Kv1.2 and Kv1.2, Kv1.4, and Kv1.5 are detected. Having shown
that all of the participants are present in heart, we tested our
hypothesis in a model cell system using heterologous expression in
Xenopus oocytes. First, we found that KChAP did not act as a
chaperone for Kv1.2 in that levels were unaltered in the presence of
KChAP. However, KChAP did prevent the effects of Kv1.2 on expression
and gating of its target Kv1.x channels. Conversely, Kv1.2 prevented
the chaperone effects of KChAP on Kv2.1 and Kv4.3. Thus KChAP may
increase K+ currents directly by its chaperone effects on
Kv2.1 and Kv4.3 and indirectly by suppressing the block of Kv1.x
channels by Kv1.2. Conversely, Kv-subunits may decrease
K+ currents directly by block of Kv1.x channels or
indirectly by suppressing the chaperone effect of KChAP on Kv2.1 and
Kv4.3. Such mechanisms would provide an additional level of control
over Kv currents and could be controlled by the relative levels of distinct but interacting modulatory proteins.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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mRNA Extraction and Northern Blot Analysis
mRNA from rat heart and brain was isolated using the Poly(A)Pure kit (Ambion) following the manufacturer's instructions. Poly(A)+ RNA (2 µg/lane) was electrophoresed on a 1.0% agarose/formaldehyde gel and transferred to Bright-Star membranes (Ambion). The blot was hybridized to a 32P-labeled riboprobe spanning the region encoding the NH2-terminal 79 residues of human Kv1.2. A T7 promoter sequence was engineered directly onto the end of the coding fragment using the Lig'nScribe kit
from Ambion and the riboprobe synthesized with the Strip-EZ T7 kit
(Ambion). The blot was hybridized with probe (106
counts · min
1 · ml1)
overnight at 44°C in ULTRAhyb buffer (Ambion), washed at 50°C in
0.1× saline-sodium citrate (SSC)-0.1% SDS, and exposed to BioMax MS
film (Kodak) at 
70°C with an intensifying screen.
PCR Amplification of Rat Heart Kv Channel
-Subunit cDNA Fragments
-subunits by RT-PCR. Primer sequences and locations in published cDNA sequences in GenBank
of the National Center for Biotechnology Information were as follows:
for Kv1.1 (accession number X70662), 5'-ATG CAA GTC TCC ATA GCC TGC
ACA GAG C-3' (sense primer; nt 332-359) and 5'-GTC ATC AGC CGT TCA
GCA ACC TCA TCT G-3' (antisense primer; nt 627-654); Kv1.3,
5'-GAC AAC AGC AAG TTT AGA AAG CAG TC-3' (sense primer) and 5'-GTC ATC
AGC CGT TCA GCA ACC TCA TCT G-3' (antisense primer); Kv2 (accession
number X76724), 5'-ATC TAC AGT ACT CGG TAT GGG AGT C-3' (sense primer;
nt 662-686) and 5'-GCC TCC ATG ATC TCC ATG GAG C-3' (antisense
primer; nt 1163-1185); Kv3 (accession number X76723), 5'-ATG
CAG GTG TCT ATC GCG TGT AC-3' (sense primer; nt 388-410) and
5'-AAG CCT CGC TCA GTT TCT GCC-3' (antisense primer; nt 881-902).
RT-PCR.
Poly(A)+ mRNA (100 ng) was denatured at 70°C for 5 min
and reverse-transcribed into first-strand cDNA by priming with random hexamers (First-Strand cDNA Synthesis Kit; Perkin-Elmer). PCR was
performed with the Advantage PCR System (Clontech) using the following
cycling program: one cycle (2 min at 95°C); 35 cycles (15 s at
94°C, 15 s at 50°C, 1 min at 68°C); one cycle (10 min at
72°C). PCR products were resolved by electrophoresis on 1.2% agarose
gels and transferred to BrightStar-Plus membranes (Ambion). Channel-specific PCR products were identified by hybridization to the
following 32P-labeled internal oligonucleotides specific
for either the Kv1 subfamily (5'-GTT TCG TGC TTG GGT CTT GGA ACA
TGG-3'), Kv2 (5'-GAG GCC AGA TCA CAG ATG AG-3'; nt 774-793), or
Kv3 (5'-GGC TCT CAG ATC TCA GAT GAG ACA G-3'; nt 691-715). The
blots were washed with 0.1× SSC-0.1% SDS at 44°C and exposed to
BioMax MS film (Kodak) at 70°C with an intensifying screen.
Antibodies
An anti-Kv1.2 antibody was raised to a fusion protein
consisting of the unique NH2 terminus (residues 1-79)
of human Kv1.2, which was PCR amplified and cloned in-frame into an
expression vector (pET-15b) encoding six histidine tags on the
NH2 terminus. The fusion protein was purified by metal
chelation chromatography using the His-Bind purification kit (Novagen)
and injected into rabbits (Research Genetics). Antisera was affinity
purified on a fusion protein-Sepharose column. An antibody to human
Kv1.5 (COOH-terminal residues D528-L613) was prepared by creating
a maltose-binding fusion protein (New England Biolabs). Polyclonal antisera from rabbits was affinity purified on a fusion-protein Sepharose column. Monoclonal antibodies against Kv1.2 and Kv1.4 were
obtained from Upstate Biotechnology. The monoclonal anti-green fluorescent protein used for Western blotting was from Clontech (no.
8362-1). The polyclonal anti-KChAP antibody was previously described (12).
Tissue Lysate and Membrane Preparation
Rat tissue lysates were prepared from freshly dissected organs. Heart, kidney, and skeletal muscle were minced and placed in ice-cold lysis or immunoprecipitation (IP) buffer (1:7 wt/vol) containing 150 mM NaCl, 50 mM Tris · HCl, 1 mM EDTA, 0.2% BSA, and 1% Triton X-100, pH 7.5, supplemented with a protease and phosphatase inhibitor cocktail (Complete; 50 mM sodium fluoride-0.2 mM sodium vanadate; Roche). Samples were homogenized with a Polytron at setting 6 for 5 s. All of the other tissue samples were homogenized in a Teflon-glass homogenizer (1:7 wt/vol) in the same lysis buffer. After 1 h of incubation on ice, the lysates were centrifuged at 3,000 g for 10 min to remove insoluble material. Human atrial appendage tissue was pooled from adult patients undergoing aortocoronary bypass surgery. A sample of human left ventricle was obtained from an explanted heart at transplant.To isolate crude cellular membranes, rat tissues were homogenized in 0.3 M sucrose and 10 mM phosphate, pH 7.4, supplemented with a protease and phosphatase inhibitor cocktail as above. After nuclei and debris were removed by centrifugation at 3,000 g for 10 min, the supernatant was centrifuged at 50,000 g for 1 h to pellet membranes. Protein concentrations were determined by the bicinchoninic acid method (Pierce). IP and immunoblotting were performed as previously described (12).
Constructs and Yeast Two-Hybrid Assay
Full-length KChAP (residues 1-619) and the KChAP NH2-terminal fragment (KChAP-N; residues 46-354), the middle fragment of KChAP (KChAP-M; residues 355-452), and the COOH-terminal fragment (KChAP-C; residues 453-619) in the GAL4 yeast two-hybrid vector pGAD424 were used as described (11). Kv1.2 in pGBT9 was tested for interaction
with individual KChAP fragments by cotransformation of host strain
Y190 as previously described (25).
Transfection
COS-1 cells were plated to ~70% confluence in 60-mm dishes 1 day before transfection. Cotransfection of either enhanced green fluorescent protein (EGFP)-C2 or KChAP-M/EGFP-C2 with Kv1.2/pCR3 was
achieved with FuGene (Roche) using 1 µg of each DNA plus 6 µl FuGene/60-mm dish. As a control, Kv1.2/pCR3 was
substituted with pcDNA3 in some experiments. At 24 h
posttransfection, cells were lysed in IP buffer (as described above).
For IP, equal amounts of protein (150 µg/500 µl) were incubated
overnight with or without anti-Kv1.2 antibody (1:200 dilution).
Expression in Xenopus Oocytes and Electrophysiology
cRNAs for expression in Xenopus oocytes were prepared using the mMESSAGE mMACHINE kit (Ambion). Plasmids encoding Kv1.2
and Kv1.5 were previously described (1) as were KChAP,
Kv2.1, and Kv4.3 (12). cRNAs were injected as previously
described (24).
Measurement of Xenopus oocyte whole cell current was
performed using the standard two-microelectrode voltage-clamp
technique. Bath solution contained (in mM) 5 KOH, 100 NaOH, 0.5 CaCl2, 2 MgCl2, 100 methanesulfonic acid, and
10 HEPES (pH 7.4). Electrodes were filled with 3 M KCl and had a
resistance of 0.3-0.6 M
. All recordings were made at room
temperature. Linear leakage and capacity transient currents were
substracted (P/4 prepulse protocol) unless specified, and data were
low-pass filtered at 1 kHz. pCLAMP software (Axon Instruments) was used
for voltage-pulse protocols and data acquisition. Data are reported as
means ± SE. Comparisons among multiple groups of oocytes were
performed by one-way ANOVA, Student's t-test, and the
Student-Newman-Keuls post hoc test. Means are considered to be
significantly different at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The molecules of interest to us in rat cardiomyocytes are KChAP,
Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.3, and the particular Kvs
represented in these cells. We have already shown that KChAP interacts
with Kv2.1 and Kv4.3 in rat heart (12). The expression of
Kv1.2, Kv1.4, and Kv1.5, and Kv2.1 and Kv4.3 in rat heart has been
reported elsewhere (8, 9, 26). Kv1.2 was cloned from
human heart (10, 13), but the complement of Kvs in rat heart is unknown.
Kv-Subunit Expression in Rat Heart
-subunits interact in heart,
we had to first identify which Kv-subunits are expressed in rat
heart. Kv1.2 was examined first, since it had been cloned previously
from human heart (10, 13), and its expression was suggested by RT-PCR from rat heart (14). To confirm its
expression in adult rat heart, we did Northern blot analysis and
compared its expression with adult rat brain. As shown in Fig.
1A, both heart and brain
exhibited a single band of ~5 kb that hybridized with a Kv1.2-
specific riboprobe. Expression of the other Kv-subunit mRNAs was
examined by RT-PCR using Kv1.1-, Kv1.3-, Kv2-, or Kv3-specific primers and rat heart and brain mRNA. Of these four Kv-subunits, only Kv1.1 mRNA was detected in rat heart and brain (Fig. 1B). Transcripts for Kv1.3, Kv2, and Kv3 were
not found in rat heart, although strong signals were detected in brain.
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Given the expression of Kv1.2 message in both rat and human heart,
we raised a polyclonal antibody to the unique NH2 terminus of Kv1.2 to examine protein levels in native tissue.
Affinity-purified anti-Kv1.2 antisera detected a single band of
~45 kDa in lysates of oocytes injected with Kv1.2 cRNA, consistent
with the predicted molecular mass of Kv1.2 (Fig. 1C,
lane 2). In adult rat tissue lysates (Fig. 1E),
anti-Kv1.2 antibody detected a band of ~45 kDa in skeletal muscle
(lane 4), atrium (lane 6), and ventricle (lane 7). In lung (lane 2) and kidney (lane
5) lysates, the antibody labeled a faint protein band of slightly
lower mobility, ~47 kDa. No signal was obtained from rat liver
(lane 3) or brain (lane 1) lysates. The lack of
Kv1.2 reactivity in brain was surprising, since Kv1.2 message was
detected by Northern blotting (Fig. 1A). Because
Kv-subunits are Kv1.x channel-associated proteins, we examined the
expression of Kv1.2 in enriched membrane fractions from rat tissues
(Fig. 1D). As shown in Fig. 1E, Kv1.2 was
detected in membranes from rat brain (lane 2), but at a much
lower level than in heart (lane 1) or skeletal muscle
(lane 3). In human heart, Kv1.2 was detected at
equivalent levels in the human atrial appendage (Fig. 1E,
lane 8) and ventricle (lane 9).
Kv1.2 Coimmunoprecipitates with
Kv1-Subunits in Rat Heart
1.2, we performed
coimmunoprecipitation experiments. In Fig.
2A, a membrane fraction from 3-day postnatal rat heart was solubilized with 1% Triton X-100 buffer,
immunoprecipitated with anti-Kv1.2, and Western blotted with
anti-Kv1.4. Kv1.4 was detected as a double band of 90 and 110 kDa in
adult rat brain (Fig. 2A, left) and 3-day
postnatal rat heart (Fig. 2A, right) membranes.
Both bands were also seen in complexes immunoprecipitated with
anti-Kv1.2. Figure 2B shows that Kv1.2 was also
immunoprecipitated with Kv1.2, in this case from adult rat heart
lysates. Anti-Kv1.2 detected a prominent band at ~76-kDa in both the
lysate and immunocomplexes. Because the anti-Kv1.5 antibody that we
raised recognizes the human channel much better than rat, we examined
association of Kv1.5 with Kv1.2 from the human atrial appendage. In
lysates from human atrial appendage, we observed a major band of ~70
kDa, which was also present in immunocomplexes with Kv1.2 (Fig.
2C). Thus Kv1.2 was found in association with all three
Kv1.x subunits that have been shown to be expressed in either rat or
human heart, Kv1.2, Kv1.4, and Kv1.5. By contrast, we were never able
to identify two other cardiac Kv channels, Kv2.1 or Kv4.3, in
immunocomplexes with Kv1.2 (data not shown).
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Kv1.2 Binds to the KChAP-M
Fragment
1.2 association with a subset of cardiac
Kv channels, we examined the interaction of Kv1.2 with KChAP. In the
yeast two-hybrid system, we showed that KChAP binds to the conserved
Kv1 COOH-terminus of Kv1.2, the same fragment to which the Kv1
NH2-terminus binds (25). To determine whether Kv1.2 interacts with KChAP in myocardium, we performed
coimmunoprecipitation experiments with anti-Kv1.2 and blotted with
anti-KChAP. We were unable to detect KChAP reproducibly in these
experiments, suggesting a weak or very transient interaction.
In a previous study, the Kv-subunit binding fragment of KChAP was
localized to a stretch of 98 amino acids in the middle of the protein,
which we called KChAP-M (12). To determine if KChAP-M also
interacted with Kv1.2, we used yeast two-hybrid assays. As shown in
Fig. 3, A and B, we
found that KChAP-M did interact with Kv1.2, but, just as was
observed with Kv NH2-termini, there was no evidence of
interaction between the other KChAP fragments (N and C) and Kv1.2.
This interaction was confirmed using coimmunoprecipitation experiments
from cotransiently transfected COS-1 cells. EGFP-tagged KChAP-M was
found in association with Kv1.2 (Fig. 3C,
left). As a control for nonspecific binding of EGFP-KChAP-M
to the secondary antibody-coated beads, we did the same experiments
with lysates from cells transfected with only EGFP-KChAP-M and no
Kv1.2 and did not detect any KChAP-M attached to the beads (Fig.
3C, middle). In a second control experiment, we
saw that EGFP did not coimmunoprecipitate with Kv1.2 (Fig.
3C, right). Thus binding sites for Kv
NH2 termini and Kv-subunits are located within the same
98-amino acid fragment of KChAP.
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KChAP Does Not Increase Kv1.2
Protein Levels
1.2, we examined steady-state protein levels after
coexpression of the two proteins in Xenopus oocytes. As shown in Fig. 3D, the amount of Kv1.2 was not changed
when Kv1.2 was either expressed alone (lane 2) or with an
equimolar ratio of KChAP cRNA (lane 3). Conversely, Kv1.2
did not alter KChAP levels either (Fig. 3E). Note the
equivalent KChAP protein levels in oocytes injected with KChAP alone
(lane 2) or with an equimolar ratio of Kv1.2 cRNA
(lane 3).
Functional Consequences of KChAP-Kv1.2
Interactions on Kv Channel Expression
1.2 bind to the NH2 termini of
Kv1.2, Kv1.4, and Kv1.5; Kv1.2 alters processing and can introduce
or accelerate open-channel block of Kv1.x subunits (10, 13,
21) while KChAP produces no obvious alteration in either
parameter (12, 25). Because both proteins bind to the
NH2 termini of Kv1.x subunits and to each other, we
hypothesized that KChAP may indirectly affect Kv1.x channel
expression through the modulation of Kv1.2/Kv1.x subunit
interactions. We tested this hypothesis by heterologous expression of
KChAP with the cardiac channel Kv1.5 and the COOH-terminus of Kv1.2
in Xenopus oocytes. We showed previously that the Kv1 COOH terminus (Kv1-C) produced a significant decrease in the expression of Kv1.5 currents (1) and have used this
construct rather than full-length Kv1.2 to analyze effects in the
absence of the fast inactivation introduced by the -ball. Kv1.5 cRNA alone, Kv1.5, and Kv1-C cRNAs or Kv1.5, Kv1-C and KChAP cRNAs were injected in Xenopus oocytes. As shown in Fig.
4, injection of Kv1-C produced
dramatic suppression of Kv1.5 current. When KChAP cRNA was added to the
injectate, we observed a significant recovery in Kv1.5 current. Five
days after injection, the steady-state currents at +70 mV potential
were 1.14 ± 0.03, 0.20 ± 0.03, and 0.61 ± 0.08 µA
for Kv1.5 alone, Kv1.5 plus Kv1-C, and Kv1.5 plus Kv1-C and
KChAP, respectively (n = 10 for each group). Averaged whole cell current traces are shown in Fig. 4A. The
disinhibition by KChAP was concentration dependent (Fig.
4B). As we reported previously, coexpression of Kv1.5 with
Kv1-C (1) or with KChAP (25) had no
effects on kinetics and voltage dependence of the Kv1.5 current.
Simultaneous coexpression of Kv1-C and KChAP with Kv1.5 also did not
affect kinetics of the Kv1.5 current (Fig. 4C). Time
constants of activation were 6.64 ± 0.29, 6.72 ± 0.38, and
6.92 ± 0.28 ms for Kv1.5 alone, Kv1.5 with Kv1-C, and Kv1.5 with Kv1-C plus KChAP, respectively, and current-voltage
relationships were scaled.
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In contrast to Kv1.5, Kv1.4 current amplitude was increased by Kv1-C
or Kv2 (1, 17). This increase was suppressed by coinjection with KChAP (Fig. 5). On the
second day after injection, oocytes coinjected with Kv1.4 and Kv2
expressed current that was two to three times larger than in oocytes
injected with Kv1.4 alone. Addition of KChAP cRNA to the injectate
suppressed the stimulatory effect (Fig. 5A). At a test
potential of +70 mV, averaged peak currents were 1.9 ± 0.2, 5.2 ± 0.4, and 2.8 ± 0.3 µA for oocytes injected with
Kv1.4 alone, Kv1.4 plus Kv2, and Kv1.4 plus Kv2 and KChAP,
respectively (n = 8-10 for each group).
Additionally, KChAP attenuated the acceleration of inactivation of
Kv1.4 current produced by Kv2. Time constants of the inactivation
were 15.02 ± 0.73, 12.31 ± 0.53, and 14.11 ± 0.49 ms
for oocytes injected with Kv1.4 alone, Kv1.4 plus Kv2, and Kv1.4
plus Kv2 and KChAP, respectively (Fig. 5B).
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We hypothesized that Kv1.2, although it does not interact directly
with Kv2.1 or Kv4.3, might suppress the stimulatory effect of KChAP
Kv2.1 or Kv4.3 currents. As we have described previously (25) and as replicated in this experiment, KChAP produced
a twofold increase of Kv2.1 current (Fig.
6 A and B). When
increasing concentrations of Kv1.2 cRNA were added, a dose-dependent
reduction in the effect of KChAP on Kv2.1 was seen. At a 2:1 ratio of
Kv1.2 to KChAP cRNA, the expression enhancement of Kv2.1 currents by KChAP was eliminated. Coexpression of Kv1.2 cRNA alone or in combination with KChAP produces no changes in voltage dependence or
kinetics of Kv2.1 currents (Fig. 6C). For Kv2.1 alone and
Kv2.1 coinjected with KChAP and Kv1.2, time constants of activation were 17.1 ± 0.2 (n = 10) and 17.4 ± 0.2 (n = 10) ms, respectively.
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Similar results were obtained when KChAP and Kv4.3 were coexpressed in
the presence of Kv1.2 (Fig. 7). KChAP
also produced an approximate twofold increase in Kv4.3 currents that
was abolished when an excess of Kv1.2 cRNA was coinjected. By
itself, Kv1.2 had no effect on Kv4.3 current amplitude or kinetics.
On the second day after injection, average peak currents at +70 mV were
1.38 ± 0.11, 4.01 ± 0.24, 2.34 ± 0.16, and 1.50 ± 0.13 µA for oocytes injected with Kv4.3 cRNA alone, Kv4.3 plus
KChAP cRNAs, Kv4.3 plus KChAP and Kv1.2 cRNAs, and Kv4.3 plus
Kv1.2 cRNAs, respectively (n = 8-12). Time
constants of inactivation for Kv4.3 alone, Kv4.3 coinjected with
Kv1.2, and Kv4.3 coinjected with KChAP and Kv1.2 were
47.2 ± 1.0, 47.3 ± 0.7, and 48.3 ± 0.3 ms,
respectively (n = 5). Because Kv1.2 does not bind to
Kv2 or Kv4 channels, these results are consistent with the
interpretation that Kv1.2 binds to KChAP and prevents its
association with the channel.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The modulation of Kv channels by accessory proteins is an
expanding area of research. Here we report that two distinct modulatory subunits may interact with each other to regulate Kv channels. The
interactions between KChAP and Kv-subunits may have important consequences for the properties, i.e., expression level and gating, of
Kv channels. Results from this model system predict that controlling the relative levels of interacting modulatory subunits within a single
cell can constitute a novel mechanism for modulating Kv currents.
Kv-subunits and KChAP are representative of the two functionally
distinct classes of cytoplasmic Kv channel modulatory proteins that
have been described to date. The first class includes those subunits
that bind tightly to the NH2 termini of Kv-subunits, exist as part of the mature cell-surface channel complexes, and alter
channel gating and/or expression. These include the Kv-subunits, which are specific for Kv1 channels (27), and the
recently described KChIP family, which binds only to Kv4 subunits
(3). KChAP belongs to a second class of modulators and
acts as a chaperone to increase a subset of Kv currents. KChAP
differs from Kv-subunits or KChIP in that it interacts only
transiently with the NH2 termini of Kv-subunits, thus
behaving more as a true chaperone (12, 25). The functional
relationship between KChAP and KChIPs is not known. It will be
interesting to determine whether these two subunits can compete for
binding sites on Kv4 channels to produce altered currents.
The present experiments show for the first time at the protein level
that Kv1.2 is expressed in both rat and human heart and that it
coimmunoprecipitates with cardiac Kv1.2, Kv1.4, and Kv1.5. The
association of Kv1.2 with the very slowly inactivating Kv1.2 and
Kv1.5 proteins in cardiomyocytes could result in NH2-type inactivation and raises the possibility that Ito
in these cells is in part a result of these associations.
We were unable to demonstrate an association between Kv1.2 and
KChAP in rat heart by coimmunoprecipitation. This result was consistent
with a lack of a detectable interaction between KChAP and Kv1.2
overexpressed in Xenopus oocytes and may reflect the transient nature of the interaction. However, KChAP-M, the middle 98-residue fragment that contains the Kv-NH2-terminal
binding sites, was found to interact with Kv1.2 by both yeast
two-hybrid assays and coimmunoprecipitation from transfected cells.
Interestingly, this is the same fragment of KChAP that binds to the
NH2 terminus of Kv-subunits. Competition for the same
binding site on KChAP could explain the interference of Kv1.2 with
the enhancement of the functional expression of Kv2.1 and Kv4.3 by
KChAP. There is no evidence that Kv-subunits bind directly to Kv2 or
Kv4 NH2 termini, so it is likely that Kv1.2 binding to
KChAP would prevent its association with these channels. Because KChAP
coimmunoprecipitates with Kv2.1 and Kv4.3 in rat cardiomyocytes, it is
possible that KChAP modulates these currents directly via its chaperone
effect and that Kv1.2 modulates them indirectly via interactions
with KChAP.
The situation with KChAP, Kv-subunit, and Kv-subunit is more
complex because Kv-subunits can also bind to the NH2
terminus of Kv1 subunits. At this point, we are not able to
distinguish whether the interference of KChAP on the modulation of
Kv1.5 and Kv1.4 by Kv-subunits is a result of 1) KChAP
binding to Kv and preventing its association with Kv1.x or
2) KChAP binding to the Kv1.x NH2 terminus and
preventing Kv binding. The latter explanation would give some
functional significance to the interactions between Kv1 subunits and
KChAP, since, with the exception of Kv1.3, KChAP does not act as a
chaperone for Kv1.x subunits.
Both Kv-subunits and KChAP are thought to interact with
Kv-subunits at early stages in channel synthesis or assembly.
Kv-subunits have been shown to assemble with Kv1 subunits while
the channels are in the endoplasmic reticulum (15, 21).
This complex promotes trafficking of the channels to the cell surface
(17). We have evidence from in vitro translation
experiments to suggest that KChAP interacts with Kv-subunits
cotranslationally (25). Furthermore, for KChAP to act
as a chaperone for Kv-subunits in oocytes, the two cRNAs must be
injected in the same pipette (25). Because the timing of
interaction of both types of subunits with Kv-subunits during
processing may be comparable, it is reasonable to assume that
preventing the initial interactions of the channel with the modulatory
subunit would eliminate the influence of this subunit entirely. It is
likely then that the extent to which different modulatory subunits
might affect Kv currents would be dependent on their levels relative to
other binding partners. For KChAP, this is especially intriguing, since
it belongs to a family of proteins known to interact with and modulate
the activity of certain transcription factors such as STAT3
(6). Regulating the availability of auxiliary subunits for
channel binding might constitute a system in which channel expression
could be rapidly altered without changing channel message levels. In
the future, experiments addressing these possibilities will be targeted
directly in native cardiomyocytes that express all of the component molecules.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. W. Dong for technical assistance with the oocytes.
| |
FOOTNOTES |
|---|
This work was supported by National Institutes of Health Grants HL-55404, HL-36930, and NS-23877 (to A. M. Brown), HL-60759 (to B. A. Wible), and HL-61436 (to D. L. Kunze for support of A. N. Ramirez).
Address for reprint requests and other correspondence: A. M. Brown, Rm. 301, Rammelkamp Center, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109-1998 (E-mail: abrown{at}metrohealth.org).
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.
Received 1 July 2000; accepted in final form 13 February 2001.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Accili, EA,
Kiehn J,
Yang Q,
Wang Z,
Brown AM,
and
Wible BA.
Separable Kvbeta subunit domains alter expression and gating of potassium channels.
J Biol Chem
272:
25824-25831,
1997
2.
Accili, EA,
Kuryshev YA,
Wible BA,
and
Brown AM.
Separable effects of human Kvbeta1.2 N- and C-termini on inactivation and expression of human Kv14.
J Physiol (Lond)
512:
325-336,
1998
3.
An, WF,
Bowlby MR,
Betty M,
Cao J,
Ling HP,
Mendoza G,
Hinson JW,
Mattsson KI,
Strassle BW,
Trimmer JS,
and
Rhodes KJ.
Modulation of A-type potassium channels by a family of calcium sensors.
Nature
403:
553-556,
2000[Medline].
4.
Barhanin, J,
Lesage F,
Guillemare E,
Fink M,
Lazdunski M,
and
Romey G.
K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current.
Nature
384:
78-80,
1996[Web of Science][Medline].
5.
Barry, DM,
and
Nerbonne JM.
Myocardial potassium channels: electrophysiological and molecular diversity.
Annu Rev Physiol
58:
363-394,
1996[Web of Science][Medline].
6.
Chung, CD,
Liao J,
Liu B,
Rao X,
Jay P,
Berta P,
and
Shuai K.
Specific inhibition of Stat3 signal transduction by PIAS3.
Science
278:
1803-1805,
1997
7.
Deal, KK,
England SK,
and
Tamkun MM.
Molecular physiology of cardiac potassium channels.
Physiol Rev
1:
49-67,
1996.
8.
Dixon, JE,
and
McKinnon D.
Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats.
Circ Res
75:
252-260,
1994
9.
Dixon, JE,
Shi W,
Wang HS,
McDonald C,
Yu H,
Wymore RS,
Cohen IS,
and
McKinnon D.
Role of the Kv4.3 K+ channel in ventricular muscle A molecular correlate for the transient outward current.
Circ Res
79:
659-668,
1996
10.
England, SK,
Uebele VN,
Shear H,
Kodali J,
Bennett PB,
and
Tamkun MM.
Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.
Proc Natl Acad Sci USA
92:
6309-6313,
1995
11.
Fink, M,
Duprat F,
Lesage F,
Heurteaux C,
Romey G,
Barhanin J,
and
Lazduski M.
A new K+ channel beta subunit to specifically enhance Kv2.2 (CDRK) expression.
J Biol Chem
271:
26341-26348,
1996
12.
Kuryshev, YA,
Gudz TI,
Brown AM,
and
Wible BA.
KChAP as a chaperone for specific K+ channels.
Am J Physiol Cell Physiol
278:
C931-C941,
2000
13.
Majumder, K,
De Biasi M,
Wang Z,
and
Wible BA.
Molecular cloning and functional expression of a novel potassium channel -subunit from human atrium.
FEBS Lett
361:
13-16,
1995[Web of Science][Medline].
14.
Morales, MJ,
Castellino RC,
Crews AL,
Rasmusson RL,
and
Strauss HC.
A novel subunit increases rate of inactivation of specific voltage-gated potassium channel subunits.
J Biol Chem
270:
6272-6277,
1995
15.
Nagaya, N,
and
Papazian DM.
Potassium channel and subunits assemble in the endoplasmic reticulum.
J Biol Chem
272:
3022-3027,
1997
16.
Nakahira, K,
Shi G,
Rhodes KJ,
and
Trimmer JS.
Voltage-gated K+ channel -subunits with -subunits.
J Biol Chem
271:
7084-7089,
1996
17.
Peri, R,
Wible BA,
and
Brown AM.
Mutations in the Kvbeta 2 binding site for NADPH and their effects on Kv1.4.
J Biol Chem
276:
738-741,
2001
18.
Rettig, J,
Heinemann SH,
Wunder F,
Lorra C,
Parcej DN,
Dolly JO,
and
Pongs O.
Inactivation properties of voltage-gated K+ channels altered by presence of -subunit.
Nature
369:
289-294,
1994[Medline].
19.
Sanguinetti, MC,
Curran ME,
Zou A,
Shen J,
Spector PS,
Atkinson DL,
and
Keating MT.
Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKs potassium channel.
Nature
384:
80-83,
1996[Medline].
20.
Scott, VES,
Rettig J,
Parcej DN,
Keen JN,
Findlay JBC,
Pongs O,
and
Dolly JO.
Primary structure of a subunit of -dendrotoxin-sensitive K+ channels from bovine brain.
Proc Natl Acad Sci USA
91:
1637-1641,
1994
21.
Shi, G,
Nakahira K,
Hammond S,
Rhodes KJ,
Schechter LE,
and
Trimmer JS.
Subunits promote K+ channel surface expression through effects early in biosynthesis.
Neuron
16:
843-852,
1996[Web of Science][Medline].
22.
Wallner, M,
Meera P,
Ottolia M,
Kaczorowki GJ,
Latorre R,
Garcia ML,
Stefani E,
and
Toro L.
Characterization and modulation by a -subunit of a human maxi KCa channel cloned from myometrium.
Receptors Channels
3:
185-199,
1995[Web of Science][Medline].
23.
Wang, W,
Xia J,
and
Kass RS.
MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled IsK channel.
J Biol Chem
273:
34069-34074,
1998
24.
Wang, Z,
Kiehn J,
Yang Q,
Brown AM,
and
Wible BA.
Comparison of binding and block produced by alternatively spliced Kv1 subunits.
J Biol Chem
271:
28311-28317,
1996
25.
Wible, BA,
Yang Q,
Kuryshev YA,
Accili EA,
and
Brown AM.
Cloning and expression of a novel K+ channel regulatory protein, KChAP.
J Biol Chem
273:
11745-11751,
1998
26.
Xu, H,
Dixon JE,
Barry DM,
Trimmer JS,
Merlie JP,
McKinnon D,
and
Nerbonne JM.
Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes.
J Gen Physiol
108:
405-419,
1996
27.
Xu, J,
and
Li M.
Auxiliary subunits of Shaker-type potassium channels.
Trends Cardiovasc Med
8:
229-234,
1998.
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) or coinjected with
Kv1.5, Kv
).
Inset: superimposition of averaged and normalized currents
from oocytes injected with Kv1.5 cRNA alone and oocytes coinjected with
Kv1.5, Kv
