2 Rammelkamp Center for Education and Research, MetroHealth Campus, and Departments of 1 Physiology and Biophysics and 3 Biochemistry, Case Western Reserve University, Cleveland, Ohio 44109-1998
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ABSTRACT |
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The concept of chaperones for K+ channels is new. Recently, we discovered a novel molecular chaperone, KChAP, which increased total Kv2.1 protein and functional channels in Xenopus oocytes through a transient interaction with the Kv2.1 amino terminus. Here we report that KChAP is a chaperone for Kv1.3 and Kv4.3. KChAP increased the amplitude of Kv1.3 and Kv4.3 currents without affecting kinetics or voltage dependence, but had no such effect on Kv1.1, 1.2, 1.4, 1.5, 1.6, and 3.1 or Kir2.2, HERG, or KvLQT1. Although KChAP belongs to a family of proteins that interact with transcription factors, upregulation of channel currents was not blocked by the transcription inhibitor actinomycin D. A 98-amino acid fragment of KChAP binds to the channel and is indistinguishable from KChAP in its enhancement of Kv4.3 current and protein levels. Using a KChAP antibody, we have coimmunoprecipitated KChAP with Kv2.1 and Kv4.3 from heart. We propose that KChAP is a chaperone for specific Kv channels and may have this function in cardiomyocytes where Kv4.3 produces the transient outward current, Ito.
Kv2.1; Kv4.3; Kv1.3; protein inhibitor of activated STAT3; rat heart
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INTRODUCTION |
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VOLTAGE-DEPENDENT POTASSIUM CHANNELS (Kv) contribute to
the electrical properties of excitable cells such as cardiomyocytes and
neurons by affecting action potential shape and duration. Kv channels
are also important in many nonexcitable cell types where they may be
involved in diverse processes such as secretion, cell volume
regulation, and activation by mitogens. Four gene families encompassing
a total of at least 16 different genes encode the majority of the
pore-forming Kv-subunits (Kv1-4) (15). Kv
-subunits assemble
as functional tetramers with multiple members of each subfamily
exhibiting tissue-specific expression. Given the extensive diversity in
Kv channels, matching native K+ currents with the correct
Kv gene product has been problematic. In some cases though, there is a
good correlation between native K+ current and Kv channel.
For example, Kv4.3 is thought to underlie the transient outward current
(Ito) in adult heart (8, 9, 16), whereas Kv1.3
has been shown to be responsible for the n-type current
characteristic of T lymphocytes (5).
The level of expression and/or functional properties of certain Kv
channels can be modulated by accessory cytoplasmic proteins. To date,
two distinct types of modulatory proteins have been described: Kv-subunits and KChAP. Multiple Kv
-subunits that modulate the kinetics and/or expression of channels in the Kv1 subfamily have been
described (1, 2, 10, 11, 20, 22, 24, 26, 27, 29, 33). These subunits
bind tightly to the amino termini of Kv
-subunits and, although they
have been described as chaperone-like (26), they are found at the cell
surface in native channel complexes. Kv1 and Kv2
-subunits also bind
a cytoplasmic Kv channel-associated protein, KChAP, which we cloned and
characterized recently (30). This association occurs at the
intracellular amino terminus of the Kv
-subunit, but, unlike
Kv
-subunits, KChAP does not remain attached to the channel at the
cell surface. Coexpression of KChAP with Kv2.1 in Xenopus
oocytes produced a significant increase in both total Kv2.1
channel protein and the number of functional Kv2.1 channels at the cell
surface. Our observations led to the hypothesis that KChAP acts as a
chaperone to enhance expression of Kv2.1 protein.
KChAP belongs to a newly described multigene family consisting of PIAS3 (6), GBP (28) or PIAS1 (19), and Miz1 (31) or ARIP3 (21). PIAS3, GBP/PIAS1, and Miz1/ARIP3 were all cloned in the yeast two-hybrid system as transcription factor interacting proteins. PIAS3, which is most homologous to KChAP, is thought to bind to activated STAT3, resulting in the inactivation of this transcription factor (6). Thus KChAP is likely to have multiple cellular binding partners in addition to Kv channels.
The present experiments were designed to 1) determine the number of Kv channels with which KChAP interacts, 2) explore the possibility of transcriptional activation by KChAP, 3) identify the KChAP domain mediating chaperone effects, and 4) examine KChAP/Kv channel interactions in native cells. The results show that KChAP enhances the functional expression, without altering gating or voltage dependence, of a subset of Kv channels, Kv1.3, Kv2.1, and Kv4.3, without increasing Kv1.1, 1.2, 1.4, 1.5, 1.6, 3.1, or Kir2.2, HERG, or KvLQT1 currents. The effects are independent of transcriptional activation, and a 98-residue fragment is able to reproduce both the binding and current enhancement effects of full-length KChAP. KChAP may chaperone Kv channels in cardiomyocytes, since complexes of KChAP and Kv2.1 or Kv4.3 were coimmunoprecipitated from rat heart.
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METHODS |
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In Vitro Transcription of cRNAs and Oocyte Injection
Kv4.3 was obtained by RT-PCR from rat brain RNA and subcloned into pSP64 for cRNA synthesis. Mouse Kv1.3 cDNA in pSP64 was purchased from the American Type Culture Collection. KChAP with an amino-terminal extension of 45 amino acids (MVMSFRVSELQVLLGFAGRNKSGRKHELLAKALHLLKSSCAPSVQ) was amplified by PCR from our original rat brain cDNA clone and subcloned into pSP64. We had originally assigned KChAP methionine-46 as the initiating residue based on the presence of an in-frame upstream stop codon (30). After a DNA sequencing error upstream of this position was discovered, which removes this stop codon, it now appears likely that the start site is 45 residues upstream. The original KChAP as well as KChAP with the amino-terminal extension exhibit no differences in their binding in yeast two-hybrid assays or in their effect on Kv channels in heterologous expression assays. cRNAs were prepared and injected as previously described (29, 30).Constructs and Yeast Two-Hybrid Assay
Full-length KChAP (residues 1-619) and the following KChAP fragments were tagged with enhanced green fluorescent protein (EGFP) at their amino termini by subcloning into the EcoR I and Sal I sites of EGFP-C2 (Clontech): KChAP-N (residues 46-354), KChAP-M (residues 355-452), and KChAP-C (residues 453-619). The same fragments were also subcloned into the GAL4 yeast two-hybrid vectors, pGBT9 and pGAD424. Fragments encoding Kv1.2-N (residues 1-164) and Kv1.3-N (residues 1-186) were also subcloned into pGBT9. Protein-protein interactions were tested in the yeast two-hybrid system by cotransformation of host strain HF7C with pairs of pGBT9 and pGAD424 fusion constructs as previously described (29). Cotransformants were selected and spotted on media with and without histidine to follow the activation of the HIS3 reporter gene.Cell Lines and Injection
Mouse L cells were grown in minimum essential medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). L cells stably transfected with either Kv1.1, Kv1.5, Kv4.3, HERG, or KvLQT1 + minK cDNAs were isolated, using methods as previously described in Ref. 7, and maintained in media containing 0.5 mg/ml G418 (Life Technologies). L cells were injected with an Eppendorf 5242 microinjector and 5171 micromanipulator (Madison, WI) as described previously (14). The bottoms of 35-mm plastic dishes were labeled with circles (1 mm diameter), and cells were plated at a density of ~20 cells/circle.Transient Transfection and Immunocytochemistry
Transient transfection. L cells stably expressing Kv4.3 (L/Kv4.3) were plated 1 day before transfection on poly-L-lysine-coated coverslips. For transient transfection with EGFP-C2, EGFP tagged full-length KChAP or KChAP fragments, cDNAs were mixed in a 1:5 ratio with Lipofectin (Life Technologies) and incubated with the cells for 4 h at 37°C. In some experiments, polyethylenimine (Fluka) was used as the lipid carrier (4).Immunofluorescence microscopy. About 48 h posttransfection, cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized for 5 min with 0.1% Triton X-100 in PBS. After blocking for 1 h in 5% nonfat milk/PBS, the cells were incubated overnight at 4°C with Kv4.3 polyclonal antibody (1:200 dilution; Alomone Labs) in 5% nonfat milk/PBS. The secondary antibody, tetramethylrhodamine B isothiocyanate (TRITC)-conjugated anti-rabbit IgG, (1:100; Jackson Labs), was added for 1 h at room temperature. Coverslips were mounted with Vectashield (Vector Labs) and examined with an Olympus BH-2 microscope. Images were obtained with a Spot 2.1 digital camera (Diagnostic Instruments).
Electrophysiology
Measurements of Xenopus oocyte whole cell currents were performed using the standard two-microelectrode voltage-clamp technique as described previously (30). In mouse L cells, K+ currents were measured using the whole cell configuration of the gigaseal recording technique (13). The pipette solution contained (in mM) 140 potassium aspartate, 5 MgCl2, 10 HEPES, 10 EGTA, 10 glucose, and 2 Na2ATP at pH 7.2. The bath solution contained (in mM) 140 NaCl, 5.4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose at pH 7.4.Data acquisition and analyses were performed with pCLAMP 5.5.1 software (Axon Instruments, CA). In oocytes, linear leakage and capacity transient currents were subtracted using a P/4 prepulse protocol. Records were low-pass filtered at 2 or 5 kHz and digitized at 10 kHz. Experiments were conducted at room temperature (20-22°C). Data are reported as means ± SE. Comparisons between two groups of cells were performed by t-test. Comparisons between multiple groups of cells were performed by one-way ANOVA test and Student-Newman-Keuls post hoc test. Means are considered to be significantly different when P < 0.05.
Tissue and Oocyte Lysate Preparation
Freshly dissected adult rat hearts (Sprague-Dawley) were minced and placed into ice-cold lysis buffer (1:7 wt/vol) containing (in mM) 150 NaCl, 50 Tris, 1 EDTA, 0.2% BSA, 1% Triton X-100, pH 7.5, supplemented with a protease and phosphatase inhibitor cocktail (Complete; Boehringer-Mannheim; plus 50 mM sodium fluoride and 0.2 mM sodium vanadate). Samples were homogenized with a Polytron at setting 6 for 5 s. After a 1-h incubation on ice, the lysates were centrifuged at 900 g for 10 min to remove insoluble material. Xenopus oocytes were homogenized with 20 strokes in a glass homogenizer in lysis buffer (20 µl/oocyte) and incubated on ice for 1 h, and insoluble debris was removed by centrifugation at 3,000 g. Protein concentrations were determined by the bichinchoninic acid method (Pierce).KChAP Antibody Production, Western Blotting, and Immunoprecipitation
Antibody production. A bacterial fusion protein consisting of maltose-binding protein (MBP; New England Biolabs) and the carboxy terminus of KChAP (residues 453-619) was purified on amylose resin and sent to Research Genetics for polyclonal antibody production. IgG was purified on a protein G Sepharose column (Pharmacia) and passed over an MBP affinity column, and anti-KChAP reactivity was immunopurified on a KChAP:MBP affinity column.Polyclonal antisera were also raised to a peptide (residues 534-549; SPSVITSLDEQDTLGH) in the carboxy terminus of KChAP (SynPep). The antibody, referred to as KChAP-Ctpep, was affinity purified on an AminoLink column (Pierce) to which free peptide was coupled.
Western blotting. Lysates and solubilized immunoprecipitated proteins were separated on 10% SDS polyacrylamide gels and blotted to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk in PBS plus 0.1% Tween 20 (PBST), blots were incubated with primary antibodies for 1 h at room temperature. The blots were washed three times in PBST at least 30 min, incubated in horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech; 1:3,000 dilution in 5% nonfat dry milk /PBST) for 1 h at room temperature and washed three times in PBST. The blots were developed with the ECL+Plus detection system (Amersham Pharmacia Biotech).
Immunoprecipitation. Immunoprecipitation reactions were performed at 4°C using rat heart lysates. For each experiment, the lysate (2-4 mg/ml) was equally divided into two tubes. Lysates were precleared by incubation with anti-rabbit IgG-conjugated magnetic beads (Dynabeads; Dynal), after which affinity-purified anti-KChAP was added to one tube (1:100 dilution). Both tubes were incubated overnight with gentle mixing at 4°C. Antigen-antibody complexes were captured on Dynabeads by gentle mixing for 1 h at 4°C. The Dynabeads were washed three times with lysis buffer, and immunoprecipitates were eluted by boiling in reducing SDS sample buffer.
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RESULTS |
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KChAP Increases the Functional Expression of Kv1.3 and Kv4.3 in Xenopus Oocytes
We showed previously that KChAP produced significant increases in Kv2.1, but not Kv1.5, currents when coexpressed in Xenopus oocytes (30). Here we examined the effect of KChAP on other Kv channels when both cRNAs were expressed in Xenopus oocytes. Kv1.3 current amplitude was increased about twofold when the channel was coexpressed with KChAP (Fig. 1A). This increase occurred in the absence of changes in channel gating or voltage dependence of activation (Fig. 1B). Activation (from
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We demonstrated previously that KChAP was able to bind to the amino terminus of both Kv2.1 and Kv1.5 channels (30). Because we saw a significant increase in Kv1.3 currents in the presence of KChAP, we tested the amino terminus of Kv1.3 for its ability to bind to KChAP. A yeast two-hybrid interaction assay indicated that Kv1.3-N was able to interact with KChAP. As shown in Fig. 1F, growth on minus histidine selection media as a result of the activation of the HIS3 reporter gene was indicative of interaction between the two proteins.
Kv1.3 is unique, among the Kv1 subfamily members that we have studied,
in its sensitivity to KChAP. A thorough examination by heterologous
expression in Xenopus oocytes revealed no enhancement of Kv1.2,
Kv1.4, Kv1.5, or Kv1.6 current amplitudes by KChAP (Fig. 2), even though the amino termini of Kv1.2,
Kv1.4, and Kv1.5 interacted with KChAP in yeast two-hybrid assays (30).
We expanded our analysis to additional Kv subfamilies and found that
Kv3.1 currents were unaffected by KChAP but that the amplitude of Kv4.3
currents increased significantly (Fig. 2). Due to nonspecific
transactivation of the reporter genes by Kv3.1-N and Kv4.3-N in
yeast two-hybrid assays, we were not able to use this
method to determine whether KChAP interacted directly with
either channel. As a further check of its specificity, KChAP did not
increase Kir2.2, HERG, or KvLQT1 currents when tested in oocytes (Fig.
2).
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An examination of the effects of KChAP on Kv4.3 currents in
Xenopus oocytes is presented in Fig.
3. In the presence of KChAP, Kv4.3 currents
were increased about twofold (Fig. 3A), while the kinetics of
activation and inactivation were unchanged (Fig. 3, B and
C). At depolarizing test potentials from 80 to +70 mV, activation constants were 1.6 ± 0.3 and 1.6 ± 0.2 ms for currents recorded from oocytes injected with Kv4.3 cRNA alone (n = 24) and Kv4.3 plus KChAP cRNAs (n = 24), respectively.
Inactivation constants were 40.1 ± 0.3 ms (Kv4.3 alone;
n = 24) and 40.8 ± 0.4 ms (Kv4.3 + KChAP; n = 24).
The values of half-maximal inactivation (V0.5) were
45.6 ± 0.6 and
46.8 ± 0.2 mV, and the slope factors (k) were
5.8 ± 0.1 and
6.0 ± 0.1 mV
for currents from oocytes injected with Kv4.3 cRNA alone (n = 5) and Kv4.3 plus KChAP cRNAs (n = 5), respectively. As with
Kv1.3, KChAP expression enhancement of Kv4.3 currents was dose
dependent and saturable with increasing amounts of KChAP cRNA (Fig.
3D). Coexpression with KChAP did not alter the time course of
Kv4.3 expression in oocytes that peaked at day 2 and decreased
thereafter (Fig. 3E), suggesting that, if there were more Kv4.3
channels at the cell surface as for Kv2.1 (30), their stability at the
surface was not altered.
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KChAP Increases Kv2.1, Kv4.3, and Kv1.3 Currents in Transfected Mammalian Cells
Thus far, all of our functional assays of KChAP have been in Xenopus oocytes. We wanted to determine whether the oocyte observations could be replicated in mammalian cells. In the first set of experiments, KChAP and Kv2.1 cRNAs were microinjected into mouse L cells. Noninjected cells or cells injected with 100 mM KCl had very small outward currents, whereas cells injected with Kv2.1 cRNA exhibited voltage-dependent outwardly rectifying current (Fig. 4A). The current density in cells coinjected with a mixture of Kv2.1 and KChAP cRNAs was significantly higher then in cells injected with Kv2.1 cRNA alone (Fig. 4, A and B).
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To examine the effect of KChAP on Kv4.3 in mouse L cells, we
transiently transfected a cell line stably expressing Kv4.3 (L/Kv4.3) with a plasmid encoding a chimeric protein consisting of KChAP fused to
the carboxy terminus of EGFP (EGFP-KChAP). As a control, the plasmid
EGFP-C2 was transfected. Cells exhibiting green fluorescence were
chosen for electrophysiological recording. L/Kv4.3 cells exhibited a
relatively small transient outward current (Fig. 4C). Kv4.3
currents were not altered in cells coexpressing EGFP (Fig. 4C).
However, cells transfected with the chimeric EGFP-KChAP construct exhibited dramatically increased (8- to 10-fold) currents with no
apparent changes in voltage dependence or channel gating (Fig. 4,
C and D). The Kv4.3 currents in cells coexpressing EGFP
or EGFP-KChAP had similar kinetics of activation and inactivation. With
pulses from 90 to +40 mV, activation time constants were 4.09 ± 0.21 ms (EGFP; n = 16) and 3.92 ± 0.36 ms
(EGFP-KChAP; n = 16). Inactivation time constants were 79.3 ± 5.4 ms (EGFP; n = 16) and 72.8 ± 4.0 ms
(EGFP-KChAP; n = 16). Parameters of steady-state inactivation
were also similar: V0.5 =
49.4 ± 0.8 mV,
k =
6.7 ± 0.2 mV (n = 5); and
V0.5 =
51.5 ± 3.0 mV, k =
7.2 ± 0.4 mV (n = 5) for EGFP and EGFP-KChAP,
respectively. Recovery from inactivation did not differ in cells
coexpressing EGFP (recovery constant at
100 mV; 217 ± 18 ms;
n = 6) or EGFP-KChAP (194 ± 25 ms; n = 4).
Because the mammalian cell environment supported the action of KChAP on Kv2.1 and Kv4.3 currents as well or better than Xenopus oocytes, we tested the effect of KChAP on Kv1.5 in L cells, a channel whose amplitude is not modulated by KChAP in oocytes. An L cell line stably expressing Kv1.5 (L/Kv1.5) was transiently transfected with either EGFP-C2 or EGFP-KChAP. As shown in Fig. 4, E and F, there was no significant alteration in the amplitude of Kv1.5 currents in any of the transiently transfected cells. Thus KChAP did not modulate Kv1.5 in either oocytes or L cells.
A summary of the effects of KChAP on K+ channels in L cells is presented in Fig. 4G. In addition to Kv2.1, Kv4.3, and Kv1.5, we also examined the effect of KChAP on four other stable L cell lines: L/Kv1.1, L/Kv1.3, L/HERG, and L/KvLQT1 + minK. As with Kv1.5, Kv1.1 currents were unaffected by the coexpression of KChAP. Neither HERG nor KvLQT1 + minK currents were altered by KChAP in L cells. However, Kv1.3 currents were increased by KChAP in mammalian cells as well as oocytes.
Kv-Subunit Binding Region Is Localized to a Stretch
of 98 Residues in KChAP
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KChAP and KChAP-M Increase Kv4.3 Current Amplitude and Total Protein in L Cells
In Xenopus oocytes, increased functional expression of Kv2.1 with KChAP was reflected in an increase in the total amount of Kv2.1 protein and the number of functional Kv2.1 channels (30). In the same experiments, we found that, 48 h after injection of KChAP cRNA in oocytes, KChAP protein was present in the nucleus and the soluble and membrane fractions. Kv2.1 protein was visualized at the cell surface but KChAP was not, suggesting that KChAP and Kv2.1 interacted transiently at some point before the channel reaches the cell surface. The evidence of increased Kv2.1 current, increased Kv2.1 protein, and direct but transient interactions between Kv2.1 and KChAP led to our hypothesis that KChAP is a chaperone for Kv2.1 in Xenopus oocytes.To determine whether KChAP behaved similarly in mammalian cells, we
examined L/Kv4.3 cells that were transfected with EGFP-KChAP with an
anti-Kv4.3 polyclonal antibody and immunofluorescence microscopy. For
comparison, cells from the same transfection experiment were recorded
and electrophysiological data compiled. L/Kv4.3 cells transfected with
EGFP-KChAP are shown in Fig. 6A.
Fluorescence from EGFP-KChAP was observed in the nuclei of transiently
transfected cells (Fig. 6, A and B, right).
When we stained EGFP-KChAP transfected cells with a polyclonal antibody
raised against a peptide in the carboxy terminus of KChAP
(anti-KChAP-CTpep), we observed both cytoplasmic and nuclear staining
in green fluorescing cells (Fig. 6B, right). Cells expressing
EGFP-KChAP exhibited dramatically increased staining with the Kv4.3
antibody compared with nontransiently transfected L/Kv4.3 cells (Fig.
6A, left). Kv4.3 staining was not restricted to the cell
surface but was especially bright in the perinuclear region, consistent
with endoplasmic reticulum staining. Thus KChAP increased Kv4.3 total
protein in L cells as it did for Kv2.1 in Xenopus oocytes.
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Having localized the Kv binding domain of KChAP to residues
355-452 (KChAP-M), we wanted to determine whether this fragment would also increase Kv4.3 protein in this assay. L/Kv4.3 cells were
transiently transfected with EGFP-tagged KChAP-M and the cells stained
with anti-Kv4.3 antibody. KChAP-M appeared to be in both the nucleus
and cytoplasm, especially in the perinuclear region (Fig. 6C,
left). In the same cell, we saw a dramatic increase in anti-Kv4.3
reactivity comparable to what was observed with full-length KChAP (Fig.
6C, right). Figure 6F presents a summary of the
K+ currents that were recorded from transiently transfected
L/Kv4.3 cells. Both KChAP and KChAP-M produced increased currents of
comparable magnitude relative to EGFP-C2 alone. We also examined the
effects of EGFP-tagged fusions of KChAP-N and KChAP-C on
Kv4.3 protein and current levels. KChAP-N (Fig. 6D,
right) showed a diffuse cytoplasmic localization with no
concomitant increase in Kv4.3 staining (Fig. 6D, left). KChAP-C
(Fig. 6E, right) appeared to be present in both the nucleus and
cytoplasm, but again produced no increase in the amount of Kv4.3
protein (Fig. 6E, right). The lack of increased Kv4.3
immunoreactivity with these two fragments was reflected in the lack of
increase in Kv4.3 current density in cells overexpressing them as well
(Fig. 6F). Thus the small fragment of KChAP that was identified
as binding to Kv
amino termini was sufficient to produce increases
in Kv4.3 in mammalian cells.
Transcription Is Not Required for KChAP Modulation of Kv Channels in Xenopus Oocytes
The partial nuclear localization of overexpressed KChAP in both oocytes and mammalian cells raises the question of whether KChAP is affecting the transcription of a gene that may influence the activity of Kv channels in our experiments. This is particularly relevant since KChAP-related proteins have been identified as functioning as transcription factor regulatory proteins (6, 19, 21, 28, 31). Despite our evidence that KChAP and certain Kv channels interact directly (30), we had to consider the possibility that nuclear KChAP was modulating Kv channels indirectly through a mechanism that involved transcription. We could test this possibility in oocyte experiments by examining KChAP effects on Kv channels in oocytes incubated with actinomycin D, an inhibitor of transcription. Figure 7 shows that KChAP increased Kv1.3 currents, irrespective of whether the oocytes were incubated with actinomycin D after injection or not (Fig. 7A). To confirm that actinomycin D could be effective, we injected Kv1.3 cDNA into oocyte nuclei and saw that, as expected, actinomycin D significantly reduced Kv1.3 currents in comparison to control (Fig. 7B). We observed similar results with Kv2.1 cRNA, cDNA, and KChAP (not shown). Thus transcription is not required for KChAP to increase Kv channel amplitudes.
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Association of KChAP With Kv2.1 and Kv4.3 in Rat Heart
To assess the physiological relevance of KChAP interactions with Kv channels, we raised a polyclonal antisera to KChAP and used it to probe for association with Kv channels in native tissue. The anti-KChAP antibody was raised against a KChAP fragment consisting of residues 453-619, was affinity purified, and was initially tested for its ability to detect overexpressed KChAP in Xenopus oocytes. A single band of ~68 kDa was detected in lysates of oocytes injected with KChAP cRNA, consistent with the predicted molecular mass of KChAP (Fig. 8A). In lysates from adult rat heart, the anti-KChAP antibody detected a band >75 kDa. This suggests that KChAP may be posttranslationally modified in heart, leading to an increased apparent molecular mass.
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Because Kv2.1 and Kv4.3 are both sensitive to the effects of KChAP in heterologous expression assays and are expressed in adult rat heart, we searched for complexes of KChAP and Kv channel proteins by coimmunoprecipitation with anti-KChAP. Adult rat heart lysate immunoblotted with anti-Kv2.1 revealed two bands of ~105 and 130 kDa (Fig. 8C). These observations are consistent with the Western blotting pattern of Kv2.1 in rat heart (32) and brain (25). The 105-kDa Kv2.1 band coimmunoprecipitated with KChAP as shown in Fig. 8C. Interestingly, the larger 130-kDa Kv2.1 band, which is thought to be hyperphosphorylated (23), was not detected in KChAP immunoprecipitates. A polyclonal antibody to Kv4.3 detected a single band of ~75 kDa in adult rat heart lysates, which was present in complexes coimmunoprecipitated with the KChAP antibody (Fig. 8D). These data indicate that KChAP interacts with Kv channels in native tissue.
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DISCUSSION |
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Our results demonstrate that KChAP modulates the functional expression of specific Kv channels without changing channel properties such as gating or voltage dependence in both Xenopus oocyte and mammalian expression systems. Following our initial characterization of KChAP and Kv2.1, we hypothesized that KChAP was a novel chaperone that interacted transiently with the channel but did not remain attached to the mature channel complex at the cell surface (30). Considered together with our previous data, the results reported here support the view that interaction of KChAP with Kv channels is responsible for the observed increase in current and protein levels. First, the coimmunoprecipitation of complexes of KChAP with Kv2.1 and Kv4.3 from rat heart lysates demonstrates that the proteins do interact in vivo. Second, yeast two-hybrid data suggest a direct interaction between KChAP and the amino termini of Kv1 and Kv2 channels as we had reported previously (30) but confirmed here for Kv1.3-N. Kv3 and Kv4 amino termini produced nonspecific transactivation of reporter genes in yeast and could not be assayed. In previous oocyte experiments, we only observed increases in Kv2.1 currents with KChAP when the two cRNAs were injected simultaneously in the same pipette, suggesting a requirement for the cotranslational association of the two proteins, and saw that a partial amino-terminal deletion of Kv2.1, although producing functional channels, was not sensitive to KChAP (30).
The evidence that KChAP is not part of the mature channel complex at the cell surface comes from heterologous expression experiments. In transiently transfected mammalian cells we have observed EGFP-tagged KChAP in both the nucleus and cytoplasm of the cell when examined 48 h after transfection. Interestingly, however, a partial fragment of KChAP, KChAP-M (98 residues), which retained the ability to bind to Kv channels, gave a punctate appearance in the cytoplasm reminiscent of Kv channel staining, suggesting a possible extended interaction of the two proteins.
KChAP belongs to a newly described multigene family consisting of PIAS3 (6), GBP (28) or PIAS1 (19), and Miz1 (31) or ARIP3 (21). KChAP is most homologous to PIAS3, and examination of the nucleotide sequences suggests that the two may be the products of alternative splicing of a single gene. The difference between the two proteins is a 35-amino acid insert in the amino-terminal region of KChAP, which is not present in PIAS3. The Kv channel binding region, KChAP-M, is present in both KChAP and PIAS3, so it is likely that both KChAP and PIAS3 will interact with channels. All of the proteins in this family were cloned in yeast two-hybrid screens as binding to proteins that at least partly reside in the nucleus. PIAS3 was cloned as a STAT3 binding protein and is thought to function as an inactivator of activated transcription factor, STAT3. Thus the partial localization of KChAP to the nucleus on overexpression may not be surprising. There is evidence that proteins in this family may affect transcription either directly or indirectly. Our results suggest, however, that the effect of KChAP on Kv channels appears to be independent of events that occur in the nucleus. Blocking transcription in Xenopus oocytes with actinomycin D did not alter the ability of KChAP to increase Kv1.3 and Kv2.1 currents. Thus, even though KChAP may serve additional roles in the nucleus, our data are consistent with its effects on ion channels occurring through a transient interaction with the channel protein.
The actions of KChAP differ from the other family of Kv channel
modulatory proteins, Kv-subunits. While Kv
-subunits have also
been described as having chaperone-like effects in that they increase
the functional expression of some Kv1 channels, they remain tightly
attached to the channels at the cell surface as with other
hetero-oligomeric channels such as the ACh receptor (17, 18) and
Ca2+ channel (3, 12). These hetero-oligomers are in marked
contrast to KChAP, for which we have no evidence that KChAP:Kv channel complexes exist at the cell surface. Kv
-subunits also bind to KChAP,
however. Our data suggest that KChAP and Kv
-subunits may compete for
binding to Kv
1 amino termini, producing alterations in current
properties (unpublished observations).
Our data show that a restricted subgroup of Kv channels is sensitive to modulation by KChAP: Kv1.3, Kv2.1, Kv2.2, and Kv4.3 (data presented here and in Ref. 30). It is not clear at present why some Kv channels are sensitive to modulation by KChAP and others are not. Among the Kv1 subfamily, only Kv1.3 currents increase on coexpression with KChAP, even though the amino termini of other family members, such as Kv1.2, also bind to KChAP in yeast two-hybrid assays. Preliminary experiments with chimeric channels of Kv1.2 and Kv1.3 suggest that neither the amino nor carboxy termini of Kv1.3 confer sensitivity to KChAP and point to the core domain as critical.
Given its homology with PIAS3, it is intriguing to speculate that KChAP may function as an integral component of a signaling pathway linking nuclear processes with the expression of ion channels at the cell surface. Perhaps by regulating KChAP binding to Kv channels, the expression of Kv currents at the cell surface can be modulated in a rapid, acute manner, independent of changes in Kv channel gene transcription. Whether KChAP is available for binding to Kv channels could depend on its binding to other targets such as STAT3. Further experiments are required to sort out the meaning of these possible multiple interactions in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Nerbonne for providing Kv4.3 polyclonal antibody for preliminary experiments; Dr. M. Keating for the KvLQT1 cDNA; and N. Kuzmin, J. Falquet, I. Kuryshev, and Dr. W. Dong for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-60759 (to B. A. Wible) and NS-23877, HL-36930, and HL-55404 (to A. M. Brown).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. A. Wible, Rammelkamp Center, 2500 MetroHealth Dr., Cleveland, OH 44109-1998 (E-mail: bwible{at}research.metrohealth.org.)
Received 17 September 1999; accepted in final form 10 December 1999.
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