Cloning and Expression of a Novel K+ Channel
Regulatory Protein, KChAP*
Barbara A.
Wible
§,
Qing
Yang,
Yuri A.
Kuryshev¶,
Eric. A.
Accili
, and
Arthur M.
Brown¶
From the Rammelkamp Center of Research, MetroHealth Campus, the
Department of Biochemistry, and the ¶ Department of
Physiology and Biophysics, Case Western Reserve University,
Cleveland, Ohio 44109-1998
 |
ABSTRACT |
Voltage-gated K+ (Kv) channels
are important in the physiology of both excitable and nonexcitable
cells. The diversity in Kv currents is reflected in multiple Kv channel
genes whose products may assemble as multisubunit heteromeric
complexes. Given the fundamental importance and diversity of Kv
channels, surprisingly little is known regarding the cellular
mechanisms regulating their synthesis, assembly, and metabolism. To
begin to dissect these processes, we have used the yeast two-hybrid
system to identify cytoplasmic regulatory molecules that interact with
Kv channel proteins. Here we report the cloning of a novel gene
encoding a Kv channel binding protein (KChAP, for
K+ channel-associated
protein), which modulates the expression of Kv2 channels
in heterologous expression system assays. KChAP interacts with the N
termini of Kv
2 subunits, as well as the N termini of Kv
1 and the
C termini of Kv
subunits. Kv2.1 and KChAP were coimmunoprecipitated
from in vitro translation reactions supporting a direct
interaction between the two proteins. The amplitudes of Kv2.1 and Kv2.2
currents are enhanced dramatically in Xenopus oocytes
coexpressing KChAP, but channel kinetics and gating are unaffected.
Although KChAP binds to Kv1.5, it has no effect on Kv1.5 currents. We
suggest that KChAP may act as a novel type of chaperone protein to
facilitate the cell surface expression of Kv2 channels.
 |
INTRODUCTION |
The electrical properties of excitable cells are determined in
large part by the voltage-gated K+ channels
(Kv)1 they possess. Multiple
Kv channels control the falling phase of the action potential in
excitable cells. Kv channels are also important in many nonexcitable
cells, where they may contribute to diverse processes such as volume
regulation, hormone secretion, and activation by mitogens. The
extensive diversity in Kv currents is matched by the multiplicity of
genes encoding the pore-forming or
-subunit of Kv channels. About 20 mammalian Kv
genes have been cloned, and most have been assigned to
one of four major subfamilies based on sequence similarities: Kv1, Kv2,
Kv3, and Kv4 (1). Each K+ channel gene encodes a single
subunit, and functional channels are formed by the tetrameric
association of individual subunits apparently mediated by specific
binding between the N-terminal domains of subunits within individual
subfamilies (2, 3). With multiple Kv channel genes whose products may
assemble as multisubunit heteromeric complexes (4-6), there may be
hundreds of functionally distinct K+ channels. Given the
great diversity and fundamental importance of K+ channels,
the cellular mechanisms regulating their synthesis, assembly, and
metabolism are of prime interest but remain largely unknown.
The identification and characterization of accessory or modulatory
subunits for Kv channels is a new and rapidly expanding area of
research. One family of modulatory proteins that interact with Kv
1
channels, Kv
subunits, has been cloned and characterized in the past
several years. Kv
subunit genes, cloned from heart (7-10) and brain
(11-13), encode cytoplasmic proteins that form stable complexes with
Kv
1 subunits and exert multiple effects on Kv
1 currents. The
three Kv
1 isoforms and Kv
3 introduce inactivation into Kv
1
subunit currents but with variable potency (12-14). A second effect of
Kv
subunits is to increase the surface expression of certain Kv
1
channels. This has been demonstrated both as an increase in the number
of dendrotoxin-binding sites (for Kv1.2 transient expression) (15), as
well as an increase in the number of functional channels (16).
Complexes between Kv
1 and Kv
subunits have been found to form in
the endoplasmic reticulum (15, 17), suggesting that Kv
subunits
assist in the folding and assembly of at least some Kv
1 subunits.
The association of Kv1.2 with Kv
subunits produces more efficient
glycosylation of Kv1.2, increases the stability of Kv1.2 through
Kv1.2·Kv
complex formation and results in an increase in
cell surface expression (15).
To gain more information about the synthesis, assembly, and metabolism
of K+ channels, we have used the yeast two-hybrid system to
identify novel cytoplasmic molecules that interact with Kv subunits.
Using Kv
1.2 as bait, we screened a rat brain cDNA library in the
GAL4 activation domain vector and isolated a novel gene that encodes a
K+ channel-binding protein that we have termed KChAP (for
K+ channel-associated
protein). In addition to Kv
subunits, KChAP also binds
to the N termini of Kv
1 and Kv
2 subunits. Coexpression of KChAP
with Kv
2 subunits results in a dramatic enhancement of both total
Kv2.1 protein and surface expression of functional Kv2 channels. The
unique sequence and properties of KChAP suggest that it may belong to a
novel class of proteins with "chaperone-like" properties.
 |
EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Library Screen--
The entire Kv
1.2 coding
sequence (amino acids 1-408) was subcloned in frame into the GAL4 DNA
binding domain vector, pGBT9 (CLONTECH) after
polymerase chain reaction-mediated addition of a 5' EcoRI
site and a 3' SalI site and used to screen a pGAD10 rat
brain cDNA library (CLONTECH). Transformants in
the yeast Y190 strain were plated on synthetic medium lacking
tryptophan, leucine, and histidine but containing 3-aminotriazole (25 mm). After incubation for 8 days at 30 °C, His+ colonies
were screened for
-galactosidase activity by a filter lift assay.
Individual pGAD10 recombinant plasmids were screened for interaction
with Kv
1.2 by repeating the yeast two-hybrid assay.
Cloning of Full-length cDNA--
The KChAP-Y/pGAD10 plasmid
contained a 1.78-kb insert with an open reading frame of 264 amino
acids. To obtain the full-length clone, the 32P-labeled
KChAP-Y insert was used to screen a rat brain cDNA library in
gt10 (CLONTECH). One of the hybridizing clones
contained an insert of 3.2 kb with a single open reading frame of 574 amino acids. KChAP cDNA without 5'- or 3'-untranslated sequences
for subcloning in frame into the yeast two-hybrid vector pGBT9 was prepared by polymerase chain reaction to include a 5' EcoRI
site and a 3' SalI site.
Analysis of Protein-Protein Interactions by the Yeast Two-hybrid
System--
Protein-protein interactions were monitored with the yeast
Matchmaker two-hybrid system (CLONTECH). The
following fragments were tested for interaction with KChAP: Kv
1.2
(amino acids 1-408), Kv
1.2 N terminus (amino acids 1-79), Kv
1C
(carboxyl-terminal 329 amino acids of the Kv
1 subfamily), Kv
2
(amino acids 1-367), Kv1.1 N terminus (amino acids 1-168), Kv1.2 N
terminus (amino acids 1-124), Kv1.4 N terminus (amino acids 1-305),
Kv1.4 C terminus (amino acids 562-654), Kv1.5 N terminus (amino acids
1-248), Kv2.1 N terminus (amino acids 1-168), Kv2.2 N terminus (amino
acids 1-185), Kv6.1 N terminus (amino acids 1-209), Kir2.2 N terminus (amino acids 1-86), and HERG N terminus (amino acids 1-396). Human Gu-binding protein (GBP) cDNA encoding the region from residues Met49 to Asp645 was obtained by reverse
transcription-polymerase chain reaction from human brain
poly(A)+ RNA. Met49 corresponds to the start
methionine residue in KChAP. Protein-protein interactions were tested
by cotransformation of plasmid pairs into the yeast host strain Y190 as
described previously (16). The appearance of a blue color within 8 h was scored as a positive interaction.
Northern Blot Analysis--
A rat multiple tissue Northern blot
(CLONTECH) was probed with a
32P-riboprobe spanning the region encoding the C-terminal
167 amino acids of KChAP. A T7 promoter sequence was engineered
directly onto the end of the 501-base pair coding fragment using the
Lig'nScribe kit from Ambion and the riboprobe synthesized with the
Maxiscript T7 kit (Ambion). The blot was hybridized with probe
(106 cpm/ml) overnight at 68 °C in NorthernMax
hybridization buffer (Ambion). Two room temperature washes in 2 × SSC, 0.1% SDS (15 min each) were followed by two washes at 70 °C in
0.1 × SSC, 0.1% SDS (20 min each).
In Vitro Translation and Immunoprecipitation--
Full-length
KChAP cDNA was removed from pGBT9 with EcoRI and
SalI and subcloned into a pCR3 vector, which we modified to
allow the cloning of EcoRI/SalI fragments in
frame behind a c-myc tag. cRNA for c-myc-KChAP
was prepared with the T7 mMESSAGE mMACHINE kit (Ambion). cRNAs for
c-myc-KChAP and Kv2.1 were translated in vitro
either separately or together in rabbit reticulocyte lysates in the
presence of [35S]methionine using the Retic Lysate IVT
kit (Ambion). A maximum of 500 ng of cRNA was used in each 25-µl
translation reaction. Canine pancreatic microsomes (Boehringer
Mannheim) (1 µl/25 µl of translation reaction) were included in
reactions in which Kv2.1 was translated. For immunoprecipitation (IP),
10-µl aliquots of each translation were diluted into 1 ml of IP
buffer (1% Triton X-100, 150 mM NaCl, 50 mM
Tris, pH 7.5, 1 mM EDTA). To monitor the ability of the two
proteins to associate after translation, 10-µl aliquots of individual
translates of Kv2.1 and c-myc-KChAP were mixed in 1 ml of IP
buffer prior to the addition of antibody. IP was performed with two
primary antibodies: anti-Kv2.1 polyclonal (1:100; Upstate
Biotechnology, Inc) or anti-c-myc monoclonal (1:400; Boehringer Mannheim). After the addition of the primary antibody, the
reactions were mixed gently overnight at 4 °C. Immune complexes were
collected on magnetic beads coupled to either anti-rabbit or anti-mouse
secondary antibodies (Dynal, Inc). After four washes in IP buffer,
bound protein was eluted by boiling in SDS sample buffer and analyzed
on 10% polyacrylamide/SDS gels. The gel was fixed, soaked in Amplify
(Amersham Pharmacia Biotech), and radiolabeled protein detected by
fluorography.
Immunofluorescence Microscopy--
Five days postinjection, two
microelectrode voltage clamp recordings were made from
Xenopus oocytes injected with either Kv2.1 cRNA alone or
coinjected with Kv2.1 plus c-myc-KChAP cRNAs. Several hours
after recording, the same oocytes were fixed and sectioned as described
previously (18). After incubation for 2 h in 1% bovine serum
albumin/phosphate-buffered saline to block nonspecific binding sites,
the oocyte sections were incubated at 4 °C overnight with primary
antibody (anti-Kv2.1 polyclonal, 1:100) in 1% bovine serum
albumin/phosphate-buffered saline. The secondary antibody (fluorescein
isothiocyanate-conjugated anti-rabbit, 1:100; Cappel Labs) was added
for 2 h at room temperature. For oocytes coinjected with
c-myc-KChAP, the same protocol was followed with
anti-c-myc monoclonal antibody (1:400) and
tetramethylrhodamine B isothiocyanate-conjugated anti-mouse secondary
antibody (1:125; Sigma). The sections were examined with an Olympus
BH-2 microscope.
Oocyte Fractionation and Western Blotting--
To isolate
membranes, oocytes were homogenized in 0.3 M sucrose, 10 mM NaPO4, pH 7.4 (20 µl/oocyte) containing a
protease inhibitor mixture (Complete, Boehringer Mannheim). After
removal of nuclei and debris by centrifugation at 3000 × g for 10 min, the supernatant was spun at 48,000 × g for 1 h to pellet membranes. Membranes from adult rat
brain were prepared using the same protocol. In some experiments,
oocyte nuclei were removed manually (19). Enucleated oocytes were
processed as described above while the nuclei were extracted for 1 h in homogenization buffer plus 1% Triton X-100. Following a 20-min
spin at 10,000 × g, the supernatant containing
solubilized nuclear proteins was collected. Protein concentrations were
determined by the BCA method (Pierce). For Western blotting, proteins
were separated on SDS-polyacrylamide gel electrophoresis and blotted to
polyvinylidene difluoride membranes. After blocking with 5% nonfat dry
milk in phosphate-buffered saline plus 0.1% Tween 20, blots were
incubated with primary antibodies, either a monoclonal Kv2.1 antibody
(Upstate Biotechnology, Inc; 1:1000) or monoclonal
anti-c-myc antibody (1:400), for 1 h at room
temperature. The blots were then incubated with secondary antibody
(anti-mouse HRP conjugate, Amersham Pharmacia Biotech; 1:3000) and
developed with the ECL-Plus detection system (Amersham Pharmacia
Biotech).
Expression in Xenopus Oocytes and Electrophysiology--
For
cRNA synthesis and expression in Xenopus oocytes,
full-length KChAP coding sequence was subcloned into the vector, pCR3 (Invitrogen). KChAP cRNA was prepared using the T7 mMESSAGE mMACHINE kit (Ambion) following linearization of the construct with
NotI. GBP cDNA was subcloned into a modified pSP64
vector (NruI site for linearization incorporated past the
poly(A)+ tail) for in vitro transcription with
SP6 polymerase. cRNAs for Kv1
subunits were prepared as described
previously (7, 14). Rat Kv2.1 in pBluescript was linearized with
NotI, and cRNA was prepared with T7 polymerase. cRNAs were
mixed and injected into Xenopus oocytes as described
previously (7). HERG cDNA was kindly provided by Dr. M. Keating.
Kv2.2 was a gift from Drs. S. Snyder and J. Trimmer. Kir2.2 cRNA was
prepared as described previously (20). We also used a cRNA encoding
Kv2.1
N in which the N-terminal 139 amino acids had been deleted
(21).
Measurement of Xenopus oocyte whole cell currents was
performed using the standard two-microelectrode voltage clamp
technique. Bath solution contained (in mmol/liter): 5 KOH, 100 NaOH,
0.5 CaCl2, 2 MgCl2, 100 methanesulfonic acid,
and 10 HEPES (pH 7.4). Solution containing 50 K+ was
prepared by replacing an equivalent concentration of Na+.
Electrodes were filled with 3 M KCl and had a resistance of 0.3-0.6 megohms. All recordings were made at room temperature. Linear
leakage and capacity transient currents were subtracted (P/4 prepulse
protocol) unless specified, and data were low pass-filtered at 1 kHz.
pClamp software (Axon Instruments) was used for generation of the
voltage-pulse protocols and data acquisition. Data are reported as
means ± S.E. Comparisons among multiple groups of oocytes were
performed by one-way analysis of variance test, Student's t
test, and Student-Newman-Keuls post hoc test. Means are
considered to be significantly different when p < 0.05.
 |
RESULTS |
Isolation of a Novel Kv
and Kv
Subunit-binding Protein with
the Yeast Two-hybrid System--
Full-length Kv
1.2 was used as bait
to screen a rat brain cDNA library in the GAL4 activation domain
vector, pGAD10. We isolated one clone that exhibited a strong positive
signal in the
-galactosidase assay. pGAD10 plasmid DNA containing a
1.78-kb insert was isolated from this clone and tested positive for
interaction with Kv
1.2. Sequence analysis of the clone, which we
termed KChAP-Y (for K channel-associated protein; Y
refers to the fragment isolated in the yeast two-hybrid screen),
revealed a novel clone with no similarity to Kv
or Kv
subunits.
We tested the specificity of interaction of KChAP-Y with a panel of
Kv
, Kv
, and other K+ channel subunit fragments with
the yeast two-hybrid assay. As shown in Fig.
1, KChAP-Y interacted with both Kv
1.2
and Kv
2 subunits. KChAP-Y interacted with the conserved Kv
1 C
terminus but not the unique N terminus of Kv
1.2, suggesting that the
protein may recognize conserved sequences among Kv
subunits. Kv
subunits interact specifically with the N terminus of Kv1
subunits,
so we tested these fragments for binding to KChAP-Y as well.
Surprisingly, a positive signal was observed between the N termini of
Kv1.1, Kv1.2, Kv1.4, Kv1.5, and KChAP-Y. Just as with the Kv
subunits, however, no interaction was evident between the Kv1.4 C
terminus and KChAP-Y. KChAP-Y also interacted with the N termini of
Kv2.1 and Kv2.2 but not with the N terminus of the electrically silent Kv2 partner, Kv6.1 (22). Further specificity for a subset of Kv
channels was apparent from the lack of interaction with the N terminus
of the inward rectifier K+ channel, Kir2.2, and the N
terminus of the delayed rectifier K+ channel, HERG. Thus,
KChAP-Y apparently interacts with both the C terminus of Kv
subunits
and the N termini of Kv1 and Kv2
-subunits.

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Fig. 1.
Yeast two-hybrid assay of the interaction of
a novel protein, KChAP, with K+ channel fragments. The
KChAP fragment isolated from the yeast two-hybrid screen (KChAP-Y in
the GAL4 activation domain (AD) vector pGAD10) was tested
for interaction with a panel of K+ channel fragments cloned
in the GAL4 DNA binding domain (BD) vector, pGBT9.
Cotransformants of KChAP-Y/pGAD10 with individual binding domain
plasmids in the yeast strain Y190 were selected by plating on medium
lacking both tryptophan and leucine ( Trp/ Leu). A
representative colony from each cotransformation was spotted onto
another Trp/ Leu plate for assay of the lacZ reporter
gene. After growth for 2.5 days at 30 °C, yeast colonies were lifted
to paper filters and assayed for -galactosidase activity.
Interaction between fusion proteins was revealed by color development
in the X-gal column resulting from transcriptional activation of the
lacZ reporter gene. Note that KChAP-Y does not produce
autonomous transcriptional activation when cotransformed with the pGBT9
vector only. All constructs expressed as binding domain fusions were
shown to be negative for autonomous transcriptional activation with the
activation domain plasmid, pGAD424, prior to cotransformation with
KChAP-Y (not shown).
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Cloning and Sequence Analysis of Full-length KChAP--
Screening
of a rat brain cDNA library with the KChAP-Y coding sequence
produced a 3.2-kb insert, which overlapped KChAP-Y and contained a
single open reading frame of 574 amino acids. The initiating methionine
was assigned as the first ATG downstream from three in frame stop
codons. Hydropathy analysis indicated no potential membrane spanning
domains in KChAP, suggesting that the protein was cytoplasmic (not
shown).
Search of the GenBankTM nonredundant data base revealed
significant homology with the mammalian gene encoding GBP (23). GBP was
isolated originally in a yeast two-hybrid screen as a protein that
binds to the Gu/RNA helicase II subunit. Alignment of KChAP with GBP is
presented in Fig. 2. GBP has an
N-terminal extension of 55-57 amino acids compared with KChAP, but
over the 574-amino acid open reading frame of KChAP, the two proteins
are 50% identical. We tested the binding of both full-length KChAP
(amino acids 1-574) and human GBP (amino acids 49-645) with
K+ channel fragments in the yeast two-hybrid assay as was
described for KChAP-Y in Fig. 1. Full-length KChAP was identical to
KChAP-Y in its interaction with protein partners in the yeast
two-hybrid assay, while GBP did not interact with any of the tested
fragments including Kv
and Kv
subunits (data not shown). Thus,
although KChAP shares significant homology with GBP, interaction with
Kv
and Kv
subunits appears to be a unique feature of KChAP.

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Fig. 2.
Comparison of KChAP peptide sequence with
GBP. The 574-amino acid open reading frame of KChAP is aligned
with the predicted peptide sequence of GBP (23). The KChAP sequence was
deposited in GenBankTM with accession number AF032872. The
first ATG after three in frame stop codons was chosen as the initiating
methionine in KChAP. The start codon in GBP has not been determined but
may be one of the two methionines (residue number 4 or 6) marked in
boldface type and indicated with asterisks (23).
The arrow above KChAP tryptophan residue 310 indicates where
the KChAP-Y fragment begins. The filled circle above KChAP
leucine 407 marks the start of the coding sequence used for
construction of a riboprobe for Northern blot analysis. KChAP and GBP
share two putative protein kinase A phosphorylation sites at KChAP
positions Ser185 and Thr309, which are in
boldface type and underlined.
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Northern Blot Analysis--
The expression of KChAP mRNA was
examined in a panel of rat tissues. The blot was probed with a fragment
of KChAP encoding amino acids Lys407-Asp574, a
region with minimal homology to GBP, to avoid detecting GBP transcripts
as well. As shown in Fig. 3, a single
band of ~3.2 kb was detected in a variety of tissues including heart
and brain with especially high levels in lung and kidney.

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Fig. 3.
Northern blot analysis of KChAP expression in
rat tissues. A rat multiple tissue Northern blot (2 µg of
poly(A)+ RNA/lane) from CLONTECH was
probed with a 32P-labeled riboprobe prepared from a
fragment of KChAP cDNA encoding the C-terminal 167 amino acids.
Hybridization was done overnight in NorthernMax hybridization buffer
(Ambion) at 68 °C. Washes in 0.1 × SSC, 0.1% SDS were done at
70 °C. Autoradiography was for 5 h at 70 °C with Kodak
Biomax MS film and intensifying screen. RNA size markers are indicated
on the left.
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Functional Characterization of KChAP-Kv Interactions--
The
surprising finding that KChAP associated with Kv
1 and Kv
2
subunits as well as Kv
subunits led us to examine the functional consequences of KChAP-K+ channel interaction upon
heterologous expression in Xenopus oocytes. Whole oocyte
currents were recorded by two-electrode voltage clamp from eggs
injected with cRNAs encoding different Kv
-subunits alone or with
saturating concentrations of KChAP. Coexpression with KChAP produced a
dramatic 3-fold increase in the amplitude of Kv2.1 currents (Fig.
4A). No change in Kv2.1
currents was apparent when the channel was coexpressed with GBP (data
not shown). At more depolarized potentials, Kv2.1 has an opening
probability of about 0.9 (24), suggesting that the increased currents
recorded when KChAP was coexpressed were probably due to an increase in the number of functional channels. KChAP also interacted with the N
terminus of Kv1.5 but, in contrast to Kv2.1, produced no significant
change in Kv1.5 currents when coexpressed in oocytes (Fig.
4B). The experiments were done so that Kv1.5 expressed whole cell currents at +70 mV in the range of 0.5-5 µA. This greatly reduced the possibility that amplitude changes might be missed as a
result of voltage clamp difficulties. Thus, while KChAP interacted with
the N termini of both Kv2.1 and Kv1.5 in the yeast two-hybrid assay,
KChAP only produced amplitude increases in Kv2.1 currents in oocyte
expression assays.

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Fig. 4.
Effect of KChAP on K+ channel
functional expression. A, averaged macroscopic currents from
eight oocytes in one injection series measured on day 6 postinjection
in oocytes injected with Kv2.1 cRNA (0.62 ng/µl) alone
(left) or one coinjected with Kv2.1 and KChAP cRNAs (0.62 and 250 ng/µl, respectively) (right). Holding potential
was 80 mV, and pulses were from 70 mV to +80 mV in 10-mV steps with
50 mM K+ in bath solution. B,
averaged macroscopic currents from 10 oocytes in one injection series
measured 5 days postinjection from oocytes injected with Kv1.5 cRNA (50 ng/µl) alone (left) or Kv1.5 plus KChAP cRNAs (50 and 500 ng/µl, respectively) (right). Holding potential was 80
mV, and pulses were from 70 to +70 mV in 10-mV steps with 5 mM K+ in bath solution. C, bar plot
showing averaged current levels in the presence of KChAP as fractions
of currents in the absence of KChAP (control current). The
numbers above each bar indicate the number of batches of
oocytes examined for each K+ channel. Oocytes were injected
with either K+ channel cRNAs or K+ channel plus
KChAP cRNAs, and currents were recorded from 8-12 oocytes in each
batch. Whole oocyte currents were measured 2 days after injection
(Kir2.2 and Kv2.1 N) or 5-6 days after injection (Kv2.1, Kv2.2,
Kv1.5, and HERG), and the ratio of means
(Icoinjected/Icontrol)
was calculated. For Kv2.1, Kv2.2, and Kv1.5, the holding potential was
80 mV. Steady-state currents were measured at a test potential of +70
mV (5 or 50 K+ in bath). Kir2.2 steady-state and HERG tail
currents were recorded with 50 K+ in the bath at test
potentials to 100 mV with a prepulse to +20 mV. Asterisks
indicate that in all injection series, current amplitudes in oocytes
coinjected with KChAP were significantly higher than in oocytes without
KChAP (t test, p < 0.05).
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Fig. 4C summarizes the effects of KChAP on the current
amplitudes of a variety of K+ channels. In 13 batches of
oocytes coinjected with both Kv2.1 and KChAP, we observed an average
increase in whole oocyte currents of about 2.5-fold compared with
oocytes expressing Kv2.1 alone. KChAP also produced comparable
increases in Kv2.2 currents. We also examined the functional expression
of a deleted Kv2.1 in which the N-terminal 139 residues were removed
(21). As shown in Fig. 4C, coexpression with KChAP did not
significantly alter current amplitudes in oocytes expressing Kv2.1
N.
This suggests that binding between the Kv2.1 N terminus and KChAP is
critical for current enhancement.
Two K+ channels that did not exhibit N-terminal binding to
KChAP, Kir2.2 and HERG, were also tested. For each channel, experiments were done with whole cell control inward currents not exceeding 10 µA
at
100 mV. As shown in Fig. 4C, neither channel exhibited altered current amplitudes in the presence of KChAP.
KChAP Increased Functional Expression of Kv2.1 without Altering
Channel Kinetics or Gating--
The expression enhancement of Kv2.1
currents in the presence of KChAP could be due to an increase in the
number of functional channels at the cell surface or an alteration in
the kinetics or gating of individual channels. To distinguish between
these mechanisms, we used both immunocytochemical and
electrophysiological methods. We examined the surface expression of
Kv2.1 protein in oocytes expressing either Kv2.1 alone or Kv2.1 plus
c-myc-KChAP by immunocytochemistry. Fig.
5 shows the whole cell currents recorded from a single oocyte injected with Kv2.1 alone (panel A) or
Kv2.1 plus c-myc-KChAP (panel B). Currents were
increased about 3-fold in the c-myc-KChAP-coinjected egg.
The same two oocytes were fixed, sectioned, and co-stained with Kv2.1
and c-myc antibodies. Kv2.1 at the cell surface was
visualized by indirect immunofluorescence with a fluorescein
isothiocyanate-conjugated secondary antibody. Fluorescence at the
oocyte surface was much brighter in the egg expressing both Kv2.1 and
KChAP (panel D) compared with the one expressing Kv2.1 alone
(panel C), suggesting that the amount of Kv2.1 protein at
the cell surface was increased when the channel was coexpressed with
KChAP. No staining with the c-myc antibody was seen,
suggesting that KChAP is not present at the cell surface with Kv2.1
(panel E).

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Fig. 5.
KChAP increases the amount of Kv2.1 protein
at the oocyte surface. Macroscopic currents recorded 5 days after
cRNA injection from an oocyte expressing Kv2.1 (1.25 ng/µl cRNA;
panel A) or one expressing Kv2.1 (1.25 ng/µl) plus
c-myc-KChAP (250 ng/µl; panel B). Recordings
were obtained by stepping from a holding potential of 80 mV with
10-mV steps from 70 to +80 mV. Below each current trace is a section
of the same oocyte stained by indirect immunofluorescence with
anti-Kv2.1 antibody and fluorescein isothiocyanate-conjugated
anti-rabbit secondary antibody (panels C and D).
Note that the intensity of fluorescence at the oocyte surface is much
greater in the cell expressing Kv2.1 and KChAP (panel D).
The oocyte section in panel D was co-stained with
anti-c-myc and a tetramethylrhodamine B
isothiocyanate-conjugated secondary antibody. No tetramethylrhodamine B
isothiocyanate fluorescence was visible at the cell surface, suggesting
that c-myc-KChAP is not present there with Kv2.1
(panel E).
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To determine whether KChAP increased the total expression of Kv2.1
protein or only altered the subcellular distribution of the channel, we
examined membrane fractions from oocytes injected with Kv2.1 alone or
Kv2.1 plus KChAP by Western blotting. As shown in Fig.
6A, the amount of Kv2.1
protein in oocyte membranes was increased in oocytes coinjected with
KChAP (compare lanes 1 and 2). Densitometry of
the blots indicated about a 2.5-fold increase in Kv2.1 protein in the
presence of KChAP. Similar results were obtained when Kv2.1 was
immunoprecipitated from homogenates of total oocyte protein (data not
shown). This value is comparable with the increase observed in Kv2.1
currents with KChAP.

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Fig. 6.
KChAP increases the total amount of Kv2.1
protein and is localized primarily to the nucleus of coinjected
Xenopus oocytes. A, membranes (10 µg) prepared
5 days postinjection from oocytes injected with cRNAs for KChAP (250 ng/µl) plus Kv2.1 (5 ng/µl) (lane 1), Kv2.1 cRNA (5 ng/µl) alone (lane 2), uninjected oocytes (lane
3), or adult rat brain (lane 4) was separated by
SDS-polyacrylamide gel electrophoresis, blotted to polyvinylidene
difluoride, and Western blotted with a monoclonal antibody to Kv2.1.
Immunoreactive bands were visualized with ECL-Plus (Amersham Pharmacia
Biotech). Molecular mass markers (kDa) are indicated on the
left, and the position of Kv2.1 is marked on the
right. As a control for the antibody, two bands are visible
in the rat brain preparation. The upper band is thought to be a
phosphorylated form of the channel (27). No Kv2.1 is detected in
uninjected oocytes. Note that there is significantly more Kv2.1
detected in oocytes expressing both Kv2.1 and KChAP. B,
anti-c-myc Western blot of c-myc-KChAP in
fractions from enucleated oocytes. Nuclei were manually removed from
oocytes expressing either Kv2.1 or Kv2.1 plus KChAP at 3 days
postinjection. Equal amounts (10 µg) of solubilized nuclear protein
from Kv2.1 oocytes (lane 1) or Kv2.1 plus KChAP oocytes
(lane 2) were compared with soluble protein (Kv2.1 oocytes
(lane 3); Kv2.1 plus KChAP oocytes (lane 4)), and
membrane fractions (Kv2.1 oocytes (lane 5); Kv2.1 plus KChAP
oocytes (lane 6)) prepared from enucleated oocytes.
Anti-c-myc antibody detected a major band of
c-myc-KChAP (~68 kDa; indicated to right of
blot) only in oocytes coinjected with c-myc-KChAP cRNA.
Molecular mass markers (kDa) are marked on the left.
|
|
Since no KChAP was detected at the cell surface of co-expressing
oocytes, we examined the cellular distribution of KChAP in oocytes by
Western blotting. We manually removed the nuclei from pools of oocytes
expressing only Kv2.1 or Kv2.1 plus c-myc-KChAP and probed
the nuclear fraction as well as the soluble and membrane fractions
prepared from the enucleated oocytes with an anti-c-myc antibody to detect tagged KChAP. Most of the KChAP protein was present
in the nuclear fraction with smaller amounts detectable in the soluble
as well as the membrane fractions (Fig. 6B). The signal was
so strong in the nuclear material compared with the other two
fractions, however, that we were not able to estimate accurately the
relative amounts in each fraction. No Kv2.1 was detected in the nuclear
fraction, indicating that contamination with nonnuclear membranes was
minimal (data not shown).
The kinetics and gating of Kv2.1 channels were not altered in the
presence of KChAP. As shown in Fig.
7A, the voltage-dependence of
activation and the kinetics of activation and deactivation of Kv2.1
channels were not changed. Coexpression with KChAP did not alter the
sensitivity of Kv2.1 channels to TEA either (data not shown). Thus,
KChAP increased the number of functional channels at the cell surface
without altering individual Kv2.1 channel kinetics. The effect of KChAP
on Kv2.1 currents was saturable as shown in Fig. 7B.
Coexpression of a constant amount of Kv2.1 cRNA with increasing amounts
of KChAP cRNA resulted in increased steady state Kv2.1 currents until
saturation was reached. The influence of KChAP on the time course of
Kv2.1 expression in oocytes over a period of 9 days postinjection is
shown in Fig. 7C. In all of the electrophysiological
experiments presented thus far, KChAP and Kv2.1 cRNAs were mixed prior
to injection into oocytes in the same pipette. Importantly, when we
injected the two cRNAs separately into the same oocyte with different
pipettes, no enhancement of Kv2.1 currents was observed (data not
shown). This result suggests that KChAP exerts its effect on Kv2.1 in
oocytes by a direct physical association with the channel, which is
facilitated by the coinjection of both cRNAs into the same space inside
the oocyte.

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Fig. 7.
KChAP increases the functional expression of
Kv2.1 in Xenopus oocytes without altering voltage
dependence or channel kinetics. A, normalized and averaged
values of steady-state currents plotted as a function of test potential
in oocytes injected with the following cRNAs: Kv2.1 alone (0.62 ng/µl), filled circles (n = 10); Kv2.1
plus KChAP (125 ng/µl), filled triangles
(n = 9). Inset, superimposition of averaged
and scaled currents (at +70 mV test potential) from oocytes injected
with Kv2.1 alone and Kv2.1 plus KChAP. Holding potential was 80 mV.
100-ms pulses were given in 10-mV steps from 70 mV to +80 mV. 50 mM K+ in bath solution. Recordings were
performed 6 days after injection and are from one batch of oocytes.
Values of activation and deactivation
when pulsing to +70 mV and then back to 80 mV were 16.7 ± 0.2 and 5.8 ± 0.1 ms (n = 10), respectively, for
Kv2.1 alone and 15.8 ± 0.5 and 6.2 ± 0.2 ms
(n = 9), respectively, for Kv2.1 plus KChAP.
B, effect of increasing amounts of KChAP cRNA on Kv2.1
expression. Current amplitudes were measured at the end of the pulse to
+70 mV. In oocytes injected with Kv2.1 cRNA alone (0.62 ng/µl), the
current was 10.7 ± 0.9 µA; in oocytes coinjected with KChAP
cRNA, the currents were 23.2 ± 2.5, 39.5 ± 6.8, 43.7 ± 4.4, and 49.7 ± 6.2 µA at KChAP cRNA concentrations of 15, 31, 62, and 125 ng/µl, respectively; numbers of oocytes are indicated
(the same batch of oocytes as in A). *, a significant
difference from control Kv2.1 (p < 0.05; one-way
analysis of variance/Student-Newman-Keuls post hoc test).
C, time dependence of KChAP effect on Kv2.1 expression in
one injection series. Currents measured at the end of a 200-ms pulse to
+70 mV from oocytes injected with cRNAs for Kv2.1 alone (0.62 ng/µl,
filled circles) or Kv2.1 plus KChAP (125 ng/µl,
filled triangles). Numbers of oocytes are indicated in
parentheses above the points. Average currents in
oocytes injected with Kv2.1 cRNA alone were 2.8 ± 0.7, 6.1 ± 1.9, and 5.3 ± 1.5 µA measured at 3, 6, or 9 days
postinjection, respectively. Oocytes coinjected with Kv2.1 and KChAP
cRNAs had average currents of 6.8 ± 2.8, 15.7 ± 4.4, and
19.9 ± 5.6 mA measured at 3, 6, or 9 days postinjection,
respectively. **, a significant difference from Kv2.1 tested on the
same day after injection (p < 0.05; t
test).
|
|
In Vitro Association of KChAP and KV2.1--
The
electrophysiological data suggest that a direct interaction between
Kv2.1 and KChAP occurs and is responsible for the enhancement in Kv2.1
currents observed in oocytes. We used an in vitro binding
assay to demonstrate the ability of the two proteins to associate.
Kv2.1 and KChAP cRNAs were translated in vitro either separately or together in a rabbit reticulocyte lysate in the presence
of [35S]methionine. We used a commercially available
anti-Kv2.1 polyclonal antiserum to immunoprecipitate Kv2.1 and analyzed
the immunoprecipitated material with SDS-polyacrylamide gel
electrophoresis and fluorography to detect the presence of associated
KChAP. Since an antibody to KChAP was not available, we used an epitope
tag fused to the N terminus of KChAP (c-myc) to allow
detection. As we had previously shown in Fig. 5, the c-myc
tag did not interfere with the functional interaction of KChAP and
Kv2.1. Control reactions with Kv2.1 translated alone showed that
in vitro translated Kv2.1 was immunoprecipitated with
anti-Kv2.1 antibody but not anti-c-myc antisera (Fig.
8, lanes 1 and 2).
Similarly, anti-c-myc antisera immunoprecipitated c-myc-KChAP but not Kv2.1 (Fig. 8, lanes 3 and
4). Kv2.1 antibody coimmunoprecipitated complexes of Kv2.1
and KChAP when the two cRNAs were cotranslated (Fig. 8, lane
5) but not when the two cRNAs were translated in separate
reactions and mixed together prior to the addition of primary antibody
(Fig. 8, lane 6). This result suggests that the association
of KChAP with Kv2.1 occurs cotranslationally, since the mature proteins
added after translation did not coimmunoprecipitate. All reactions
involving translation of Kv2.1 cRNA shown here included canine
pancreatic microsomes to allow the channel to insert into membrane as
it was synthesized. When microsomes were omitted from cotranslation
reactions, no coimmunoprecipitation of the two proteins was observed
(data not shown).

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Fig. 8.
Coimmunoprecipitation of Kv2.1 and KChAP from
in vitro translation reactions. Kv2.1 and
c-myc-KChAP cRNAs were translated in vitro in
rabbit reticulocyte lysates with [35S]methionine. Immune
complexes were analyzed by SDS-polyacrylamide gel electrophoresis and
fluorography. The following in vitro translation reactions
were used for immunoprecipitation: Kv2.1 translated alone (lanes
1 and 2), c-myc-KChAP translated alone
(lanes 3 and 4), Kv2.1 and c-myc-KChAP
translated in the same reaction (lane 5), and Kv2.1 and
c-myc-KChAP translated separately but mixed together prior
to immunoprecipitation (lane 6). The antibody used for
immunoprecipitation in each sample is indicated at the
bottom. The positions of in vitro translated
Kv2.1 and c-myc-KChAP are indicated on the right,
and mobilities of the molecular weight markers are marked on the
left. KChAP and Kv2.1 coprecipitate with anti-Kv2.1 antibody
when the two proteins are cotranslated (lane 5) but not when
the two proteins are translated in separate reactions and mixed prior
to immunoprecipitation (lane 6). Note that the Kv2.1
antibody does not cross-react with c-myc-KChAP (lane
4).
|
|
 |
DISCUSSION |
We have cloned a novel gene, kchap, which encodes a
K+ channel regulatory protein. All of our data point to a
direct interaction between KChAP and Kv2.1 as responsible for the
current enhancement observed in Xenopus oocytes. Yeast
two-hybrid assays showed an interaction between KChAP and the N
terminus of Kv2.1. When an N-terminally truncated Kv2.1 was expressed
in oocytes, no expression enhancement was observed. Also, Kv2.1 and
KChAP were co-immunoprecipitated from in vitro translations
in which the two proteins were translated in the same reaction, but not
when they were translated separately and mixed together before
immunoprecipitation. This suggests a cotranslational association of the
two proteins, which is also supported by oocyte data. In order to
observe Kv2.1 current enhancement in oocytes, the KChAP and Kv2.1 cRNAs
had to be mixed together and coinjected with the same pipette. No
changes in Kv2.1 currents were noted when the two cRNAs were injected
from two separate pipettes, suggesting that the two proteins must be
translated in close proximity. However, in the oocyte, KChAP might bind
only transiently to Kv2 channels and not remain stably attached to the
mature channel as evidenced by the lack of KChAP staining at the oocyte
surface.
In Xenopus oocytes, KChAP does not appear to alter the time
course of Kv2.1 current expression. Without KChAP, the amplitude of
whole oocyte Kv2.1 currents continues to increase slowly from the time
of injection through about 6 days, at which time the current levels
plateau and remain fairly constant through 9 days postinjection. This
would suggest that transit of Kv2.1 channels to the oocyte surface is a
rather slow process. KChAP does not change this profile but increases
the amplitude of Kv2.1 currents recorded at each time point.
One possible explanation for the effect of KChAP on Kv2.1 expression is
that KChAP may act as a chaperone to facilitate either the translation,
assembly, or stability of Kv2.1 channels. An increase in the amount of
Kv2.1 protein in oocyte membranes in the presence of KChAP as seen by
Western blotting is consistent with this interpretation. Kv
subunits
also exhibit chaperone-like effects on Kv channels. However, Kv
subunits (Kv
1 and Kv
2) form stable complexes with Kv
subunits,
and although they are cytoplasmic proteins, travel to the cell surface
with pore-forming Kv
subunits and remain tightly attached there as
part of mature channel complexes (28-30). KChAP defines a different
structural gene from Kv
subunits, however, and there are significant
differences between Kv
and KChAP action as well. Unlike Kv
subunits 1-3, KChAP modulates both Kv2.2 and Kv2.1 currents but has no
effect on Kv1.5 currents.
The significance of KChAP binding to Kv
or Kv
1 subunits is
currently unknown. KChAP binds to the conserved C terminus of Kv
subunits. This is the same domain to which the N termini of Kv
1
subunits have been shown to bind (14, 25, 26). One could envision that
KChAP might modulate the association of Kv
1 and Kv
subunits and,
thus, indirectly affect the expression and/or kinetic properties of Kv1
currents, but this is speculative at present.
Although KChAP and Kv
subunits share no sequence homology, KChAP is
50% identical to the human GBP (23). GBP is a novel protein that was
cloned by interaction with the Gu/RH-II helicase in the yeast
two-hybrid system. GBP did not interact with either Kv
or Kv
subunits in yeast two-hybrid assays and did not modulate Kv2.1 currents
in coexpression assays in oocytes. GBP is a nuclear protein that has
been shown to produce proteolytic cleavage of Gu/RH-II (23). The
significance of the sequence similarity between the two proteins is
unclear at present. Interestingly, however, KChAP protein was primarily
detected in the nuclei of injected oocytes. The relationship of the
nuclear localization of KChAP with its effects on Kv2 channels is
presently unclear. If a direct interaction between KChAP and Kv2.1 is
required for expression enhancement, then this may occur transiently
prior to KChAP moving to the nucleus. We cannot rule out, however, that
KChAP may play an as yet unknown role in the nucleus, which results in
enhanced Kv2.1 protein and current levels.
To summarize, we have described a novel gene product, KChAP, which
binds to Kv
and Kv
subunits. KChAP increases currents expressed
by Kv2.1 and Kv2.2 but not Kv1.5, and the expression enhancement is due
to an increase in total protein as well as in the number of functional
Kv2 channels at the cell surface. We speculate that KChAP may act as a
novel type of chaperone protein for Kv2 channels.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. Post for Kv2.1, Kv2.2, and
Kv6.1 fragments in pGBT9; Dr. E. Ficker for sectioning oocytes; Drs. S. Snyder and J. Trimmer for the Kv2.2 cDNA clone; Dr. M. Keating for
the HERG cDNA clone; and T. Carroll and Dr. W. Dong for expert
technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL-57146 (to B. A. W.), NS-23877, HL-36930, and HL-55404 (to A. M. B.) and a grant from the American Heart Association, Northeast Ohio Affiliate (to B. A. W.).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/EMBL Data Bank with accession number(s) AF032872.
§
To whom correspondence should be addressed: Rammelkamp Center, 2500 MetroHealth Dr., Cleveland, OH 44109-1998. Tel.: 216-778-8984; Fax:
216-778-8282; E-mail: bwible{at}research.mhmc.org.
Present address: School of Kinesiology, Simon Fraser
University, Burnaby, British Columbia V5A 1S6, Canada.
1
The abbreviations used are: Kv channel,
voltage-gated K+ channel; kb, kilobase pair(s); GBP,
Gu-binding protein; IP, immunoprecipitation.
 |
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