From the Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received for publication, June 1, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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Auxiliary Kv Voltage-gated K+ channels are multimeric proteins that
consist of four pore-forming Kv Although many studies have shown that Kv Constructions--
Rat Kv4.3 short (14) and long (15) isoform
cDNAs were subcloned into pcDNA3 (Invitrogen, Carlsbad CA). Rat
Kv2.1, Cell Culture and Transfection--
HEK 293 cells (American Type
Culture Collection, Manassas, VA) were maintained at 37 °C under 5%
CO2 atmosphere in high glucose Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. Transient
transfection was carried out by the calcium phosphate-DNA
coprecipitation method (Transfinity, Life Technologies, Inc.).
For patch clamp recording, pCMV-Kv4.3 alone (0.3 µg for 60-mm dish)
or in combination with 5×-excess pCMV-Kv
For immunoblot analysis, cells on 100-mm plates were transfected with
expression constructs at the same ratio as for patch clamp recording
(0.9 µg of pCMV-Kv4.3/dish). Transfected cells were divided into five
60-mm plates at various densities 5 h after transfection and used
for immunoblot analysis at various days after transfection. Cell
extracts were prepared by suspending the collected cell pellet in 100 µl of lysis buffer (20 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 1 mM iodoacetamide,
0.2 mM phenylmethylsulfoxide, and 1 mM EDTA).
The suspension was kept on ice for 10 min and centrifuged at
10,000 × g for 5 min to remove nuclear debris.
Electrophysiological Recording--
Whole-cell voltage-clamp
recording (17) was performed with an EPC-9 patch-clamp amplifier using
the Pulse program (HEKA Electronik, Lambrecht, Germany) on a Power
Macintosh computer. Patch pipettes were filled with a solution
containing 140 mM KCl, 1 mM MgCl2,
1 mM EGTA, and 10 mM HEPES (pH 7.4). Bath
solution contained 155 mM NaCl, 5 mM KCl, 2 mM MgCl2, 20 mM glucose, 10 mM HEPES (pH 7.4). Series resistance compensation was set
at 70%. Peak currents were converted into conductance (G)
by the formula G = I/(Vm Biochemical Association Assays--
Two days after transfection,
transfected HEK 293 cells on 100-mm dishes were harvested with ice-cold
phosphate-buffered saline. Triton extract was prepared by suspending
the pelleted cells in 0.4 ml of solution containing 1% Triton X-100,
20 mM Tris-HCl (pH 7.9), 50 mM NaCl, and 5 mM imidazole. The extract was mixed with 100 µl (50%
slurry) of preactivated His-bind resin (Novagen, Milwaukee WI) for
2 h with gentle shaking. The resin was washed 5× with the same
solution, except that the imidazole concentration was 40 mM. The bound materials were then eluted with 0.1 M EDTA.
Immunoprecipitation--
Immunoprecipitation was performed with
polyclonal anti-panKv
Whole rat brain tissue was homogenized in 0.32 M sucrose
solution supplemented with 1 mM iodoacetamide, 0.2 mM phenylmethyl sulfonate, and 1 mM EDTA. The
homogenate was centrifuged at 1,000 × g for 10 min to
remove nuclear debris. The supernatant was transferred to a new tube
and centrifuged at 100,000 × g for 1 h. Protein concentration was determined using Bio-Rad protein assay reagent with
bovine serum albumin as a standard. The nuclei-free membrane fraction
was suspended in a solution containing 2% Triton X-100, 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride
and 1 mM iodoacetamide at a protein concentration of ~10
mg/ml. Triton extract was then obtained by centrifugation of the
suspension at 100,000 × g for 30 min. After
preclearing with fixed protein A-containing Staphylococcus
aureus cells (Pansorbin, Calbiochem), Triton extract was incubated
overnight with monoclonal anti-Kv4.3 or polyclonal anti-pan Kv Immunoblot Analysis--
Proteins were separated on a 7.5%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
The membrane was coated with 5% nonfat dry milk in phosphate-buffered
saline containing 0.1% Tween 20 and probed with primary antibody
followed by incubation with horseradish peroxidase-conjugated secondary
antibody. Primary antibodies against Kv4.3, GFP, and Express tag were
purchased from Alomone labs (Jerusalem, Israel), MBL
International Corp. (Watertown MA), and Invitrogen,
respectively. Anti-Kv1.4 (18), anti-Kv2.1 (19), and polyclonal
anti-panKv Confocal Microscopy--
Confocal Images of GFP fluorescence
were taken on a Molecular Dynamic 2001 scanning laser confocal
microscope with a 60× oil immersion objective lens (1.4 NA) using
488-nm excitation and 510-nm emission filters with 3% maximal laser
intensity. Cell surface localization was evaluated by comparing the
location of fluorescence with bright field images of cells.
Kv4.3 Proteins Are Present in Association with Kv Kv
To test if this elevation in current density was correlated with an
increase in Kv4.3 protein level, we measured channel proteins by
immunoblot analysis (Fig. 3A).
The Kv4.3 protein level significantly increased when coexpressed with
Kv Effects of Kv
The most profound effect produced by Kv
We also determined whether association with Kv The C Terminus of Kv4.3 Proteins Is Required for Association with
Kv
Kv1 family channels interact with Kv
We also used protein biochemical assays to test association. Histidine
(His6)-tagged wild type and chimeric channel proteins were
expressed with Kv Kv Association of Kv In contrast to specific alterations in channel gating, association of
Kv Despite the similarity between Kv4 and Kv1 family polypeptides, our
data indicate that the two family proteins exhibit distinct requirements for interaction with Kv Recently identified Ca2+-binding subunits (KChIP) are
likely to play important roles in controlling the expression and
function of Kv4 family channels (28). In addition, our results indicate that Kv4.3 channels are present, at least in part, in association with
Kv subunits form complexes with Kv1
family voltage-gated K+ channels by binding to a part
of the N terminus of channel polypeptide. This association influences
expression and gating of these channels. Here we show that Kv4.3
proteins are associated with Kv
2 subunits in the brain. Expression
of Kv
1 or Kv
2 subunits does not affect Kv4.3 channel gating but
increases current density and protein expression. The increase in Kv4.3
protein is larger at longer times after transfection, suggesting that
Kv
-associated channel proteins are more stable than those without
the auxiliary subunits. This association between Kv4.3 and Kv
subunits requires the C terminus but not the N terminus of the channel
polypeptide. Thus, Kv
subunits utilize diverse molecular
interactions to stimulate the expression of Kv channels from different families.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits in association with auxiliary subunits. Unlike
subunits that exhibit overall structural
similarity to various voltage-gated ion channels, auxiliary subunits
are diverse and show specificity for association with particular
classes of K+ channels. Kv
subunits belong to the
NAD(P)H-dependent oxidoreductase superfamily (1) and
contain a conserved catalytic domain with a NADPH-binding site (2).
These proteins by themselves form a tetramer (3), making an
4
4 channel complex (4-6). There are at
least four mammalian genes that encode Kv
subunits. Previous studies
have established that Kv
1, -2, and -3 gene products are components
of Kv1 family channels. Indeed, all products from the four genes
contain a conserved core region with variable N-terminal peptides. The
core region of
subunits was found to be sufficient for association
with a part of the N terminus of Kv1 family
subunits that is highly
conserved within this family (5, 6). However, more recent studies
indicate that Kv
subunits can interact with heterologously expressed
Kv4.2 (7, 8) and EAG1
family (9) channels. Furthermore, it appeared that Kv
4 subunits are
present as complexes with Kv2.2 proteins in rat brain (10). Likewise,
plant Kv
subunits (KAB) are associated with KAT1 channels (11). Hence, auxiliary Kv
subunits are structurally well
characterized, yet the specificity and mechanism of interaction between
Kv
and
subunits remain obscure.
subunits influence expression and function of K+
channels. Specifically, distinct Kv
subunits differentially affect
heterologously expressed Kv1 family channels. A long-stretched
N-terminal peptide in Kv
1 and Kv
3 gene products produces rapid
inactivation on most of Kv1 family channels by a mechanism similar to
the action of a ball peptide present at the N terminus of some channel
subunits (12). Furthermore, Kv
2 subunits have been shown to increase stability and cell surface expression of Kv1 family channels (13) without producing rapid inactivation. Thus, expression of distinct
Kv
subunits controls excitability by differentially affecting the
expression and gating of Kv1 family channels.
1 and Kv
2 subunits can
associate with heterologously expressed channels from diverse families,
it remains unclear whether these auxiliary subunits are present as
complexes with non-Kv1 family channels in native cells. Furthermore,
structural features of the interaction between Kv
subunits and
non-Kv1 family channel polypeptides remain unknown. To address these
questions, we examined complexes consisting of Kv
and Kv4.3 channel
subunits. We show here that Kv4.3 proteins are associated with Kv
2
subunits in the brain and that this interaction requires the C terminus
of the channel polypeptide.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.1. and
2.1 expression constructs were previously
obtained (7). Chimeric constructs between Kv4.3 short splicing form and
Kv2.1 were made using a two-step overlapped polymerase chain reaction with primers that corresponded to the border region of Kv4.3 and Kv2.1
sequences. Kv4.3-Kv2.1N contains amino acids 1-184 of rat Kv2.1
polypeptide (16) linked to amino acids 183-636 of rat Kv4.3
polypeptide (14). Kv4.3-Kv2.1C consists of amino acids 1-406 of the
Kv4.3 polypeptide connected to amino acids 413-853 of the Kv2.1
polypeptide. For coisolation experiments, wild type and chimeric
channel cDNAs were subcloned in-frame into pcDNA3.1/HisC vector
(Invitrogen) using a polymerase chain reaction-based method. GFP-tagged
Kv
subunit constructs were prepared by subcloning the whole coding
region of Kv
1.1 and Kv
2.1 cDNAs at the end of enhanced green
fluorescent protein-coding sequence of EGFP-C1 (CLONTECH, Palo Alto CA). All the obtained
constructs were verified by DNA sequencing.
1.1 or pCMV-Kv
2.1 were
used. In addition, EGFP-C1 plasmid (50 ng/60-mm dish) was cotransfected
to aid in the identification of transfected cells. Transfected cells in
60-mm plates were split into 35-mm dishes 5 h after transfection
and used for whole-cell recordings 48-72 h after transfection.
Vrev) assuming a reversal potential
Vrev of
84 mV, where Vm is
the membrane voltage of depolarization pulses. Using the first-order
Boltzmann equation G/Gmax = 1/(1 + exp[(V1/2
V)/slope
factor]), the half-maximal voltages (V1/2) and the slope factors were
acquired. Statistical analysis was carried out using the Mann-Whitney
two-tailed test. All the data in the text are presented as means ± S.E.
antibody (18) or monoclonal anti-Kv4.3
antibody. The latter antibody was generated against a synthetic peptide
corresponding to a part of the N terminus of rat Kv4.3 polypeptide
(amino acids 25-40)
CPMPLAPADKNKRQDE.2
(20)
antibody and Pansorbin. The bound materials were collected by
centrifugation and washed 4× with the same Triton-containing solution.
The bound materials were eluted by heating in 2× SDS sample buffer and
subjected to immunoblot analysis.
(20) antibodies were previously generated. Bound antibody
was detected by chemiluminescence method (PerkinElmer Life Sciences).
Immunoreactivity was quantified using densitometry of the developed films.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 Subunits in
the Brain--
To test for the presence of Kv4.3·Kv
subunit complexes, we first used anti-Kv4.3 monoclonal antibody for
immunoprecipitation from rat brain extract. Anti-Kv4.3 antibody
effectively and specifically precipitated its targeted channel proteins
but not Kv1.4 proteins (Fig.
1A). Importantly, the
immunoprecipitated material was found to contain significant
immunoreactive Kv
subunit proteins detected with polyclonal
anti-panKv
antibody (20). This antibody detects two bands on the
blot with distinct sizes. The larger and smaller bands are known to
correspond to Kv
1 and Kv
2, respectively (13, 19, 20). In
addition, the larger band may also contain Kv
3 subunits. We found
that only smaller molecular weight Kv
2 subunits were significant in
the precipitated material. To further obtain evidence for the presence
of Kv4.3·Kv
complexes in the brain, anti-panKv
antibody was
used for immunoprecipitation from the brain extract (Fig.
1B). The antibody precipitated significant Kv4.3 proteins in
addition to its targeted proteins. In contrast, no detectable Kv2.1
proteins were found in the precipitated material. Hence, brain Kv4.3
channel proteins are present in association with Kv
2 subunits.
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Fig. 1.
Kv4.3 proteins are associated with
Kv 2 subunits in rat brain. Triton extract
of whole rat brain was subjected to immunoprecipitation (IP)
with monoclonal anti-Kv4.3 (A) or polyclonal anti-panKv
(B) antibody. Brain extract, unbound fraction
(sup), and precipitated material (ppt) were
examined by immunoblot (IB) analysis with anti-Kv1.4,
anti-Kv2.1, anti-panKv
, and polyclonal anti-Kv4.3 antibodies. The
larger and smaller bands detected with anti-panKv
antibody have been
shown to correspond to Kv
1/3 and Kv
2 subunits, respectively (13,
19, 20). Note that a fraction of immunoreactive Kv
subunits (Kv
2)
is recovered in the anti-Kv4.3 antibody-precipitated material.
Anti-panKv
antibody also precipitated a portion of Kv4.3
proteins.
Subunits Increase Kv4.3 Current Density and
Proteins--
Functional consequences of Kv
subunit association on
Kv4.3 channels was examined in transfected HEK 293 cells. We first
examined the effect of Kv
subunits on Kv4.3 current density.
Coexpression of either Kv
1.1 or Kv
2.1 subunits led to a 2-3-fold
increase in the peak current density (Fig.
2); at +50 mV, peak current density was
0.5 ± 0.05 nA/pF (n = 6) for Kv4.3 alone,
1.7 ± 0.4 nA/pF (n = 5) for Kv4.3 + Kv
1.1, and
1.8 ± 0.5 nA/pF (n = 5) for Kv4.3 + Kv
2.1.
Increases in current density produced by Kv
1.1 or Kv
2.1 subunits
were significant at test pulses higher than
20 mV (p < 0.05). No significant change in HEK 293 cell endogenous current was
detected; peak current density at +50 mV was 39.2 ± 8.7 pA/pF
(n = 4) for mock transfection, 29.3 ± 9.9 pA/pF
(n = 4) for Kv
1.1, and 39.7 ± 7.1 pA/pF
(n = 4) for Kv
2.1. Thus, Kv
subunits increase
functional cell surface Kv4.3 channels.
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Fig. 2.
Kv subunits increase
Kv4.3 channel current density. HEK 293 cells were transfected with
expression constructs for Kv4.3 alone, Kv4.3 + Kv
1.1 or Kv4.3 + Kv
2.1 (
:
= 1:5). Outward currents were elicited by 200-ms
depolarization pulses with 10-mV increments from a holding potential of
70 mV. Current density was obtained with cellular capacitance to
account for deviation in cell size. A, representative
current traces are shown. Scale bars: horizontal, 50 ms;
vertical, 250 pA/pF. B, current-voltage
relationships are presented. Points and error
bars represent mean and S.E., respectively. n
4 for each transfection condition. Current density is significantly
larger with expression of Kv4.3 + Kv
1.1 or Kv4.3 + Kv
2.1 than
that of Kv4.3 alone at a membrane depolarization above
20 mV
(p < 0.05).
2.1 subunits 3 or 4 days after transfection (Fig. 3B,
n = 6, p < 0.05). In contrast,
coexpression of these auxiliary subunits did not produce changes in
Kv2.1 protein levels. Similar increases in Kv4.3 proteins were also
produced by Kv
1.1 at 3 days after transfection (Fig. 3C).
Hence, Kv
subunits increase total cellular Kv4.3 protein level.
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Fig. 3.
Kv subunits increase
Kv4.3 channel protein level. HEK 293 cells were transfected with
expression constructs for Kv4.3 or Kv2.1 channels with Kv
2.1 or
control vector (
:
= 1:5). Various days after transfection,
cells were harvested for immunoblot analysis. A, immunoblots
with anti-Kv4.3 antibody (top) and anti-Kv2.1 antibody
(bottom) are shown. B, densitometry was used to
quantify immunoreactivity. Kv4.3 (top) and Kv2.1
(bottom) immunoreactivities were normalized with total
protein amounts. n = 6 for each time point.
Points and error bars represent mean and S.E.,
respectively. An asterisk indicates p < 0.05. C, cells were transfected expression constructs for
Kv4.3 channel proteins with Kv
1.1, Kv
2.1, or control vector.
Three days after transfection, cell extracts were prepared, and channel
immunoreactivities were measured. An asterisk indicates
p < 0.05. w/o, without.
Subunits on Kv4.3 Channel Gating--
Next we
examined the gating properties of Kv4.3 channels with or without
coexpression of Kv
subunits. Excess Kv
subunits (
:
= 1:5) were used to enhance formation of Kv4.3·Kv
complexes. Kv
subunits have been shown to shift voltage dependence of activation of
Kv1 family channels to the left. However, we found that Kv
1.1 or
Kv
2.1 subunits produce no significant change in voltage dependence of activation of Kv4.3 channels (Fig.
4A). The voltage for
half-maximal activation and the slope factor were
7.00 ± 1.08 mV and 14.0 ± 0.99 mV (n = 5) for Kv4.3 alone,
7.83 ± 1.01 mV and 15.8 ± 0.96 mV (n = 4)
for Kv4.3 + Kv
1.1, and
8.43 ± 1.13 mV and 14.4 ± 1.05 mV (n = 4) for Kv4.3 + Kv
2.1, respectively. Thus,
Kv
subunits do not influence activation of Kv4.3 channels.
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Fig. 4.
Kv subunits produce
no marked effects on Kv4.3 channel gating. Outward currents were
measured in HEK 293 cells transfected with expression constructs for
Kv4.3 alone, Kv4.3 + Kv
1.1, or Kv4.3 + Kv
2.1 (
:
= 1:5). Conductance-voltage relation (A) was obtained
using pulse paradigm shown in Fig. 2. Currents at 50-mV depolarization
pulses were normalized, and the averages of these traces
(n
4) are shown in B. Steady-state
inactivation (D) and recovery from inactivation
(F) were determined using pulse paradigms shown in
C and E, respectively. n
4 for
each point. Points and error bars represent mean
and S.E., respectively. No significant differences in any of the three
gating properties between any two conditions were detected.
subunits on Kv1 family
channels is acceleration of inactivation, due to the ball and chain
mechanism. Thus, the effect of Kv
1.1, which contains a ball peptide,
as well as Kv
2.1 on inactivation properties of Kv4.3 channels was
examined. Coexpression of these auxiliary subunits did not
significantly affect time constant of inactivation (Fig. 4B); time constant for inactivation at +50 mV was 50.5 ± 5.1 ms for Kv4.3 alone (n = 5), 52.6 ± 4.3 ms
(n = 4) for Kv4.3 + Kv
1.1, and 57.2 ± 2.2 ms
(n = 4) for Kv4.3 + Kv
2.1. We also measured the
steady-state inactivation using a test pulse to +40 mV after a
conditioning prepulse at various voltages (Fig. 4, C and
D). The voltage for half-maximal inactivation and the slope
factor were
48.4 ± 0.33 mV and
5.2 ± 0.3 mV
(n = 5) for Kv4.3 alone,
41.5 ± 0.23 mV and
6.8 ± 0.2 mV (n = 4) for Kv4.3 + Kv
1.1, and
47.2 ± 0.45 mV and
6.85 ± 0.4 mV (n = 4) for Kv4.3 + Kv
2.1. Therefore, Kv
subunits, regardless of the
presence of a ball peptide, do not influence inactivation of Kv4.3 channels.
1.1 or Kv
2.1 might
influence the recovery from inactivation (Fig. 4, E and F). A protocol of two consecutive depolarizing test pulses
interrupted by variable interpulse intervals at
70 mV was used to
determine the time course of recovery from inactivation (Fig.
4E). The recovery from inactivation was fitted by a single
exponential function. Time constant for recovery from inactivation was
181 ± 24 ms for Kv4.3 alone (n = 6), 169 ± 11 ms for Kv4.3 + Kv
1.1 (n = 5), and 176 ± 38 ms for Kv4.3 + Kv
2.1 (Fig. 4F, n = 5).
Thus, Kv
subunits do not affect recovery from inactivation. Taken
together, Kv
1.1 and Kv
2.1 subunits produce no marked effects on
Kv4.3 channel gating.
Subunits--
To assess association of Kv channel
subunits
with Kv
subunits, we first examined localization of GFP-tagged Kv
subunits upon coexpression of various channel proteins. Confocal
microscopy revealed that GFP-Kv
2.1 (Fig.
5A) or GFP-Kv
1.1 (data not
shown) were predominantly present in the cytosol in the absence of
channel
subunits. Coexpression of Kv4.2 or Kv4.3 proteins as well
as Kv1.4 proteins, but not Kv2.1 proteins, localized the fluorescence to plasma membrane (Fig. 5A). Similarly, a splicing variant
of Kv4.3, which contains a 19-amino acid insertion at the C terminus (15), targeted GFP-Kv
fusion proteins to plasma membrane. Thus, Kv4
family channel proteins regardless of the presence or absence of the
insertion can associate with Kv
subunits.
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Fig. 5.
The C terminus of Kv4.3 is required for
localizing GFP-tagged Kv subunits to plasma
membrane. A, HEK 293 cells were transfected with
expression constructs for GFP-tagged Kv
2.1 and indicated Kv channel
subunits at the ratio of
:
= 5:1. Kv4.3S and Kv4.3L
represent short and long splicing isoforms of Kv4.3
subunits,
respectively. Confocal images were obtained 2 days after transfection.
Coexpression of Kv4.2, Kv4.3S, Kv4.3L, or Kv1.4 alters the location of
the fluorescence to cell surface (a ring-like structure), whereas the
fluorescence is predominantly seen in cytosol with GFP-Kv
2.1 alone
or GFP-Kv
2.1 + Kv2.1. B, wild type or chimeric channel
subunits were expressed with GFP-Kv
2.1 fusion proteins into HEK
293 cells at the expression construct ratio of
:
= 5:1.
Confocal images 2 days after transfection are shown. Kv4.3-2.1N and
Kv4.3-2.1C indicate chimeric Kv4.3 proteins containing the N terminus
and C terminus of Kv2.1 polypeptide instead of their own, respectively.
Note that replacing the C terminus, but not the N terminus, eliminated
the ability of Kv4.3 proteins to transfer GFP-Kv
subunits to plasma
membrane.
subunits via a highly conserved
region of the N terminus. Although the corresponding region of Kv4
family polypeptides exhibits significant sequence homology, this
peptide itself was insufficient for association with Kv
subunits (5,
6). To identify the region important for association, we generated
chimeric channel proteins consisting of Kv4.3 and Kv2.1 polypeptides.
If chimeric proteins are capable of interacting with Kv
subunits,
the fluorescence would be expected in plasma membrane or other
membrane-associated compartments. Replacing the N terminus of Kv4.3
protein with that of Kv2.1 polypeptide (Kv4.3-Kv2.1N) did not affect
the ability to localize Kv
2.1 (Fig. 5B) and Kv
1.1
(data not shown) subunits to plasma membrane and other
membrane-associated regions. In contrast, substituting the C terminus
of Kv4.3 protein with that of Kv2.1 protein (Kv4.3-Kv2.1C) eliminated
plasma membrane localization of the fluorescence. This chimeric channel
(Kv4.3-Kv2.1C) was functional as confirmed by patch clamp recording
(data not shown). Thus, the C terminus, but not the N terminus, of
Kv4.3 polypeptide is required for localizing Kv
subunits at plasma membrane.
1.1 or Kv
2.1 subunits. After purification with
His-binding beads, copurified Kv
subunit proteins were examined by
immunoblot analysis (Fig. 6).
Significantly higher levels of immunoreactive Kv
1.1 or Kv
2.1
proteins were recovered from cells coexpressed with Kv4.3 or
Kv4.3-Kv2.1N than those with Kv2.1 or Kv4.3-Kv2.1C. These results
demonstrate that the C terminus, but not the N terminus, of Kv4.3
channels is necessary for association with Kv
subunits.
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Fig. 6.
The C terminus of Kv4.3 proteins is necessary
for association with Kv subunits.
(His)6- and Express epitope-tagged wild type and chimeric
subunits were expressed with Kv
1.1 (left) or Kv
2.1
(right) subunits into HEK 293 cells at the expression
construct ratio of
:
= 1:1. Two days after transfection,
cell lysate was prepared and subjected to isolation with His-bind
beads. Input and ppt represent immunoblots
obtained with cell lysate and His-bind bead-bound fraction,
respectively. A small amount of Kv
subunit proteins similar to the
Kv2.1 and Kv4.3-2.1C lanes was also obtained without
expression of any channel
subunits.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits have been suggested to interact with various
K+ channels including Kv2.2 (10), Kv4.2 (7, 8), and several EAG family (9) and plant KAT1 (11) channels in addition to Kv1 family
channels. In particular, Kv4.2 channels were found to interact with
Kv
1 and -2 subunits in heterologous expression systems (7, 8),
suggesting that the same Kv
subunits might form complexes with
K+ channels from the two different families. However,
previous studies had not addressed whether such association actually
occurs with native channels and did not identify a physiological effect
of the association. In this study, we have shown that Kv4.3 proteins are associated with Kv
2 subunits in the brain. We also found that
coexpression of Kv
subunits leads to increases in Kv4.3 current
density and protein level without altering gating properties. Finally,
this association requires the C terminus, but not the N terminus, of
the channel polypeptide. Thus, the same Kv
2 subunits influence
expression and function of channels from the two different families by
distinct interaction mechanisms.
subunits appeared to produce different effects on
interacting channels. Our results indicate that the ball peptide of
Kv
1.1 does not alter inactivation of Kv4.3 channels. Similarly, it
has been shown that Kv
1 does not markedly influence inactivation
kinetics of Kv4.1 (21), Kv4.2, (8) and Drosophila Shal (5)
channels. Likewise, Drosophila Kv
subunit HK
was unable to produce rapid inactivation on EAG or ERG channels (9). Thus, the effect of Kv
subunit ball peptide depends on associating channels. The lack of the ball peptide effect may be due to differences in the ball peptide-accepting structure of channels. Recent work indicated that a difference in the amino acid sequence in a small region of the linker between S4 and S5 affects the interaction with the
Kv
1 ball peptide (22). To support this possibility, one of the
important amino acids identified in the above-mentioned study, Arg-324
in Kv1.1, is altered to serine at the corresponding position (Ser-421)
in Kv4.3. Another possibility is that Kv
subunits are positioned in
such a way that the ball peptide cannot access the internal pore region
of Kv4.3 channels. Since we found that structural requirement for
association with Kv
subunits differs between Kv1 family and Kv4.3
channels, it is possible that the relation of a Kv
tetramer to a
Kv4.3
tetramer does not allow the ball peptide to act on its
receptor region of the channel. In addition to the difference in rapid
inactivation, a recent study revealed that sensitivity to
O2 tension differs between Kv
subunit-associated Kv4.2
and Shaker channels (8). In analogy to the difference in
rapid inactivation, this difference in hypoxia response may arise from
distinct ability of channels to respond O2 tension signals.
Alternatively, specific interaction of Kv4.2 channels with Kv
subunits may be essential for the regulation. Further structural and
functional information of channel complexes may resolve these issues.
subunits commonly increases current amplitude or density of
various channels. This has been observed with Kv1 family (13), Kv2.2
(10), and EAG family (9) channels. Our results also revealed that Kv
subunits increase Kv4.3 current density and proteins. In addition to
Kv
subunits, other channel auxiliary subunits for K+
channels as well as Na+ and Ca2+ channels have
been shown to increase associating channel current density. It is
assumed that the exit from endoplasmic reticulum is the rate-limiting
step for plasma membrane protein targeting. Therefore, the generally
observed increase in current density by various auxiliary subunits may
be due to masking of endoplasmic reticulum retention signals present in
channel proteins. This mechanism has been implicated for controlling
selective cell surface expression of heteromeric ATP-sensitive
K+ channel complexes (23, 24) and voltage-gated
Ca2+ channel (25). Thus, it is possible that some of the
Kv
subunit effect on Kv4.3 channel current density and proteins may
be due to masking potential endoplasmic reticulum retention signals in the channel polypeptide. However, our previous study found that GFP-tagged Kv1.4 and Kv1.5 channels are efficiently transported to
plasma membrane in the absence of Kv
subunits (26, 27). Similarly,
we found efficient plasma membrane localization of GFP-tagged Kv4.3
(data not shown). Furthermore, coexpression of Kv
subunits produced
no apparent changes in localization of these GFP-tagged channel
proteins. Thus, it is likely that Kv
subunits increase Kv4.3 as well
as Kv1 family proteins in endoplasmic reticulum and at the plasma
membrane. This stabilization effect is further supported by our finding
that the Kv
effect on Kv4.3 protein level is larger at longer times
after transfection. Hence, Kv4.3·Kv
complexes are likely more
stable than those without these auxiliary subunits.
subunits. A part of the N
terminus of Kv1 family polypeptide is sufficient for association (5,
6). In contrast, our results demonstrated that the corresponding region
of Kv4 family peptide is not necessary. Instead, the association requires the C terminus of Kv4.3 polypeptide. The importance of the C
terminus for interaction with Kv
subunits was also suggested in
Kv2.2·Kv
4 complex formation; a part of the C terminus of Kv2.2 protein is required for the increase in current density produced by
Kv
4 coexpression in Xenopus oocytes (10). Thus, the C
terminus of Kv2.2 and Kv4 family polypeptides is likely to be involved in association with Kv
subunits. The apparent lack of sequence similarity between the N terminus of Kv1 family and the C termini of
Kv2.2 or Kv4.3 polypeptides suggest that the interaction between Kv
and -
subunits may be more complex than previously assumed. To
further elucidate interaction mechanisms, we generated a chimeric Kv2.1
channel containing the C terminus of Kv4.3 polypeptide. We found that
this chimera does not efficiently associate with Kv
subunits (data
not shown), suggesting that the Kv4.3 C terminus may not be sufficient
for association. However, this chimera was found to be nonfunctional.
Therefore, misfolding of this chimeric channel protein might be
responsible for the observed lack of interaction. Thus, a simple
explanation for the requirement of the C terminus is that this peptide
interacts with a site of Kv
polypeptide that is distinct from one
for the Kv1 family N terminus. Alternatively, the C terminus may
indirectly participate in interaction. For example, the C-terminal
peptide interacts with other part of the channel protein to place an
association site in a position for efficient interaction with these
auxiliary subunits. More detailed analyses are required to
differentiate these possibilities.
2 subunits in brain. The association of non-Kv1 family channels
with Kv
subunits may have pronounced effects under physiological conditions. Although mutations in Drosophila Kv
subunit
HK and Kv1 channel Shaker resulted in almost
identical electrophysiological changes in some giant neurons,
alterations in other types of neurons caused by these HK
mutations were distinct from those by Shaker mutations (29).
Thus, the interaction of Kv
subunits with non-Kv1 channels is likely
to play important roles in controlling neuronal excitability. Our
results indicate that the major effect produced by Kv
subunits on
Kv4 family channels is to increase the number of functional channels.
The association of Kv
subunits may also have other regulatory
functions on these channels, such as sensing redox state of the cell. A
novel interaction mechanism between Kv4 family channels and Kv
subunits may be important for these regulatory functions. Hence, Kv
subunits control neuronal excitability by influencing expression and
function of Kv1 and Kv4 family channels.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. J. E. Dixon and D. McKinnon for rat Kv4.3 cDNA and Dr. J. S. Trimmer for rat
Kv1.1 and Kv
2.1 cDNAs and anti-Kv4.3, anti-Kv2.1, and
anti-panKv
antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL55312 (to E. S. L.) and HL63123 (to K. T.), a grant-in-aid from the American Heart Association Pennsylvania affiliate (to K. T.), and a postdoctoral fellowship from the Korean Science and Engineering Foundation (to E.-K. Y.).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.
An Established Investigator of the American Heart Association.
§ To whom correspondence should be addressed: E1355 Biomedical Science Tower, Dept. of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-383-7325; Fax: 412-648-1945; E-mail: koichi+@pitt.edu.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M004768200
2 J. S. Trimmer, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EAG, ether-a-gogo; GFP, green fluorescent protein; pF, picofarads; HEK, human embryonic kidney.
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