From the Rammelkamp Center for Education and
Research, MetroHealth Campus and the ¶ Departments of Physiology
and Biochemistry, Case Western Reserve University, Cleveland, Ohio
44109
Received for publication, September 14, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Kv Voltage-gated K+
(Kv)1 channels are integral
membrane proteins that regulate the electrical properties of excitable
cells. Channels in the Kv1 subfamily consist of hetero-oligomeric
protein complexes comprising four pore-forming The conserved C-terminal core region of Kv In this study, we investigated the contribution of these residues to
Kv Site-directed Mutagenesis--
Site-directed mutations (D85A,
Y90F mutant 1; S188A, R189L mutant 2) were made in Kv Analysis of Protein-Protein Interaction by the Yeast Two-hybrid
System--
Protein-protein interactions were monitored with the yeast
Matchmaker two-hybrid system (CLONTECH). Kv In Vitro Transcription of RNA and Expression in Xenopus
Oocytes--
cRNA was prepared using the T7 mMESSAGE mMACHINE kit
(Ambion), and the concentrations were estimated on denaturing agarose gels stained with ethidium bromide by comparison with an RNA mass ladder. Xenopus oocytes were injected with 46 nl of cRNA
solution. Final concentrations of cRNA were 2 ng/µl Kv1.4 and 50 ng/µl Kv Electrophysiology--
Whole cell current was measured at room
temperature (20-22 °C) using the standard two-electrode voltage
clamp technique. Electrodes were filled with 3 M KCl and
had a resistance of 0.2-0.5 megohms when immersed in bath
solution containing (in mM): 50 KOH, 55 NaOH, 0.5 CaCl2·2H2O, 100 methanesulfonic acid, 2 MgCl2·6H2O, and 10 HEPES, pH 7.3. All
chemicals were purchased from Sigma. We used 50 mM
K+ in bath solution to slow C-type inactivation and reduce
its contribution to the N-type inactivation of Kv1.4. All currents were
measured 6 days after injection. Data acquisition and analysis were
performed using pCLAMP software (Axon Instruments). Data were low
pass-filtered at 2 kHz before digitization at 10 kHz. Data are reported
as means ± S.E.
Xenopus Oocyte Fractionation and Western Blotting--
Plasma
membranes were prepared from Xenopus oocytes using a
previously published procedure (12). Oocytes were homogenized (20 µl/oocyte) in buffer containing 0.25 mM sucrose,
10 mM HEPES 1 mM EGTA, 2 mM
MgCl2·6H2O plus 1 µg/ml
phenylmethylsulfonyl fluoride with five strokes in a Dounce homogenizer
using a loose-fitting pestle. Sheets of plasma membrane were allowed to
settle by gravity for 15 min and then washed and resedimented three
times. Washed plasma membranes were solubilized in a buffer containing
1% Triton X-100 prior to determination of protein concentration. The
supernatant from the initial homogenization was spun at 3,000 × g for 10 min to remove debris, and the internal membranes
were pelleted by centrifugation at 50,000 × g for 60 min. Protein concentrations were estimated by the BCA method. For
Western blotting, proteins were separated by SDS-polyacrylamide gel
electrophoresis, blotted to polyvinylidene difluoride membranes, and
blocked with 5% nonfat dry milk in phosphate-buffered saline plus
0.1% Tween 20. Blots were incubated with primary antibodies
(monoclonal anti-Kv1.4, Upstate Biotechnology, Inc., 1:500; or
polyclonal anti-Kv Interaction of Kv1.4 N Terminus with Kv Effect of Kv
Yeast two-hybrid data indicated that the mutant Kv Kv
Because Kv Kv Our results do not show that NADPH binding by Kv2 enhances the rate of inactivation and
level of expression of Kv1.4 currents. The crystal structure of Kv
2
binds NADP+, and it has been suggested that Kv
2 is
an oxidoreductase enzyme (1). To investigate how this function might
relate to channel modulation, we made point mutations in Kv
2 in
either the NADPH docking or putative catalytic sites. Using the yeast
two-hybrid system, we found that these mutations did not disrupt the
interaction of Kv
2 with Kv
1 channels. To characterize the Kv
2
mutants functionally, we coinjected wild-type or mutant Kv
2
cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv
2
increased both the amplitude and rate of inactivation of Kv1.4
currents. The cellular content of Kv1.4 protein was unchanged on
Western blot, but the amount in the plasmalemma was increased.
Mutations in either the orientation or putative catalytic sites for
NADPH abolished the expression-enhancing effect on Kv1.4 current.
Western blots showed that both types of mutation reduced Kv1.4 protein.
Like the wild-type Kv
2, both types of mutation increased the rate of
inactivation of Kv1.4, confirming the physical association of mutant
Kv
2 subunits with Kv1.4. Thus, mutations that should interfere with
NADPH function uncouple the expression-enhancing effect of Kv
2 on
Kv1.4 currents from its effect on the rate of inactivation. These
results suggest that the binding of NADPH and the putative
oxidoreductase activity of Kv
2 may play a role in the processing of
Kv1.4.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits in
permanent association with four modulatory
subunits (1). Three
different Kv
subunit genes (
1,
2, and
3) have been cloned
from different tissues in several species (2-6). Kv
subunits
coassemble with Kv
subunits in the endoplasmic reticulum and enhance
biosynthesis, maturation, and surface expression of voltage-gated
K+ channel complexes. Kv
2 increases Kv1.2 (7) and Kv1.4
(8) currents and also increases the cell surface dendrotoxin binding site of Kv1.2 (9). The N terminus of Kv
1.x (
1.1,
1.2, and
1.3) acts as a ball peptide and rapidly inactivates open channels. This fast inactivation resembles the N-type inactivation produced by
the N-terminal ball peptide of Kv
subunits. The Kv
2 subunit while
lacking the inactivation peptide accelerates the N-type inactivation of
Kv1.4 and in addition increases Kv1.4 expression (8).
subunits shares a
remarkable structural homology to aldo-keto reductases (10). The
crystal structure of the conserved core of Kv
2 subunit depicts a
4-fold symmetrical TIM barrel structure with the bound cofactor NADP+ (1). The structure shows that residues Ser-188 and
Arg-189 are important for the orientation of the nicotinamide ring of NADP+ and that Asp-85 and Tyr-90 are the putative catalytic
site residues of Kv
2. The phenolic moiety of Tyr-90 positioned near
the C-4 of nicotinamide ring would be a proton donor for reduction of a
putative substrate aldehyde or ketone. The Asp-85 residue is involved
in extensive hydrogen bonding and together with Tyr-90 positions the
catalytic site as for other aldo-keto reductases. Despite this
structural information, it is not known whether Kv
subunits function
as oxidoreductases and, if so, what their physiological substrates may be.
2 function. We made site-directed point mutations in both the
NADPH orientation and putative catalytic sites. The putative catalytic
mutant (D85A, Y90F) is referred to as Kv
2 Mut1 and the
orientation mutant (S188A, R189L) is referred to as Kv
2 Mut2. We
tested the effects of these mutants on both the inactivation properties
and the expression levels of Kv1.4 currents in Xenopus
oocytes. We report that wild-type Kv
2 increased Kv1.4 expression by
enhancing its trafficking to the cell surface. Mutations at either site
eliminated the ability of Kv
2 to enhance Kv1.4 expression. However,
mutant Kv
2 subunits still interacted with Kv1.4 to increase the rate
of inactivation. Thus, the dual effects of Kv
2 regulation of Kv1.4
were shown to be independent by mutating residues involved in
NADP+ binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 cDNA
using the overlapping polymerase chain reaction method, from
which the products were subcloned into the TOPO shuttle vector
(Invitrogen) for sequencing. Wild-type and mutant Kv
2 cDNAs were
subcloned into the pCR3 vector (Invitrogen), which we modified to
contain a poly(A)+ tail and a unique NruI site
for linearizing the constructs prior to cRNA synthesis.
2
wild-type and Kv
2 mutant cDNAs were subcloned into the yeast
shuttle vector, pGBT9 (DNA binding domain vector). The N
terminus of Kv1.4 (amino acids 1-305) was fused to the activation
domain vector, pGAD424. Protein-protein interactions were tested in the
yeast strain Y190 by cotransformation with pairs of pGBT9 and pGAD424
constructs as described previously (11). Cotransformants were selected
on medium lacking tryptophan (trp
) and
leucine (leu
) after growth for 2-3 days at
30 °C. Representative colonies were replated on
trp
/leu
medium to
allow for direct comparison of individual colonies. Transcription of
the reporter gene, lacZ, was tested by a
-galactosidase filter assay.
2 or Kv
2 mutants.
2, QCB, 1:5000) for 1 h at room temperature,
washed three times (10 min/wash) with phosphate-buffered saline, 0.1%
Tween 20, and incubated with secondary antibody (anti-mouse or
anti-rabbit horseradish peroxidase conjugate, Amersham Pharmacia
Biotech, 1:3000) for 1 h. After three washes in phosphate-buffered
saline plus 0.1% Tween 20, the blots were developed with the ECL-Plus
detection system (Amersham Pharmacia Biotech). Total membranes were
prepared from oocytes by methods previously established in our
laboratory (13). The oocytes were ground by 20 strokes in a Dounce
homogenizer and spun at 3,000 × g for 10 min to remove
debris, and the total membranes were pelleted by centrifugation at
50,000 × g for 60 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 Mutants--
We first
tested whether the mutations in the NADPH orientation and NADPH
oxidoreductase catalytic sites affected interaction with Kv1.4
channels. Using the yeast two-hybrid assay, we found that neither set
of mutations diminished the binding of Kv
2 with the Kv1.4 N
terminus, as evidenced by activation of the lacZ reporter gene (Fig. 1).
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Fig. 1.
Yeast two-hybrid interaction of
Kv 2 mutants with
Kv1.4N. The yeast strain Y190 was cotransformed
with the GAL4 binding domain (BD, pGBT9) and activation
domain (AD, pGAD424) fusion plasmids as
indicated and plated on medium lacking trp and
leu. All cotransformants grew on
trp
/leu
medium and
were tested for activation of the reporter gene, lacZ, by a
filter assay. The control reaction did not result in activation of
lacZ, but Kv
2 as well as both Kv
2 mutants interacted
with Kv1.4N as seen by the positive
-galactosidase assay.
2 Mutants on Kv1.4 Currents--
Kv
2 has been
reported to increase both the amplitude and the rate of inactivation of
Kv1.4 currents. We investigated the effects of Kv
2 mutations on
Kv1.4 currents by coexpressing cRNAs encoding Kv1.4 with wild-type or
mutant Kv
2 in Xenopus oocytes. Six days after injection,
we observed that Kv
2 increased Kv1.4 current amplitude by 1.6-fold
at +80 mV (compare Fig. 2,
panels A and B). The current amplitude
measured at +80 mV was 15.35 ± 0.78 µA (n = 9)
(Fig. 2B) in oocytes expressing a combination of Kv1.4 and
Kv
2 compared with 9.55 ± 0.88 µA (n = 7) for
oocytes expressing Kv1.4 alone (Fig. 2A). The increase in current was manifest at all potentials above threshold (Fig.
3A). When Kv
2 mutants were
examined, the expression-enhancing effect of Kv
2 on Kv1.4 was
abolished. In fact, coexpression with Kv
2 mutants produced a
decrease in the amplitude of Kv1.4 current, and the decrease occurred
at all potentials above threshold (Fig. 3A). At +80 mV,
mutations in the NADPH oxidoreductase catalytic site (D85A, Y90F)
decreased currents by 63.3% to 4.46 ± 0.24 µA
(n = 5) (Fig. 2C), whereas mutations in the
NADPH docking site (S188A, R189L) reduced currents by 36.6% to
6.05 ± 0.45 µA (n = 6) (Fig. 2D).
The double mutant in which all four residues were simultaneously disrupted also showed a reduction in current amplitude to 7.24 ± 0.36 µA (n = 6), a decrease of 24% (data not shown).
The differences in mean current amplitude for Kv1.4 compared with mean
current amplitude for Kv1.4 in combination with either Kv
2 or Kv
2
mutants (p < 0.01, one-way analysis of variance) were
statistically significant.
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Fig. 2.
Mutations in NADPH docking and oxidoreductase
catalytic sites abolish the expression enhancing effects of
Kv 2. In Xenopus oocytes, whole
cell currents were recorded 6 days post-injection. Currents were evoked
from a holding potential of
80 mV by 125-ms depolarizing step pulses
to +80 mV in 10 mV increments (shown in insert). The oocytes
were either injected with Kv1.4 (2 ng/µl) alone (A) or
coinjected with Kv
2 (50 ng/µl) (B), Kv
2 mut1
(50 ng/µl) (C), or Kv
2 mut2 (50 ng/µl)
(D). E, peak current amplitudes at +80 mV are
depicted in the bar diagram from the current traces shown in
A-D. F, normalized current traces at +80 mV
depict enhanced N-type inactivation in the presence of Kv
2 and
Kv
2 mutants, indicating their interaction with Kv1.4.
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Fig. 3.
Mutations in NADPH docking and oxidoreductase
catalytic sites do not significantly alter the biophysical properties
of Kv 2. A, current-voltage
relationship of currents recorded from Xenopus oocytes.
Whole cell currents were evoked from a holding potential of
80 mV by
125-ms depolarizing step pulses to +80 mV in 10 mV increments (current
traces shown in Fig.2, A-D). B, coexpression of
Kv1.4 with Kv
2 or Kv
2 mutants did not alter the voltage
dependence of activation. I/Imax
ratios were calculated by dividing the peak current at each potential
with the maximal current at +80 mV and plotted as a function of
potential. C, steady state inactivation levels of
Kv1.4 in the presence and absence of Kv
2 and Kv
2 mutants were
similar. Oocytes were held at
90 mV and pulsed to
10 mV in 10 mV
increments for 1 s followed by a 450-ms test pulse to +70 mV and
repolarization to
90 mV for 6 s (n = 4). Steady
state inactivation curves were generated by normalizing the currents at
each potential to the maximal currents at +70 mV. The data were fit to
the Boltzman equation I/Imax = 1/[1 + exp(V0.5
V)/k)], where V0.5
is the potential for half-inactivation and k is the slope
factor. D, plot of inactivation time constants
versus test potential. Inactivating current traces of Kv1.4
in the presence of Kv
2 and Kv
2 mutants were fitted with a single
exponential function. The time constants were plotted as a function of
potential (n = 4). Inactivation time constants are
significantly smaller when Kv
2 or wild-type Kv
2 mutants are
coexpressed with Kv1.4, indicating enhanced inactivation; however, they
are not significantly different from each other. E, plot of
recovery from inactivation. From a holding potential of
90 mV, the
oocyte was depolarized to +70 mV for 1 s and subsequently
repolarized to
90 mV for time intervals ranging from 100 ms to
2.4 s. This was followed by a 400-ms test pulse to +70 mV
(n = 4). The recovery curve is the plot of the ratio of
inactivated current during the second pulse, normalized to the amount
of inactivating current during the first pulse versus the
inter-pulse interval. Kv
2 slowed the recovery from inactivation
compared with Kv1.4 alone, whereas the Kv
2 mutants produced an even
slower recovery from inactivation.
subunits were
still able to interact with the Kv1.4 N terminus (Fig. 1). As a further
test of association, we examined Kv1.4 currents for evidence of
increased inactivation produced by association with Kv
2 subunits. In
an earlier study (8), we had shown that Kv
2, although absent the
inactivation peptide of Kv
1.x, increased the rate of N-type
inactivation of Kv1.4. Normalized current traces of Kv1.4 when
coexpressed with either Kv
2 or Kv
2 mutants showed that wild-type
and mutant Kv
2 enhanced the intrinsic inactivation of Kv1.4 currents
to a similar extent (Fig. 2F). Plots of inactivation time
constants as a function of membrane potential were also similar (Fig.
3D). At +80 mV, the mean inactivation time constants for Kv1.4, Kv1.4+
2, Kv1.4 +
2 mut1, and Kv1.4 +
2 mut2 were
49.55 ± 2.3, 25.52 ± 0.37, 21.67 ± 0.18, and
22.32 ± 0.28, respectively. Neither wild-type nor mutant Kv
2
subunits altered the voltage dependence of activation (Fig.
3B) or steady state inactivation (Fig. 3C) of
Kv1.4. Recovery from steady state inactivation, which was decreased
when Kv
2 was coexpressed with Kv1.4, was slowed further in the
presence of the Kv
2 mutants (Fig. 3E). At
90mV, the
mean time for half-recovery from inactivation for Kv1.4, Kv1.4 +
2,
Kv1.4 +
2 mut1, and Kv1.4 +
2 mut2 were 3.35, 3.42, 4.23, and
5.82 s, respectively. To eliminate the possibility of nonspecific effects of Kv
2 mutants on Kv1.4 in oocytes, Kv
2 wild-type and mutants were also coexpressed with Kv2.1, a Kv
subunit with which they do not interact. Neither Kv
2 nor the Kv
2 mutants had any effect on Kv2.1 current amplitude or kinetics (data not shown).
2 Enhances Trafficking of Kv1.4 to the Plasma
Membrane--
Our electrophysiological data showed that the
expression-enhancing effect of Kv
2 on Kv1.4 was distinct from its
effects on the biophysical properties of the channel. The enhancement
of Kv1.4 expression by Kv
2 could be caused by: 1) an increase
in total Kv1.4 protein, 2) an increase in the number of functional channels at the cell surface without an increase in total Kv1.4 levels,
or 3) an increase in opening probability and/or single channel
conductance. The third possibility was considered unlikely because the
conductance-voltage relationship of Kv1.4 was unaltered by wild-type
and mutant Kv
2s (Fig. 3B). To determine whether coexpression of Kv
2 increased total Kv1.4 protein levels, we used
Western blotting to examine membrane-enriched fractions from oocytes.
Multiple bands of Kv1.4 were detected in oocytes injected with Kv1.4
cRNA: an immature or core-glycosylated ~78-kDa band; and a mature,
glycosylated, 105-kDa band, which resolved as a doublet in some
experiments (Fig. 4). Coexpression with
Kv
2 did not alter the amounts of Kv1.4 protein in the total membrane
fraction. However, coexpression with either of the Kv
2 mutants did
show a reduction in total Kv1.4 protein.
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Fig. 4.
Kv 2 coexpression did
not alter the total membrane associated Kv1.4 protein in
Xenopus oocytes, whereas Kv
2
mutations in the NADPH docking and putative oxidoreductase catalytic
sites decreased Kv1.4 protein levels. Total membranes were
isolated 6 days post-injection from Xenopus oocytes either
injected with Kv1.4 (2 ng/µl) alone or coinjected with Kv
2 or
Kv
2 mut1 or Kv
2 mut2 (all at 50 ng/µl). Proteins were separated
by SDS-polyacrylamide gel electrophoresis, blotted to polyvinylidene
difluoride membranes, and probed with either polyclonal anti-Kv
2
(1:5000, QCB; panel A) or monoclonal anti-Kv1.4 (1:500,
Upstate Biotechnology, Inc.; panel B). Immunoreactive bands
were visualized with ECL-Plus (Amersham Pharmacia Biotech). Kv1.4
appeared as three bands, a 78-kDa core-glycosylated band and a doublet
at 105 kDa representing mature-glycosylated bands). Coexpression with
wild-type Kv
2 did not increase the total Kv1.4 expression, whereas
both of the Kv
2 mutants decreased total Kv1.4 protein levels. Kv
2
blot shows that the mutation in the catalytic site led to decreased
2 protein synthesis.
2 did not increase total Kv1.4 protein levels, we explored
the possibility that Kv
2 increased currents by enhancing trafficking
of Kv1.4 channels to the plasma membrane by physically separating
oocyte plasma membranes from internal membranes (i.e. endoplasmic reticulum and Golgi). Western blot analysis of Kv1.4 in
these fractions showed an enhanced expression in the plasma membrane
when coexpressed with Kv
2 (Fig. 5,
right). As expected, only the mature, fully glycosylated
~105-kDa band (which was not resolved into a clear doublet in this
gel) was detected in the plasma membrane. In the internal membrane
fraction, we found both the immature and mature Kv1.4 bands with no
apparent change in expression in the presence or absence of Kv
2
(Fig. 5, left). Consistent with decreased currents,
coexpression with Kv
2 mutants showed a reduction of Kv1.4 in both
the plasma and internal membrane fractions (Fig. 5). Both wild-type and
mutant Kv
2 subunits were detected in both the internal and plasma
membrane fractions, providing further evidence for the interaction of
mutant Kv
2 subunits with Kv1.4. Although equal amounts of cRNAs were
injected for each Kv
cDNA, we consistently observed a lower
level of expression of Kv
2 mutant 1. It is not known whether this
finding reflects an increased degradation of mutant protein, decreased
synthesis, or a combination of both.
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Fig. 5.
Kv 2 enhanced the
surface expression of Kv1.4, whereas Kv
2
mutations in the NADPH docking and oxidoreductase catalytic sites
decreased Kv1.4 protein levels. Plasma membrane and internal
membrane fractions were isolated 6 days post-injection from
Xenopus oocytes injected with Kv1.4 (2 ng/µl) alone or
coinjected with Kv
2, Kv
2 mut1, or Kv
2 mut2 (all at 50 ng/µl). Proteins were separated by SDS-polyacrylamide gel
electrophoresis, blotted to polyvinylidene difluoride membranes, and
probed with either with monoclonal anti-Kv1.4 (1:500, Upstate
Biotechnology, Inc; panels A and B) or polyclonal
anti-Kv
2 (1:5000, QCB; panels C and D).
Immunoreactive bands were visualized with ECL-Plus (Amersham Pharmacia
Biotech). Kv1.4 appeared as two bands in the internal membrane fraction
(A), a 78-kDa core-glycosylated band and a 105-kDa
mature-glycosylated band. Only the mature, glycosylated, 105-kDa band
was seen in the plasma membrane fraction (B). Note that
coexpression with wild-type Kv
2 increased Kv1.4 expression in the
plasma membrane fraction, whereas both of the Kv
2 mutants decreased
Kv1.4 protein levels in the internal as well as the plasma membrane
fractions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 increased Kv1.4 current and its rate of inactivation (8).
From the present results it appears that the increased Kv1.4 current
may be due to a redistribution of Kv1.4 protein to the plasmalemma
rather than an increase in the total amount of membrane-associated Kv1.4 protein. Mutating wild-type Kv
2 at the orientation site for NADPH (Mut2) or the putative catalytic site for aldo-keto reductase
activity (Mut1) abolished the ability of Kv
2 to increase Kv1.4 current without changing the ability of Kv
2 to increase inactivation. The latter result, together with the persistent yeast
two-hybrid interactions, showed that the mutant Kv
2 subunits were
still permanently associated with Kv1.4 subunits. However, the Kv
2
mutants produced a reduction in both the plasmalemmal and total
membrane-associated Kv1.4 protein, which probably explains the
reduction in Kv1.4 currents associated with their coexpression (Fig.
2E).
2 has been
abolished, nor do they show whether the effects of the mutations are
specific for orientation of the nicotinamide ring or the putative catalytic site for aldo-keto reductase activity. Such studies are
possible now that the crystal structure of Kv
2 (1) and the assembly
of Kv
1.x with the T1 domain of Kv1.x channels (14) have been
resolved. Keeping these assumptions in mind, it may be possible that
the aldo-keto reductase activity of Kv
2 is important for the
processing and trafficking of Kv1.4
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL55404 and HL36930 (to A. M. B.) and HL60759 (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.
§ Current address: ChanTest Inc., 4440 Warrensville Center Rd., Cleveland, OH 44118. To whom correspondence may be addressed: Tel.: 216-586-3626; E-mail: rperi@chantest.com.
To whom correspondence may be addressed. Rammelkamp
Center for Education and Research, R301, MetroHealth Drive, Cleveland, OH, 44109. Tel.: 216-778-5960; E-mail:
abrown@research.metrohealth.org.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M008445200
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ABBREVIATIONS |
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The abbreviation used is: Kv, voltage-gated K+.
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REFERENCES |
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