Departments of 1 Pharmacology, 2 Molecular Physiology and Biophysics, and 3 Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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The Kv1.3 subunit confers a voltage-dependent, partial
inactivation (time constant = 5.76 ± 0.14 ms at +50 mV), an
enhanced slow inactivation, a hyperpolarizing shift in the activation
midpoint, and an increase in the deactivation time constant of the
Kv1.5 delayed rectifier. Removal of the first 10 amino acids from
Kv
1.3 eliminated the effects on fast and slow inactivation but not
the voltage shift in activation. Addition of the first 87 amino acids of Kv
1.3 to the amino terminus of Kv1.5 reconstituted fast and slow
inactivation without altering the midpoint of activation. Although an
internal pore mutation that alters quinidine block (V512A) did not
affect Kv
1.3-mediated inactivation, a mutation of the external mouth
of the pore (R485Y) increased the extent of fast inactivation while
preventing the enhancement of slow inactivation. These data suggest
that 1) Kv
1.3-mediated effects involve at least two distinct domains of this
-subunit,
2) inactivation involves open
channel block that is allosterically linked to the external pore, and
3) the Kv
1.3-induced shift in the
activation midpoint is functionally distinct from inactivation.
Shaker-like potassium channel; N-type inactivation; C-type inactivation
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INTRODUCTION |
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VOLTAGE-GATED K+
channels represent a structurally and functionally diverse group of
membrane proteins. These channels establish the resting membrane
potential and modulate the frequency and duration of action potentials
in nerve and muscle (18). In addition, K+ channels are involved in
processes not usually associated with electrically excitable membranes,
such as T lymphocyte activation, cell volume regulation, and pancreatic
-cell function (5, 22). Multiple
Shaker-like
K+ channel
-subunit genes have
been cloned from mammalian brain, heart, skeletal muscle, pancreas, and
smooth muscle and functionally expressed in heterologous systems (6).
The recent discovery of
-subunits, some of which convert delayed
rectifiers into rapidly inactivating channels, has revealed additional
mechanisms of voltage-gated K+
channel modulation (9, 12-14, 17, 26, 28, 32, 33). Functional
analysis has shown that four of these
-subunits, Kv
1.1, Kv
1.2,
Kv
1.3, and Kv
3.1, confer varying degrees of rapid inactivation on
certain members of the Kv1 family of delayed rectifiers (12, 13, 16,
17, 26, 28, 32, 33). In addition, Kv
1.2, Kv
1.3, and Kv
2.1
modify the voltage dependence of Kv1.5 channel opening (8-20 mV
hyperpolarizing shift in the midpoint of activation) (12, 13, 40).
However, it has been suggested that the apparent shift in the Kv1.2
activation curve following coexpression with Kv
1.2 is completely
derived from the
-subunit-induced N-type or fast inactivation (10).
In this respect, it is important to note that Kv
2.1, which does not
induce fast inactivation, does shift the activation midpoint and only
enhances slow or C-type inactivation of the Kv1.5 delayed rectifier
(40). Each Kv
family (Kv
1, Kv
2, and Kv
3) appears to derive
from a separate gene, and additional variability in the Kv
1 family
results from alternative splicing in the amino-terminal region, thus
yielding the Kv
1.1, Kv
1.2, and Kv
1.3 subunits (12, 27). The
variable amino-terminal domains are responsible for the functional
differences (16, 29, 32), whereas the conserved carboxy-terminal
domain most likely governs assembly with the
-subunit
(16, 30, 34, 43, 45).
The rapid inactivation induced by Kv1.1 when coexpressed with the
delayed rectifier, Kv1.1, is thought to occur by the same "ball-and-chain" mechanism reported for the
Shaker channel (20, 32). For example,
exposure of the cytoplasmic face of Kv1.1 to a peptide corresponding to
the 24 amino-terminal amino acids of Kv
1.1 causes inactivation (32).
One interpretation of this finding is that the peptide induces
inactivation by binding within the open pore. Alternatively, the
peptide could bind to a site removed from the pore and allosterically
modulate ion permeation. Indeed, Morales and co-workers (29) have
presented evidence that Kv
1.2 confers inactivation on a delayed
rectifier by enhancing primarily C-type inactivation (29),
suggesting that Kv
1.2-induced inactivation occurs by an allosteric
mechanism. Thus no a priori assumptions should be made about the
inactivation mechanism of other Kv
subunits.
The Kv1.3 subunit converts Kv1.5 from a delayed rectifier with a
modest degree of slow inactivation to a channel with both fast and slow
components of inactivation. The rapid inactivation has the
characteristics of open channel block and is similar to block of Kv1.5
by antiarrhythmic agents such as quinidine. Mutagenesis of
- and
-subunits followed by kinetic analysis of channel gating was used in
the present study to examine the mechanism by which Kv
1.3 modulates
Kv1.5 function. The data indicate that Kv
1.3-mediated fast
inactivation likely occurs in part by an open channel block mechanism that is sensitive to both membrane potential and external K+. Both the
-subunit-enhanced slow inactivation and the modulation of
-subunit-induced fast inactivation by an external, but not an
internal, pore mutation reveal the importance of allosteric mechanisms
in K+ channel
-
interactions. Results presented here clearly indicate that the Kv
1.3
subunit directly alters
-subunit activation, for Kv
1.3 mutants
that produce no inactivation still induce a hyperpolarizing shift in
the voltage dependence of Kv1.5 activation.
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MATERIALS AND METHODS |
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Materials. Enzymes and buffers were from New England Biolabs (Beverly, MA), Boehringer Mannheim (Indianapolis, IN), and Promega (Madison, WI). The Sequenase 2.0 kit was purchased from United States Biochemical (Cleveland, OH). The origins of other materials are specified below.
Mutagenesis.
The series of amino-terminal deletions in Kv1.3 shown in Fig.
1 was created by PCR
mutagenesis. The 5' oligonucleotide primer replaced amino acids
10, 37, 68 and 91, respectively, with an Xba I restriction site and a consensus
translation initiation site (GGTCTAGAATG. . .). The 3'
oligonucleotide annealed downstream from a unique endogenous
Kpn I (
N10 and
N37) or
Pst I (
N68 and
N91) restriction
site. The 5' sequence of the previously described Kv
1.3
construct in the modified pSP64T vector (12) was excised
with the appropriate restriction enzymes, and the amplified mutant PCR
products were ligated in its place. Two additional mutants were made in
which the first 19 or 87 amino acids of Kv
1.3 were linked to the
amino terminus of Kv1.5. The 5' region of Kv
1.3 was PCR
amplified to contain an Sph I site at
the 3' end in the same reading frame as an endogenous
Sph I site in the 5'
untranslated region of Kv1.5. Use of this site introduced seven amino
acids (His-Ala-Leu-Cys-Ser-Arg-Ala) that are not part of either
wild-type subunit. The tandem constructs were then ligated into a
modified pSP64T vector for oocyte expression. These constructs will be referred to as tandems even though only portions of the amino terminus
of Kv
1.3 are appended to the full-length Kv1.5 (Fig. 1). All
PCR-generated mutants were verified by double-stranded sequencing. The
point mutations in the Kv1.5
-subunit have been described previously
(44). These mutants were subcloned into the pSP64T vector for oocyte
expression. It was necessary to add ~710 base pairs of the 3'
untranslated sequence of Kv1.5 to constructs containing the
-subunit
to achieve expression levels in excess of 1.5 µA.
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Expression in Xenopus oocytes and electrophysiological recording. Templates were linearized with EcoR I before in vitro cRNA synthesis using the SP6 mMessage mMachine kit (Ambion) according to the manufacturer's instructions. Defolliculated Xenopus oocytes were prepared as described previously (12, 13) and injected with ~40 nl (4-20 ng) of in vitro-transcribed cRNA. The cRNA was diluted to yield peak currents of 1-10 µA.
Oocytes were bathed in one of two extracellular solutions, normal K+ or high K+. Normal K+ contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5 with NaOH). High K+ contained (in mM) 2 NaCl, 96 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5 with KOH). Equilibration of the bath solution was monitored by the stability of the direction and magnitude of the deactivating tail current atPulse protocols and data analysis.
Activation curves were obtained from the tail current amplitude 6 ms
after repolarization to 40 mV. The voltage dependence of channel
opening ("activation curve") was fit with the Boltzmann equation
I/Imax = {1 + exp[
(Em
Eh)/k]}
1,
in which I is current,
Imax is maximum
current, Em is
membrane potential,
Eh is the voltage
at which 50% of the channels are open, and
k is the slope factor. The impact of
inactivation on the generation of activation curves is reviewed in
DISCUSSION. The time course of
deactivating tail currents was fit with a single exponential by a
nonlinear least squares algorithm. Goodness of the fit was judged by
visual inspection for nonrandom trends in the residuals of the fit. For
steady-state current values, raw data points were averaged over a small
time window (2-5 ms).
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RESULTS |
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The present study examined the mechanism by which Kv1.3 alters Kv1.5
function and determined regions within each subunit that affect their
functional interactions. In the presence of the Kv
1.3 subunit, the
Kv1.5 current phenotype was changed from a delayed rectifier to one
that displayed a rapid, but partial, inactivation (time constant = 5.76 ± 0.14 ms at +50 mV, n = 17; Table
1) that was apparent only at membrane
potentials more positive than ~0 mV (see Fig.
2). A slower rate of channel deactivation was also observed in the presence of Kv
1.3 relative to Kv1.5 alone
(32.6 ± 1.2 ms vs. 13.7 ± 0.5 ms at
40 mV; Table 1).
Additionally, the threshold for Kv1.5 channel activation occurred at
more negative membrane potentials, with a parallel 13-mV
hyperpolarizing shift in the voltage dependence of activation (Table
1).
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Role of the Kv1.3 amino-terminal sequence in fast
inactivation.
The various Kv
1 subfamily members confer different functional
effects on Kv1.5 (12, 13, 16, 26, 41). The amino termini of these
subunits vary considerably, whereas the 329 carboxy-terminal amino
acids are identical. Therefore, we constructed several mutations within
the Kv
1.3 amino terminus and examined the effect of these alterations on Kv
1.3-induced inactivation. Mutant Kv
1.3 subunits lacking the first 10, 37, 68 and 91 amino acids of the variable amino
terminus were constructed. Two tandem mutants were also made in which
the first 19 or 87 amino acids of the amino terminus of Kv
1.3 were
linked to the amino terminus of Kv1.5 (Fig. 1). Representative outward
currents recorded from oocytes expressing Kv1.5, Kv1.5 + Kv
1.3,
Kv1.5 + Kv
1.3(
N10), and tandem(N87) are shown in Fig. 2. Kv1.5
displayed a delayed rectifier phenotype when expressed alone (Fig.
2A). Coexpression of the Kv1.5
subunit with wild-type Kv
1.3 resulted in currents that exhibit fast
inactivation at membrane potentials positive to +10 mV but failed to
show any apparent inactivation at more negative potentials (Fig.
2B). At +70 mV, the extent of
inactivation at 200 ms, defined as the decline from the peak current
level, averaged 54.4 ± 1.1% (n = 16; Table 1). The time constant of Kv
1.3-mediated inactivation at
+70 mV was 5.7 ± 0.1 (n = 17;
Table 1) and that for recovery from fast inactivation at
80 mV
was 5.1 ± 0.1 ms (n = 7). Removal of as few as 10 amino acids from the amino terminus of Kv
1.3 prevented fast inactivation (Fig.
2C). The other more extensive Kv
1.3 amino-terminal deletions also did not induce fast inactivation (data not shown). In contrast to the currents observed with Kv1.2 and
amino-terminal-truncated Kv
1.2 (1), no significant increase in
outward current was observed following the removal of Kv
1.3-induced inactivation. Therefore, the ratio of the peak to steady-state current
shown in Fig. 2B is likely to
represent a reasonable approximation of the percentage of Kv1.5
channels inactivated by Kv
1.3. Increases in the ratio of
- to
-subunit cRNA failed to further enhance inactivation and often
suppressed current. This suppression occurred with both the wild-type
and mutant
-subunits and is in agreement with the data of Accili et.
al. (1) showing that overexpression of the conserved Kv
1 core can
decrease Kv1.5 current.
The hyperpolarizing shift in the midpoint of activation does not
require an intact amino terminus.
Figure 3 shows activation curves
constructed from normalized deactivating tail current magnitudes as
described in MATERIALS AND METHODS.
Coexpression of Kv1.5 with the wild-type Kv1.3 subunit produced the
greatest leftward shift compared with Kv1.5 expressed alone.
Coexpression of Kv1.5 with Kv
1.3(
N10) also shifted the curve
leftward but to a slightly lesser extent. These data demonstrate the
functional independence of the Kv
1.3-mediated effects on inactivation and the change in the midpoint of activation. As shown in
Fig. 3, the 68-amino acid deletion still caused a shift in the
activation curve. The Kv
1.3(
N91) construct did not induce a shift
in the voltage dependence of activation (data not shown). Neither
tandem construct (N19 or N87) shifted the midpoint of activation
relative to wild-type Kv1.5 (Table 1). All of these data suggest that
the
-subunit domain involved in inactivation is distinct from that
governing the shift in the voltage dependence of activation.
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Prolonged depolarization revealed a slow component of inactivation.
Fast inactivation occurred with a time constant of 5.7 ms, suggesting
that this process is essentially at steady state within 25 ms (Table 1
and Fig.
4B,
inset). However, the current
continued to decay at a slower rate after this time, indicating a slow
component of inactivation. To examine the slow component of
inactivation, oocytes expressing Kv1.5 or Kv1.5 + Kv1.3 were
depolarized for 5 s. Unscaled currents resulting from 5-s
depolarizations to +70, +50, and +30 mV are shown in Fig. 4,
A and
B, for Kv1.5 and Kv1.5 + Kv
1.3,
respectively. The magnitude of current measured at 1 s increases with
membrane potential in the absence of Kv
1.3 (Fig. 4A). However, in the presence of
Kv
1.3, the K+ current measured
at 1 s decreases with depolarizations positive to +30 mV (Fig.
4B). Because fast inactivation is
complete within 50 ms (Fig. 4B,
inset), normalization to the current
magnitude at 100 ms allows qualitative comparison of slow inactivation
in the absence and presence of Kv
1.3 (Fig. 4,
C and
D). The normalized tracings in the
absence of Kv
1.3 superimposed (Fig.
4C), indicating that the extent and
rate of slow inactivation of Kv1.5 was independent of membrane
potential, i.e., the tracings reach a plateau at
10 mV (36). In
contrast, the tracings at each membrane potential in the presence of
Kv
1.3 did not superimpose (Fig.
4D), indicating that the extent of
slow inactivation depends on the membrane potential.
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Characterization of Kv1.3 interaction with internal
and external Kv1.5 pore mutations.
One possible mechanism for the fast component of inactivation is that
the amino terminus of Kv
1.3 acts as an open channel blocker that
binds within the open pore. Because the antiarrhythmic agent quinidine
probably interacts by such a mechanism (44), we tested whether a Kv1.5
point mutant that affects quinidine block (V512A) would also affect
Kv
1.3-mediated inactivation. The Kv1.5(V512A) mutation is located in
the internal mouth of the ion-conducting pore and has three major
effects on Kv1.5 function (44): a shift in the midpoint of activation
(Table 1), a slowing of deactivation, and a fourfold increase in the
affinity for quinidine. If the binding site for the inactivation
particle of Kv
1.3 overlaps that of quinidine, then the V512A
mutation may also show an enhanced affinity for the
-subunit
blocking particle (and hence display more inactivation). Currents
elicited from oocytes expressing the Kv1.5(V512A) mutant
-subunit in
the absence and presence of Kv
1.3 are shown in Fig.
5, A and
B. The internal pore mutation (V512A)
did not affect either the Kv
1.3-mediated inactivation or the
hyperpolarizing shift in the midpoint of activation (Fig. 5 and Table
1). Another internal pore mutation of Kv1.5, T505I, which enhances
quinidine block ~10-fold (44), also did not affect Kv
1.3 function
(data not shown).
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Effect of high external
K+ on
inactivation.
Sensitivity of block to concentrations of the conducting ion has been
used to support an open pore model of block by both pore-blocking drugs
and the inactivation particle (11). Increasing the external
K+ concentration also reduces the
slow component of inactivation (2, 24, 31). To test whether
Kv1.3-mediated inactivation was sensitive to external
K+ concentration, we compared the
Kv
1.3-mediated fast and slow inactivation of Kv1.5 and Kv1.5(R485Y)
in the absence and presence of 96 mM extracellular
K+ (Fig.
6). High external
K+ concentrations reduced the
extent of both fast and slow inactivation. Figure 6,
A and
B, shows representative current
tracings during depolarizations to +70 mV in oocytes
expressing Kv1.5 + Kv
1.3 and Kv1.5 (R485Y) + Kv
1.3, either in
normal K+, in high
K+, or after return to normal
K+ (wash). Currents were
normalized to the peak to emphasize the difference in the extent of
inactivation. For Kv
1.3 with either the wild-type or R485Y
-subunit, the presence of high external K+ reduced the extent of
inactivation at 200 ms (Table 1). To compare the time course of
inactivation, currents from Fig. 6, A
and B, were further normalized to the
current level at 1 s as shown in Fig. 6,
C and
D. For the wild-type subunit,
increasing external K+ eliminated
the slow component of inactivation without affecting the rate of fast
inactivation (see also Table 1). The slow component of inactivation
returned on washout of external
K+. Figure
6D illustrates that the rate of slow
inactivation of Kv1.5(R485Y) in the presence of Kv
1.3 was not
affected by the presence of 96 mM
K+.
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Voltage dependence of the inactivation induced by
Kv1.3.
Two lines of evidence suggested that Kv
1.3-mediated inactivation was
voltage dependent. First, macroscopic inactivation was apparent only at
large depolarizations. Second, the steady-state current level from
depolarizations more positive than +20 mV does not increase with a
corresponding increase in the membrane potential (12),
which would occur if the open channel probability were decreasing as
the driving force increased. To quantitate this voltage dependence, we
used a two-pulse protocol in which the membrane potential of the first
pulse was varied as shown in Fig. 7A. Figure
7B shows normalized data plotted vs.
the membrane potential of the 25-ms first pulse. Because fast
inactivation is essentially at steady state and slow inactivation is
negligible at 25 ms, this approach isolates the fast component of
inactivation. Data were obtained in both the absence and presence of 96 mM extracellular K+. The
fractional inactivation increased rapidly in the voltage range
corresponding to channel activation, indicating that Kv
1.3-mediated inactivation required the channel to enter the open state. Note that
the
10 mV tracing in Fig. 7A
shows no apparent inactivation in the first pulse, whereas there was an
obvious reduction in the peak current of the second pulse, i.e.,
channels inactivate at
10 mV even though inactivation is not
apparent in the macroscopic current. Positive to potentials at which
the channels are maximally activated (+10 mV), the fractional
inactivation continued to increase with membrane potential, indicating
that Kv
1.3-mediated fast inactivation was voltage dependent (Fig.
7B). The Woodhull model (42) was
used to quantitate the voltage dependence, and the derived
z ·
values are
indicated in Fig. 7. Similar curves and z ·
values were
obtained with tandem(N87) and the
-subunit point mutants (Fig.
7C). These data suggest that the
voltage dependence of Kv
1.3-mediated block is independent of
individual
-subunit properties, consistent with the localization of
the voltage-dependent inactivating particle to the amino terminus of
Kv
1.3 (Fig. 2). The fits are extrapolated to negative membrane
potentials to predict the fractional block of open channels at those
voltages. Note that elevating external
K+ to 96 mM reduces the extent of
fast inactivation but does not alter its voltage dependence
(z ·
).
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DISCUSSION |
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Separation of Kv1.3-mediated functional effects.
Other investigators have suggested that the hyperpolarizing shift in
the activation curve observed following coexpression of Kv1.5 with
Kv
1.2 is specifically linked to inactivation and that the
-subunit does not directly alter activation properties (10). Precise
quantitation of the voltage dependence of activation is difficult in
the presence of the voltage-dependent inactivation observed with open
channel block. Once current values are scaled, an artifactual shift in
the activation curve can be generated. Because analysis of peak current
levels is especially prone to artifact, activation curves were obtained
from the tail current amplitude 6 ms after repolarization to
40
mV. Because the time constant for recovery from fast inactivation at
80 mV was 5.1 ± 0.1 ms, these tail current measurements are
not perfect. However, even before the experiments reported here with
the Kv
1.3 mutants, it seemed likely that, at least in the case of
Kv1.5 and Kv
1.3, the
-subunit-induced activation shift was
independent of inactivation. Indeed, current was observed at membrane
potentials as low as
30 mV, a potential generating no current in
the absence of the
-subunit. In addition, the Kv
2.1 subunit
shifts the Kv1.5 activation curve without inducing fast inactivation
(16, 40).
Deactivation is affected by fast inactivation.
Prolonged deactivation has been observed in the presence of open
pore-blocking drugs and N-type inactivation (11, 35, 37). Blockade of
the pore by either the drug or inactivation particle is thought to
prevent deactivation (11, 35). Thus the drug or inactivating particle
must dissociate before the channel can close. The fact that the open
channel is subject to reblock further complicates the deactivating
current. An open channel will conduct current until it deactivates or
is reblocked. If the channel becomes reblocked, deactivation is again
prevented, and the particle must dissociate before the channel can
deactivate. Therefore, reblock by the drug or inactivating particle can
significantly contribute to prolonging the deactivating current (11).
Shifting the midpoint of activation also results in altered kinetics of deactivation (39). The tandem(N87) construct, which conferred fast
inactivation without altering the midpoint of activation, and the
Kv1.3(
N10) truncation, which hyperpolarized the midpoint of
activation without inducing fast inactivation, demonstrated deactivation time constants intermediate between those observed with
Kv1.5 alone and Kv1.5 plus the wild-type Kv
1.3. These data indicate
that the prolonged deactivation observed in the wild-type subunit may
be the result of the combined effects of a shift in the midpoint of
activation and reentry into the inactivated state.
External, not internal, Kv1.5 pore mutations alter
Kv1.3-induced inactivation.
It was conceivable that the inactivation particle of Kv
1.3 interacts
with the
-subunit at the same cytoplasmic binding site as quinidine,
since internal TEA derivatives can compete with N-type inactivation in
Shaker (7). However, the two
-subunit point mutants, V512A and T505I, which increase quinidine
block (44), failed to alter Kv
1.3-induced inactivation. Thus the molecular determinants in Kv1.5 for Kv
1.3-induced fast inactivation and open channel drug block appear to be distinct. However, it is still
possible that there is overlap in the
-subunit domains involved in
quinidine block and Kv
1.3-induced inactivation.
Inactivation is dependent on both external K+ and voltage. A model introduced by Baukrowitz and Yellen (2-4) for the Shaker channel proposes that occupancy of an external K+ binding site inhibits slow inactivation. This model proposes that the enhancement of slow inactivation in the presence of internal blockade (drug or inactivation ball) is a direct result of depletion of K+ from this external site. Increasing the external K+ concentration allows occupancy of this site, despite internal blockade, returning the extent of slow inactivation to the level observed in the absence of the blocking agent. In agreement with this model, elevating external K+ inhibited the slow component of inactivation, as seen in Fig. 6C. However, it also clearly inhibited the extent of fast inactivation, as shown in Figs. 6B and 7B. Because the extent of slow inactivation appears to be coupled to the extent of fast inactivation (Fig. 4F), we cannot discern whether the reduction in slow inactivation is the result of reduced fast inactivation or a direct effect of K+ on slow inactivation.
The voltage dependence of Kv ![]() |
ACKNOWLEDGEMENTS |
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We thank Brady Palmer and Michelle Choi for technical assistance in oocyte preparation and injection.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-49330 (to M. M. Tamkun), HL-47599 (to D. J. Snyders), and HL-46681 (to D. J. Snyders, P. B. Bennett, and M. M. Tamkun) and by a United Negro College Fund-Merck Postdoctoral Science Research Fellowship (to S. K. England).
P. B. Bennett is an Established Investigator of the American Heart Association.
Present addresses: S. K. England, Dept. of Physiology and Biophysics, University of Iowa School of Medicine, Iowa City, IA 52242-1109; V. N. Uebele, Merck Research Labs, WP26-265, West Point, PA 19486.
Address for reprint requests: M. M. Tamkun, Dept. of Physiology, Colorado State University, Fort Collins, CO 80523.
Received 18 December 1997; accepted in final form 17 February 1998.
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