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INTRODUCTION |
Voltage-gated K+ channels play a critical role in the
normal physiology of excitable cells. K+ currents
contribute to action potential repolarization in cardiac cells (1, 2)
while in vascular smooth muscle, regulation of membrane potential by
K+ currents is a major determinant of vascular tone (3, 4). Kv1.5 is one of the more cardiovascular-specific K+ channel
isoforms identified to date, although it has been found in other
tissues (1, 5-7). Upon heterologous expression, Kv1.5 generates a
rapidly activating delayed rectifier K+ current which is
sensitive to block by 4-aminopyridine (1). Substantial data indicate
that Kv1.5 forms at least in part the molecular basis for an
ultra-rapid delayed rectifier K+ current (IKur)
that is present in human atrium but not ventricle (1, 8, 9), indicating
that the current may be atrial specific. However, Kv1.5 mRNA and
protein are detected in ventricular tissue, suggesting that the subunit
composition of the Kv1.5 complex varies between atrium and ventricle.
IKur is modulated by
-adrenergic stimulation which
activates cAMP-dependent protein kinase
(PKA),1 with an increase in
K+ current amplitude (10). In addition, Kv1.5 likely
encodes a channel responsible for a 4-aminopyridine-sensitive, delayed
rectifier K+ current (IKdr) in vascular smooth
muscle, which is also enhanced by stimulation of PKA (3, 7, 11).
Kv
subunits such as Kv1.5 coassemble as homo- or heterotetramers
with
subunits to form functional K+ channels (1). In
addition, smaller
subunits modify channel function (1, 12). Two
subunits cloned from ferret and human heart (13-17), Kv
1.2 and
Kv
1.3, represent splice variants from the same gene. These two
proteins are identical in the carboxyl 329 amino acids, and this
portion appears to be responsible for physical interaction or binding
with the
subunit (12, 18). In contrast, the NH2 termini
show little identity (~25%), consistent with the concept that this
region accounts for differences in
subunit function (1, 12).
Coexpression of either Kv
1.2 or Kv
1.3 with Kv1.5 alters channel
function, with the development of rapid although partial inactivation,
slowed deactivation, and a hyperpolarizing shift in the voltage
sensitivity of activation (13, 16). Additional studies have
demonstrated that as with other Shaker-type K+ channel
-
interactions, both of these
subunits bind to a specific
region on the Kv1.5 NH2 terminus (17-19).
The amino acid sequences of both Kv
1.2 and Kv
1.3 contain
consensus sites for phosphorylation by PKA. The purpose of this study
was to determine whether this
-
interaction is modulated by PKA
stimulation. We found that coexpression of Kv
1.3, but not Kv
1.2,
enabled a response to kinase activation, with marked slowing of fast
inactivation and an increase in K+ current. These effects
can be attributed to phosphorylation of a specific consensus site by
PKA in the NH2 terminus of the Kv
1.3 subunit.
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EXPERIMENTAL PROCEDURES |
Materials--
Reagent grade chemicals, as well as
8-chlorophenylthio cAMP, 3-isobutyl-1-methylxanthine, and forskolin
were obtained from Sigma, while 8-bromo-cAMP was purchased from
Calbiochem. Tissue culture media and reagents, including LipofectAMINE,
were obtained from Life Technologies, Inc. (Grand Island, NY). Enzymes
and buffers were obtained from Roche Molecular Biochemicals
(Indianapolis, IN), Promega (Madison, WI), and New England Biolabs
(Beverly, MA). The source of other materials is specified below.
K+ Channel Expression
Xenopus laevis Oocytes--
DNA constructs of Kv1.5, Kv
1.2,
and Kv
1.3 (each in a modified pSP64T vector (13, 16)) were
linearized with EcoRI and cRNA transcribed using the SP6 RNA
polymerase (SP6 Cap-Scribe, Roche Molecular Biochemicals, Indianapolis,
IN). Defolliculated Xenopus oocytes were prepared as
described previously and injected with approximately 40 nl of RNA (20).
Kv1.5 cRNA was diluted with RNase-treated water so that currents for
experimentation did not exceed 8 µA. This was combined with an excess
of undiluted RNA in ratios which achieved maximal effect as assessed by
K+ current characteristics during electrophysiological
recordings (13, 16, 21).
HEK 293 Cells--
For coexpression of Kv1.5 and Kv
1.3 in HEK
293 cells, human Kv1.5 (
22-1894 nucleotides) and human Kv
1.3
(
53-1500 nucleotides) were inserted in tandem into a modified pBKCMV
(m-pBK) vector that had the
-galacosidase ATG at position 151 removed to increase expression efficiency. The Kv1.5 subunit was placed
3' to Kv
1.3 and behind an internal ribosome entry sequence (IRES),
thus generating a dual cistronic mRNA. A 590-base pair IRES was
subcloned into EcoRI/EcoRV prepared pBSKS+. The
above mentioned subunit fragments were blunted and subcloned into
blunted XbaI and ClaI sites of the pBS polylinker
that flanked the subcloned IRES. This construct was then digested with
SalI, blunted, and then digested with NotI to
release the Kv
1.3/IRES/Kv1.5 sequence. This fragment was then inserted into NotI/SmaI digested m-pBK. This
construct consistently generated currents showing complete Kv
1.3
effects. Kv
1.3 mutants were also expressed using this m-pBK IRES construct.
Recently thawed HEK 293 cells (ATCC number 1573-CRL) were maintained in
culture and transiently transfected using LipofectAMINE as described
previously (22), with coexpression of green fluorescent protein to
identify cells for voltage clamp analysis. For transfections, 2.5 µg
of hKv1.5-pBK, 4 µg of Kv
1.3/IRES/hKv1.5-pBK, and 0.5 µg of
GFP-pCICMV were mixed with 25 µl of LipofectAMINE reagent and applied
overnight, after which the standard culture medium was restored. Cells
were removed from the dish using brief trypsinization, washed twice
with maintenance medium, and stored at room temperature for recording
within the next 12 h.
Electrophysiological Recording and Data Analysis
Oocyte recordings were performed using the two-microelectrode
voltage clamp technique as described previously (20, 21). Membrane
potentials were controlled by a high-compliance voltage-clamp amplifier
(Clampator, Dagan Instruments, Minneapolis, MN) with voltage command
potentials generated by a 12-bit digital-to-analog converter controlled
by customized by pClamp software (Axon Instruments, Foster City, CA).
Pipettes were filled with 3 M KCl, and a standard extracellular bath solution was utilized (in mM: NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.5).
Oocytes that demonstrated endogenous currents greater than 1% of
expressed currents were not utilized. Data were sampled at 10-20 kHz
and filtered at 2-5 kHz. The holding potential was
80 mV and the
cycle time for all pulse protocols was 10 s or slower to allow
full recovery from inactivation between pulses unless otherwise
specified. To calculate cell membrane electrical capacitance, the
capacitive transient was recorded during a small voltage step (
80 to
70 mV) during which K+ currents were not activated.
Integration of the leak-corrected transient yielded the charge
(Q) transferred during the voltage step of V from
which capacitance (C) was calculated: C = Q/V. All experiments were conducted at room
temperature (22 ± 2 °C).
K+ current recordings in HEK 293 cells were obtained with
an Axopatch 200B amplifier (Axon Instruments) using the whole cell configuration of the patch clamp technique (22). The intracellular pipette solution contained (in mM): KCl 110, HEPES 10, K4BAPTA 5, K2ATP 5, and MgCl2 1 (pH
7.2); the extracellular bath solution contained (in mM):
NaCl 130, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 10, glucose 10 (pH 7.35). Currents were sampled at 1-10 kHz and filtered
at 0.5-5 kHz. Data acquisition and command potentials were controlled
by pClamp software.
Analysis of data was performed using either custom programs that were
designed to read and analyze pClamp data files or Clampfit 6.04. Activation curves were constructed from deactivating tail currents and
were fitted with a Boltzmann equation. The time course of macroscopic
K+ current inactivation was fitted with an exponential
function using a nonlinear least squares algorithm. Comparison of the
voltage-dependent and kinetic properties of K+
currents after PKA stimulation to control values was performed using a
paired t test. Results are presented as mean ± S.E.
Mutagenesis
For some experiments, Kv1.5 was coexpressed with an
NH2-terminal deletion mutant of Kv
1.3 (
N50Kv
1.3).
This mutant was constructed as described previously (21) by changing
the serine at position 50 to a methionine. A COOH-terminal deletion
mutant of Kv1.5 (
C57Kv1.5) (23) was generated by transfer from pGEM
into wild-type pBK using EcoRI and HindIII,
followed by transfer into the IRES construct using StuI.
Point mutations were constructed in the Kv
1.3 sequence in which
serines were converted to alanines at position 24 (S24A), 39 (S39A), or
164 (S164A) using either overlap extension polymerase chain reaction
mutagenesis (24) or the Quick-Change Site-directed mutagenesis kit from
Stratagene. Multiple independent recombinant clones were analyzed by
restriction digestion to verify correct assembly and to screen for the
presence of the mutation (creation of a new restriction site). Mutant
inserts were fully sequenced and clones lacking polymerase errors were
chosen for electrophysiologic studies.
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RESULTS |
Co-expression of Kv1.5 and Kv
1.3: Effects of PKA
Activation--
In Xenopus oocytes, coexpression of
Kv
1.3 with Kv1.5 produced K+ currents that displayed
partial inactivation and slowed deactivation as previously reported
(16, 21) (Fig. 1A). With
sustained depolarization to +50 mV, peak K+ current
declined by 44 ± 2% over a 50-ms period, with a rapid time
course (
= 4.2 ± 0.1 ms; n = 17). The voltage
dependence of channel activation was shifted to hyperpolarized
potentials (midpoint or V1/2 =
22.0 ± 1.3 mV) compared with Kv1.5 alone
(V1/2 =
5.2 ± 1.0 mV;
n = 19).

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Fig. 1.
Modulation of K+ current derived
from Kv1.5 + Kv 1.3 by PKA stimulation.
A, K+ currents are shown following coinjection
of cRNA for Kv1.5 and Kv 1.3 in Xenopus oocytes under
control conditions (left panel) and after bath superfusion
of PKA activators (right panel; voltage is stepped from 80
mV to a maximal potential of +50 mV, with repolarization to 30 mV).
Stimulation of PKA caused marked slowing of macroscopic inactivation
associated with an increase in K+ current. B,
similar data are presented following coexpression of Kv1.5 and
Kv 1.2. Under these conditions, there was no effect of kinase
stimulation on K+ currents. C, the time course
of normalized peak current (at +50 mV) is shown after the start of bath
superfusion of PKA activators (arrow, time 0). The increase
in K+ current amplitude seen with Kv 1.3 coexpression
( ) is not present with Kv 1.2 ( ).
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In order to stimulate PKA, cells were perfused with a combination of
kinase activators (8-chlorophenylthio-cAMP, 200 µM;
3-isobutyl-1-methylxanthine, 1 mM; and forskolin, 10 µM) which activates the cystic fibrosis conductance
regulator in a rapid, potent manner (25). As illustrated in Fig.
1A, activation of PKA caused marked slowing of macroscopic inactivation for K+ current derived from Kv1.5 + Kv
1.3
(
= 4.2 ± 0.1 ms before and 6.2 ± 0.3 ms after PKA
activation; p < 0.01), with a reduction in the overall
extent of K+ current inactivation induced by the
subunit (21 ± 2% at +50 mV, compared with 44 ± 2% before
kinase activation). In addition, K+ current deactivation
became more rapid, consistent with reduced
-mediated effects
(deactivation
at
40 mV was 63 ± 6 ms before and 47 ± 4 ms after PKA). The loss of K+ current inactivation was
associated with a significant, sustained increase in current amplitude
(+40 ± 3% at +50 mV in 15 min; n = 17). The time
course of these effects was rapid, as shown by normalized
K+ current values during an individual experiment in Fig.
1C.
To further investigate the mechanisms of these effects by PKA,
activation curves were constructed from deactivating tail currents. Stimulation of PKA did not change the midpoint of this curve
(V1/2 =
22.0 ± 1.3 mV before and
22.8 ± 1.1 mV after PKA), indicating that the voltage
dependence of channel opening was not affected. In addition, there was
no associated change in cell membrane electrical capacitance with
kinase activation (+1 ± 2% at 15 min), suggesting that
significant changes in cell surface area were not involved in the
effect observed.
K+ currents derived from coexpression of Kv
1.3 with
Kv1.5 demonstrate not only development of fast
voltage-dependent inactivation, but also enhancement of the
slow or C-type inactivation that is present with Kv1.5 alone (21). The
extent of slow inactivation was studied with a twin pulse protocol
during which the duration of the first pulse (+70 mV) was progressively
lengthened, followed by a 50 ms hyperpolarizing step to
80 mV prior
to the second pulse (+70 mV) in order to permit full recovery of fast
inactivation while minimizing recovery of slow inactivated channels
(21). The fraction of slow inactivation was calculated as 1
(P2/P1), where P2 is the peak
current of the second pulse and P1 is the peak current of
the first pulse. With a prepulse of 2 s, this value was 0.32 ± 0.2 under control conditions, and 0.30 ± 0.02 after PKA
stimulation (data not shown). These data indicate that despite the
effect of PKA to reduce fast inactivation, the extent of slow
inactivation was not altered.
Lack of PKA Response following Coexpression of Kv
1.2 with
Kv1.5--
Because Kv
1.2 can also coassemble with Kv1.5, we
investigated whether coexpression of this subunit led to a similar
effect with PKA activation. As demonstrated in Fig. 1B,
K+ currents following coinjection of Kv
1.2 with Kv1.5 in
Xenopus oocytes demonstrated partial inactivation (50 ± 2% at +50 mV; n = 11), slowed deactivation, and an
altered voltage dependence of channel opening (V1/2 =
20.4 ± 2.0 mV) compared with Kv1.5 alone. With stimulation of
PKA, there was no effect on either K+ current amplitude
(+3 ± 2% at +50 mV in 15 min; n = 11), kinetics of inactivation (
= 4.0 ± 0.1 ms before and 4.0 ± 0.1 ms
after PKA at +50 mV), or extent of inactivation (50 ± 3% after
PKA). A comparison of the typical response to kinase activation when Kv1.5 was coexpressed with the two different
subunits is
demonstrated in Fig. 1C.
Effects of PKA in Mammalian Cells--
In order to confirm the
relevance of our findings for K+ channel regulation in
mammalian cells, we also examined the effects of PKA activation in HEK
293 cells. Similar results were obtained when the K+
channel subunits under study were expressed in this mammalian cell
line. Following exposure to the cAMP analog 8-bromo-cAMP, there was
marked slowing in the time course of macroscopic fast inactivation for
cells coexpressing Kv1.5 and Kv
1.3 (
= 3.7 ± 0.2 ms before
and 5.9 ± 0.3 ms after PKA stimulation; n = 7), so that current at the end of a 250-ms voltage step was increased by
39 ± 8% (Fig. 2A).
Analogous to results in oocytes, there was no significant effect on
slow inactivation (
slow = 402 ± 100 ms before and
468 ± 78 ms after PKA). On the other hand, there was greater
variability in the effect of PKA to increase K+ current
amplitude in HEK 293 cells than was seen in oocytes. Peak
K+ current increased in most but not all cells (+29 ± 4% in 5 of 7 cells) expressing Kv1.5 + Kv
1.3 (Fig. 2A),
with no significant change for the group as a whole (+1 ± 4%).
As in oocytes, there was no change in the voltage dependence of channel
opening as assessed by deactivating tail currents (Fig. 2B;
V1/2 =
22 ± 1 mV before and
23 ± 1 mV after kinase stimulation).

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Fig. 2.
Effect of PKA activation following
coexpression of Kv1.5 and Kv 1.3 in HEK 293 cells. A, following co-transfection of Kv1.5 and
Kv 1.3 in HEK 293 cells, the cAMP analog 8-bromo-cAMP caused relief
of macroscopic fast inactivation with little effect on the slow
component of inactivation (see text) and, in this case, an increase in
K+ current amplitude as illustrated by 250-ms pulses to +50
mV before and after PKA activation. B, averaged activation
curves derived from deactivating tail currents are shown before and
after PKA activation (V1/2 = 22 ± 1 mV
before and 23 ± 1 mV after 8-bromo-cAMP).
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Role of Serine 24 in Kv
1.3 in the PKA Response--
Kv
1.2
and Kv
1.3 share a common consensus PKA phosphorylation site in the
conserved COOH-terminal region of the proteins (serine 164 in
Kv
1.3). However, two additional sites are present in the unique
NH2 terminus of Kv
1.3 (serine 24 and serine 39) which
are not present in Kv
1.2. Additional experiments were performed to
test the hypothesis that one of these potential sites in the variable
NH2 terminus of the protein was responsible for the effects of PKA activation which occurred with Kv
1.3 but not Kv
1.2. The NH2-terminal 50 amino acids of Kv
1.3 were removed and
the mutant construct (
N50Kv
1.3) was coexpressed with Kv1.5 in
Xenopus oocytes. Deletion of this portion of the Kv
1.3
NH2 terminus eliminated
-mediated inactivation as
described previously (21). Moreover, the PKA response seen with
wild-type Kv
1.3 was abolished, with no effect on K+
current amplitude (
4 ± 2% at +50 mV in 15 min;
n = 6) or voltage dependence of channel opening
(V1/2 =
18 ± 2 mV before and
19 ± 2 mV after PKA) following stimulation of PKA (data not shown). These
results implicated the Kv
1.3 NH2 terminus in the effects
of kinase activation and suggested that the observed increase in peak
current with PKA activation was probably due to decreased inactivation.
To determine if one or both unique NH2-terminal consensus
PKA sites in Kv
1.3 were responsible for the effects of PKA, each site was removed individually by changing serine to an alanine (Kv
1.3S24A and Kv
1.3S39A). K+ currents following
coexpression of Kv
1.3S24A with Kv1.5 in oocytes resembled wild-type
currents, except that both the time course and extent of fast
inactivation were enhanced (Fig.
3A). At +50 mV, inactivation
was 3.4 ± 0.2 ms (versus 4.2 ± 0.1 ms for
wild-type) with a fall in peak K+ current of 58 ± 2%
over 50 ms (versus 44 ± 2% for wild-type; n = 7). In addition, activation of PKA had no effect on
the extent of inactivation (54 ± 2% after PKA), the speed of
macroscopic current decay (
= 3.5 ± 0.1 ms after PKA), or
K+ current amplitude (+0 ± 1% at +50 mV in 15 min)
in the presence of this mutation (Fig. 3, A and
B). K+ currents generated by the Kv
1.3S39A
mutation demonstrated current characteristics and response to PKA
stimulation (Fig. 3B) which was similar to that seen with
wild-type Kv
1.3. Upon exposure to PKA activators, K+
currents demonstrated slowing of fast inactivation (
= 4.5 ± 0.2 ms before and 5.9 ± 0.3 ms after PKA) and a reduction in the extent of this process (41 ± 2% before and 23 ± 3% after
PKA), associated with a rapid increase in peak K+ current
amplitude (+36 ± 3% at +50 mV in 15 min; n = 8).
Taken together, these data indicate that coexpression of Kv
1.3
modulates the response of Kv1.5 to PKA activation by phosphorylation at a specific site, serine 24, on the NH2 terminus of the
subunit protein. Because inactivation is enhanced with the Kv
1.3S24A mutant, it is likely that wild-type Kv
1.3 is phosphorylated to some
extent under basal or unstimulated conditions.

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Fig. 3.
Involvement of serine 24 in
Kv 1.3 and effect of charge mutations at this
position. A, using site-directed mutagenesis, each of
the two unique NH2-terminal putative PKA sites in Kv 1.3
was removed individually by changing the serine in the consensus site
to alanine (Kv 1.3S24A and Kv1.3 S39A). Coexpression of
Kv 1.3S24A with Kv1.5 produced inactivating K+ currents
(left panel) which did not respond to activation of PKA
(right panel). B, the time course of normalized
peak current (+50 mV) for representative experiments with each mutant
is shown in the lower panel. PKA activation had no effect on
K+ current resulting from Kv1.5+Kv 1.3S24A (+0 ± 1% at 15 min; n = 7), while coexpression of the
Kv 1.3S39A mutant with Kv1.5 produced currents which retained the
wild-type response to PKA (+36 ± 3% at 15 min; n = 8). C, upon substitution of a negatively charged amino
acid for serine 24 (Kv 1.3S24D), fast inactivation was dramatically
reduced (left panel), while replacement with a positively
charged amino acid (Kv 1.3S24K) caused enhancement of the time course
and extent of macroscopic inactivation (right panel)
compared with wild-type Kv 1.3.
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As demonstrated in Fig. 4, data obtained
in HEK 293 cells further confirmed the mutagenesis experiments
conducted in Xenopus oocytes. There was no effect of PKA
activation on K+ currents derived from Kv1.5 + Kv
1.3S24A
(Fig. 4A) with respect to fast inactivation
(
fast = 4.8 ± 0.3 ms before and 5.1 ± 0.3 ms
after PKA; n = 7). On the other hand, stimulation of
PKA caused substantial relief of inactivation when Kv1.5 was
coexpressed with the Kv
1.3S39A mutant (Fig. 4B;
fast = 4.7 ± 0.2 ms before and 7.6 ± 0.4 ms
after PKA; n = 6). A similar effect was observed with a
mutation which removed the conserved COOH-terminal Kv
1.3 PKA
consensus site, serine 164, as shown in Fig. 4C
(inactivation
fast = 5.9 ± 0.4 ms before and
7.3 ± 0.4 ms after PKA; n = 6). Finally, because
previous reports showed that phosphorylation of sites in the COOH
terminus of the Kv1.1
subunit modulate Kv
1.1-mediated
inactivation (26, 27), a Kv1.5 mutant lacking the COOH-terminal 57 amino acids and the two PKA consensus sites (serine 555 and serine 578)
in the channel was studied. When this mutant was coexpressed with
Kv
1.3, a wild-type response to PKA activation was observed (Fig.
4D; inactivation
fast = 5.0 ± 0.3 ms
before and 10.0 ± 0.4 ms after PKA; n = 5). These
results further substantiate the data in oocytes which implicate
phosphorylation of serine 24 in Kv
1.3 as the sole molecular
mechanism responsible for the PKA reduction in fast inactivation.

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Fig. 4.
Effects of mutations in
Kv 1.3 and Kv1.5 on the PKA response following
coexpression in HEK 293 cells. A, K+
currents elicited by 250 ms voltage steps to +50 mV are shown before
and after exposure to 8-bromo-cAMP after transfection of Kv1.5 and
Kv 1.3S24A into HEK 293 cells. In the presence of the subunit
mutation, the cAMP analog had no effect on fast inactivation.
B and C, experiments similar to that shown in
panel A for the Kv 1.3S39A and Kv 1.3S164A mutations,
respectively. When coexpressed with Kv1.5, K+ currents
retained the wild-type response to stimulation of PKA, with marked
slowing of fast inactivation. D, expression of the
COOH-terminal 57-amino acid deletion mutant of Kv1.5 with wild-type
Kv 1.3 produced K+ currents which also responded to
activation of PKA with relief of fast inactivation.
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Role of Charge at Position 24 in Kv
1.3-mediated
Inactivation--
To determine whether the effects of PKA resulted
directly from the negative charge imparted by phosphorylation, the
serine at position 24 in Kv
1.3 was mutated to the negatively charged amino acid aspartate (Kv
1.3524D). Co-expression of Kv1.5 with Kv
1.3S24D in Xenopus oocytes produced K+
currents in which inactivation was substantially reduced (19 ± 3%; n = 5) compared with wild-type Kv
1.3 as shown
in Fig. 3C (left panel). Nonetheless, the
-mediated hyperpolarizing shift in the voltage dependence of channel
activation was largely preserved (V1/2 =
15 ± 1 mV), indicating that the loss of inactivation associated
with this mutation was not due to loss of
subunit expression. To
further assess the role of charge at this location, serine 24 was then
mutated to the positively charged residue lysine (Kv
1.3S24K). This
mutation had the opposite effect of Kv
1.3524D, with enhancement of
the extent (65 ± 1%; n = 5) and time course (
= 2.6 ± 0.1 ms) of inactivation with respect to wild-type
Kv
1.3 (Fig. 3C, right panel). These data indicate that
the mechanism whereby phosphorylation relieves
-mediated
inactivation is electrostatic in nature, and that the local charge at
this position in Kv
1.3 is critical to the
-
interaction which
confers inactivation to Kv1.5 current.
 |
DISCUSSION |
Our findings demonstrate that activation of PKA modulates
K+ current derived from coexpression of Kv1.5 and Kv
1.3,
with relief of inactivation that is associated with an increase in
current size. A reduction in fast inactivation would be predicted to
cause some increase in K+ current amplitude. While PKA
activation always enhanced K+ current in Xenopus
oocytes, this response was somewhat more variable in HEK 293 cells,
with an increase in current in most (5 of 7) but not all cells. A
possible explanation for this discrepancy is the fact that voltage
clamp of the K+ currents under study is faster in HEK cells
than in oocytes. Activation is rapid for Kv1.5 currents and in the
presence of
subunit coexpression, the inactivation which is induced
is also fast. If initial clamp of activating currents is slower in
oocytes, there could be more overlap in time between the processes of
activation and inactivation. Under these conditions, any change in the
degree of inactivation is more likely to alter current amplitude
because of concomitant activation. This discrepancy was the only
experimental difference between results in the two expression systems,
with other findings in oocytes essentially duplicated in HEK 293 cells.
Removal of the PKA phosphorylation site at serine 24 in the Kv
1.3
subunit abolished the PKA modification of fast inactivation. Moreover,
this effect was mimicked by substitution of an acidic amino acid which
caused reduced inactivation, while mutation to a basic amino acid
enhanced it. Thus, we conclude that the molecular basis of the PKA
effect is direct phosphorylation of the Kv
1.3 subunit
NH2 terminus at this position. The location of the
phosphorylation site responsible for the PKA effect is not surprising,
since our previous work has shown that it is the NH2
terminus of Kv
1.3 which confers inactivation upon Kv1.5 current
through a possible open channel blocking mechanism (21). Removal of the
first 10 amino acids of Kv
1.3 abolishes fast inactivation, while
attaching the first 87 amino acids of Kv
1.3 to the NH2
terminus of Kv1.5 restores it (21). Our data suggest that when the
NH2 terminus of Kv
1.3 assumes a three-dimensional
structure, serine 24 must be positioned in a region critical for
-mediated inactivation, given the effect of charge mutations at this
location. It is possible that serine 24 is positioned close to one or
more basic residues which are important for the pore blocking effect of
the NH2 terminus, and that phosphorylation or negative
charge can shield these positively charged residues. This mechanism has
been demonstrated previously for kinase modulation of Kv3.4, which
encodes a rapidly inactivating or A-type channel (28-30). Stimulation
of protein kinase C removes fast inactivation by phosphorylation of
NH2-terminal residues which are in close proximity to a
group of basic amino acids in the inactivation particle.
Our results here indicate that despite the effect of PKA to reduce
Kv
1.3 fast inactivation, the extent of slow inactivation was not
altered. We have previously proposed that the enhanced slow
inactivation observed with Kv
1.3 was directly linked to the
-induced fast inactivation, since NH2-terminal deletions in Kv
1.3 that removed fast inactivation also removed the enhanced slow inactivation (21). However, the data presented here suggest the
effect of Kv
1.3 to promote slow inactivation is more complex and not
solely coupled to
-induced fast inactivation. If it was, one
would predict that PKA relaxation of fast inactivation would also
reduce slow inactivation.
As noted above, prior studies have shown that the inactivation
conferred onto Kv1.1 by Kv
1.1 is modulated by
subunit
phosphorylation (26, 27). Based upon our data, it is now apparent that
phosphorylation of the
subunit itself also regulates
-mediated
effects. The functional response of different K+ channel
subunits to activation of protein kinases represents not only a means
to modulate subunit interactions, but also another mechanism for
K+ current diversity in vivo. Modulation of
-
function by kinase systems also further complicates the
correlation of native currents with those obtained from cloned proteins
in heterologous systems.