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INTRODUCTION |
Inward rectifier K+ channels (Kir) play an important
role in controlling membrane excitability (1, 2). Kir2.3 is a member of
the Kir superfamily and is expressed in several tissues including the
central nervous system, heart, and kidney (3-5). Unlike other members
in the Kir2 family, Kir2.3 is directly coupled to G proteins, a
coupling that enables this channel to contribute to neurotransmission and cell-cell communications (6). Also, Kir2.3 is known to be modulated
by several intra- and extracellular signal molecules including
Mg2+, polyamines, protons, and protein kinase C
(PKC)1 (7-9). The modulation
of channel activity by PKC is remarkable not only because PKC
activators strongly inhibit channel activity by almost 50% but also
because the signal transduction pathway of PKC is so common that a
large number of extracellular messengers can act on this K+
channel through the activation of PKC. This is particularly important when Kir2.3 channel modulation is considered in the central nervous system, because the control of neuronal membrane excitability by
numerous neurotransmitters and hormones can occur via this K+ channel.
Although Kir2.3 activity is affected by PKC activators and inhibitors,
the critical PKC phosphorylation site is unidentified (8). Without this
information, it remains debatable whether the modulation is mediated by
a direct phosphorylation of the channel protein or indirectly by other
molecules that are activated by PKC. To determine the molecular
substrate for the PKC phosphorylation, we perform experiments in which
channel sensitivity to a PKC activator PMA was studied in several
chimeras constructed between Kir2.3 and Kir2.1, another member in the
Kir2 family that is insensitive to PMA (8). The PKC phosphorylation
site was identified using site-directed mutagenesis.
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MATERIALS AND METHODS |
Oocytes from female frogs (Xenopus laevis) were used
in these studies. Frogs were anesthetized by bathing them in 0.3%
3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed
after a small abdominal incision (~5 mm) was made. The surgical
incision was closed, and the frogs were allowed to recover from the
anesthesia. Xenopus oocytes were treated with 2 mg/ml
collagenase (type I, Sigma) in OR2 solution (82 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) for 90 min at room temperature. After 3 washes (10 min each) of the oocytes with OR2 solution, cDNA (40-50
ng in 50 nl of double distilled water) was injected into the oocytes. The oocytes were then incubated at 18 °C in ND-96 solution
containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,
5 mM HEPES, and 2.5 mM sodium pyruvate with 100 mg/liter geneticin added, pH 7.4.
Kir2.1 (IRK1) and Kir2.3 (HIR) cDNAs were generously provided by
Drs. Lily L. Jan and Carol A. Vandenberg. These cDNAs were subcloned in a vector for eukaryotic expression (pcDNA3.1,
Invitrogen Inc., Carlsbad, CA) and used for expressions in the
Xenopus oocytes. Chimerical constructs between Kir2.1 and
Kir2.3 were prepared by overlap extension at the junction of the
interested domains using the polymerase chain reaction using the
Pfu DNA polymerase (Stratagene, La Jolla, CA). The resulting
polymerase chain reaction products were subcloned into pcDNA3.1.
Site-specific mutations were made using a site-directed mutagenesis kit
(Quickchange, Stratagene, CA). Correct mutations in both the
overlapping polymerase chain reaction and site-directed mutagenesis
were confirmed with DNA sequencing. All plasmids for oocyte injection
were prepared with a QIAfilter Plasmid Midi Kit (QIAGEN, Chatsworth, CA).
Whole-cell currents were studied on the oocytes 2-5 days after
injection. The two-electrode voltage clamp procedure was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City,
CA) at room temperature (22-24 °C). The extracellular solution (KD-90) contained: 90 mM KCl, 3 mM
MgCl2, and 5 mM HEPES (pH 7.4). Cells were
impaled using electrodes filled with 3 M KCl. One of the
electrodes (1.0-2.0 M
) was used as a voltage recording, which was
connected to an HS-2 ×1L headstage (input resistance, 1011
), and the other electrode (0.3-0.6 M
) was used for current recording connected to an HS-2 ×10MG headstage (maximum current, 130 µA). Oocytes were accepted for further experiments only if their leak
currents measured as the difference before and after a leak subtraction
were less than 10% of the peak currents. The leak subtraction was not
applied. Current records were low-pass filtered (Bessel, 4-pole filter,
3 db at 5 kHz), digitized at 5 kHz (12-bit resolution), and stored on
computer disk for later analysis (pClamp 6.0.3, Axon Instruments) (10,
11).
PMA was purchased from Sigma and dissolved in dimethyl sulfoxide
(Me2SO) for a stock concentration of 10 mM.
Immediately before experiments, PMA was diluted to a concentration of
100 µM in the KD-90 solution. Thirty microliters were
added to the recording chamber to reach a concentration of 1 µM. The final concentration of Me2SO was
0.01% or less in the present study (8). PKC inhibitor chelerythrine
(RBI, Natich, MA) was dissolved in double distilled water with a final
concentration of 10 µM. All data for PMA and chelerythrine chloride were obtained with an exposure period of 8-20 min.
Data are presented as means ± S.E. (n, number of
oocytes), and differences in the mean were tested with the Student's
t test or ANOVA (analysis of variance) and were accepted as
significant if p
0.05.
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RESULTS |
Kir2.3 but Not Kir2.1 Is Sensitive to PMA--
Whole-cell currents
were studied in Xenopus oocytes that had received an
injection of either Kir2.3 or Kir2.1 cDNA 2-5 days earlier. A high
concentration of K+ (90 mM) was applied to the
extracellular solution. The membrane potential was held at 0 mV and
stepped to a series of depolarizing and hyperpolarizing potentials.
Under such an experimental condition, strong inward rectifying currents
were observed in both Kir2.3- and Kir2.1-injected oocytes. Exposure of
these oocytes to PMA (1 µM) produced an inhibition of
Kir2.3 currents by 40.9 ± 4.8% (mean ± S.E.,
n = 6) (Fig. 1). The
inhibition of PMA had a long lasting effect. As has been observed
previously by Henry et al. (8), we did not see evident
recovery of channel activity in 20-30 min of washout. The inhibition
of Kir2.3 was voltage-independent. The profile of the I/V
relationship was identical in the presence and absence of PMA after
they were scaled to the same size (Fig. 2). Unlike the Kir2.3, however, a similar
exposure caused only modest inhibition of Kir2.1 currents by 10.1 ± 4.4% (n = 4). This small change in current
amplitude is insignificant in comparison with that of the vehicle
control using 0.01% Me2SO in the KD-90 solution
(p > 0.05, n = 4). These results are
therefore consistent with previous observations on these two
K+ channels.

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Fig. 1.
Effects of PMA on Kir2.3 activity.
A, whole-cell currents were recorded from an oocyte 3 days
after an injection of Kir2.3 cDNA using a two-electrode voltage
clamp. Membrane potential (Vm) was held at 0 mV. A
series of command pulse potentials from 160 mV to 140 mV with a 20-mV
increment were applied to the cell. Currents were inhibited when the
oocyte was exposed to 1 µM PMA. B, the profile
of current amplitude changes shows that the currents decreased rapidly
when PMA was present in the extracellular solution, reached the maximum
in about 4-5 min, and maintained at this level even after a washout of
PMA from the bath solution.
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Fig. 2.
Voltage independence of Kir2.3 inhibition by
PMA. A, currents were recorded from an oocyte in the
same condition as Fig. 1. B, these currents were inhibited
by exposure to 1 µM PMA (8 min). C, I/V plots
show that only the inward rectifying currents were affected by PMA.
D, when currents in A and B are scaled
to the same magnitude at 160 mV, the I/V relationship of the currents
recorded in these two conditions becomes identical, indicating that the
effect of PMA on Kir2.3 currents is voltage-independent. Open
circles, control; solid triangles, PMA exposure.
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The PMA-sensitive Motif Is Located in the N-terminal Region of
Kir2.3--
To identify the PKC phosphorylation site in Kir2.3, we
took advantage of the following established experimental results and constructed several chimeras between Kir2.3 and Kir2.1. 1) The effect
of PKC on Kir2.3 and Kir2.1 has been well documented, and there is a
clear difference in PMA sensitivity between Kir2.3 and Kir2.1. Thus,
extensive pharmacological manipulations using PKC activators and
inhibitors can be avoided. 2) Kir2.3 has a high homology with Kir2.1 in
their peptide sequences, suggesting that recombinant proteins are
likely to produce functional channels. 3) It is known that PKC
phosphorylation sites are only located in the cytosolic side of the
plasma membranes. 4) According to the widely accepted transmembrane
topology, both Kir2.3 and Kir2.1 have their N and C termini inside the
membrane. Hence, it is possible that the N- and/or C-terminal region
contains the critical motif(s) for PKC phosphorylation.
When the entire N-terminal region in Kir2.3 was replaced with a Kir2.1
counterpart (NI-HIR), the mutant channel lost its sensitivity to PMA
(5.0 ± 3.9%, n = 6). In a chimera with its
N-terminal sequence from Kir2.3 and the rest of the sequence from
Kir2.1 (NH-IRK), we found that this mutant channel gained sensitivity
to PMA (38.4 ± 2.3%, n = 4, p < 0.01 compared with wild-type Kir2.1), suggesting that the PMA
phosphorylation site(s) is located in the N-terminal region of Kir2.3
(Fig. 3).

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Fig. 3.
The PMA phosphorylation site is located in
the N-terminal region of Kir2.3. Percentage inhibition of the
inward rectifier currents of HIR, IRK, and their mutants created by the
addition of 1 µM PMA (10-20 min exposure) is plotted.
These chimerical and mutant channels are clearly distributed in two
groups with one showing a similar PMA sensitivity to Kir2.3 and the
other to Kir2.1. Dotted lines indicate levels of PMA
sensitivity of Kir2.3 and Kir2.1. Open circles,
Kir2.3-based mutations; open triangles,
Kir2.1-based mutations; closed diamond,
experimental control with 0.01% Me2SO alone.
HIR, Kir2.3 (n = 6); IRK, Kir2.1
(n = 4); NI-HIR, the N-terminal region of
HIR was replaced with that in IRK (n = 6);
NH-IRK, the N-terminal region of IRK was substituted with
that in HIR (n = 4); N1-19HIR, the first 19 residues in the N-terminal end of Kir2.3 were substituted with the
first 45 residues in the N-terminal end of Kir2.1 (n = 4); N25-44HIR, residues 25-44 in Kir2.3 were replaced with
residues 51-70 of Kir2.1 (n = 3);
S36G/S39G, serine 36 and serine 39 in Kir2.3 were mutated to
glycine (n = 4); N53-60HIR, residues 53-60
in Kir2.3 were replaced with residues 79-86 in Kir2.1
(n = 4); T53I-HIR, threonine 53 in HIR was
mutated to isoleucine (n = 4); N79-86IRK,
residues 79-86 in Kir2.1 were replaced with residues 53-60 in Kir2.3
(n = 4); I79T-IRK, isoleucine 79 in IRK was
changed to threonine (n = 4); DMSO, control
using Me2SO alone in the same concentration as used in all
other experiments (n = 4). Data are presented as
mean ± S.E.
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Threonine 53 Is the PKC Phosphorylation Site--
Four potential
PKC phosphorylation sites have been found in the N-terminal region of
Kir2.3. To identify which one of them is the true PKC phosphorylation
site, we focused on these potential PKC phosphorylation sites and
generated several chimeras and site-specific mutations in which one or
two of these sites were eliminated. We found that the serine residue at
position 5 was not involved in PKC phosphorylation, because a removal
of this residue by replacing the first 19 residues with the first 45 residues in IRK (N1-19HIR) did not significantly change the PMA
sensitivity (34.7 ± 4.4%, n = 4, p > 0.05 compared with wild-type Kir2.3) (Fig. 3).
Serine 36 and serine 39 had been previously proposed to be the PKC
phosphorylation sites (8). Hence, these two residues were examined.
Substitution of the middle part of the N-terminal region, including
these two serine residues and the other 18 amino acids (N25-44HIR),
did not reduce the PMA sensitivity of the chimerical channel (46.2 ± 6.2%, n = 3, p > 0.05 compared
with wild-type Kir2.3) (Fig. 3). Site-specific mutation of both of
these serine residues together (S36G,S39G) also had no effect on the
inhibition of this channel by PMA (38.1 ± 1.5%,
n = 4, p > 0.05 compared with
wild-type Kir2.3) (Fig. 3). This suggests that neither of these two
serine residues is the PKC phosphorylation site. Threonine 53 is
another residue that appears to be a PKC phosphorylation site. It has a
motif (VDTRWR) and is located near the first transmembrane
(M1) domain. Site-specific mutation of this threonine into isoleucine (T53I-HIR) with all of the rest of the sequence intact completely abolished the channel sensitivity to PMA (1.6 ± 1.0%,
n = 4, p < 0.001 compared with
wild-type Kir2.3) (Figs. 3 and 4).
Interestingly, the amino acid sequence around this residue is quite
similar between Kir2.3 and Kir2.1. To see if the creation of this
threonine can affect Kir2.1 sensitivity to PMA, a sequence including
eight amino acid residues surrounding this threonine was introduced to
Kir2.1 by substituting a corresponding sequence in the same region
(N79-86IRK). We found that the chimerical channel became PMA-sensitive
(37.8 ± 5.7%, n = 4, p < 0.01 compared with wild-type Kir2.1), although only three residues in this
segment are different between Kir2.3 and Kir2.1. We further mutated the
isoleucine residue to threonine in Kir2.1 at the same position as the
threonine 53 in Kir2.3. This mutation (I79T-IRK) led to an emergence of
the PMA sensitivity in the Kir2.1-based channel (42.6 ± 3.7%,
n = 4, p < 0.05 compared with
wild-type Kir2.1) (Fig. 4). This inhibitory effect of PMA was totally
blocked with 10 µM chelerythrine, a PKC inhibitor (0.1 ± 2.1%, n = 3, 20 min of chelerythrine
preincubation followed by 20 min of PMA exposure). Thus, these results
indicate that threonine 53 is the only PKC phosphorylation site
involved in the modulation of Kir2.3 channel activity.

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Fig. 4.
The PKC phosphorylation site is identified at
threonine 53 in Kir2.3. Left panel, when the threonine
residue was mutated to isoleucine (T53I-HIR), the PMA (1 µM) sensitivity was completely abolished in the mutant
Kir2.3. Right panel, when isoleucine 79 at the corresponding
position to the threonine 53 in Kir2.3 was replaced with threonine, the
mutant Kir2.1 (I79T-IRK) obtained PMA sensitivity. Note that the PMA
exposure periods were 18 and 20 min for T53I-HIR and I79W-IRK,
respectively. M1, transmembrane 1; M2,
transmembrane 2; P, pore zone.
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DISCUSSION |
A number of neurotransmitters and hormones has been shown to
modulate inward rectifier K+ channels via phosphorylation
of the channel protein by PKC (12-16). For example, substance P and
neurotensin cause a slow excitation of neurons of the nucleus basales
and substantia nigra. This is mediated by inhibition of inward
rectifying K+ channels through the activation of PKC (17,
18). In cardiac myocytes and renal epithelial cells, activation of PKC
also produces an inhibition of Kir currents, although detailed
functions of this inhibition are not fully understood (12, 19-23). In
addition to the covalent modulation of channel activity, PKC has also
been shown to affect the expression of these Kir channels (15, 24).
Characterization of specific Kir channels that are modulated by PKC has
been carried out recently. Macica et al. (23) have shown
that exogenous protein kinase C inhibits the wild-type but not the S4A
mutant Kir1.1 channel. Fakler et al. (25) have found that
N-heptyl-5-chloro-1-naphthalenesulfonamide, a specific PKC stimulator, causes Kir2.1 rundown. Henry et al. (8) have
indicated that Kir2.3 is strongly inhibited by PKC. The latter
investigators have also shown no effect of PKC activators on Kir2.1. In
the present study, we have observed a strong inhibition of Kir2.3 when
the oocytes are exposed to PMA. However, the same exposure causes only
a small and insignificant change in Kir2.1 currents, a result
consistent with previous observations (8). Therefore, Kir2.3 but not
Kir2.1 appears to be modulated by PKC.
Identification of the PKC phosphorylation site in Kir2.3 was attempted
previously by Henry et al. (8) who studied Ser-36 and Ser-39
as potential PKC phosphorylation sites. By replacing these two serine
residues with cysteine and alanine (S36C, S39A), they could not see
channel expression and thus were unsuccessful in demonstrating the PKC
phosphorylation site in Kir2.3 (8). In the present study, we have
mutated Ser-36 and Ser-39 to glycine residues located in the
corresponding positions in Kir2.1. These mutations have no effect on
channel expression and its sensitivity to PMA, indicating that they are
not PKC phosphorylation sites. The other serine residue at position 5 is not involved in PKC phosphorylation, because mutation of this
residue does not change the PMA sensitivity as shown in our current
study. Our results indicate that threonine 53 is the only PKC
phosphorylation site that controls channel activity, because a
substitution of this residue with isoleucine completely eliminates
channel sensitivity to PMA. This is further strengthened by our results
showing that a creation of this threonine residue in Kir2.1 at the same
location leads to a strong inhibition of the mutant channel by PMA.
In conclusion, the modulation of Kir2.3 by PKC phosphorylation of the
channel protein has been demonstrated in our current studies. The PKC
phosphorylation site is located in the N-terminal region. Threonine 53 with a VDTRWR motif is the substrate of PKC.