From the Department of Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261 and the
Vollum Institute, Oregon Health Sciences University,
Portland, Oregon 97201
Received for publication, August 23, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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We previously demonstrated that hIK1 is activated
directly by ATP in excised, inside-out patches in a protein
kinase A inhibitor 5-24 dependent manner, suggesting a
role for phosphorylation in the regulation of this
Ca2+-dependent channel. However, mutation of
the single consensus cAMP-dependent protein kinase
phosphorylation site (S334A) failed to modify the response of hIK1 to
ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor,
D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca2+-dependent K+
channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated
a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not
activate a chimera containing the N terminus plus S1-S4 from
hIK1. In contrast, ATP activated a chimera containing the hIK1
C-terminal amino acids His299-Lys427.
Furthermore, truncation of hIK1 at Leu414 resulted in an
ATP-dependent channel, whereas larger truncations of hIK1
failed to express. Additional hIK1/rSK2 chimeras defined the
minimal region of hIK1 required to confer complete ATP sensitivity as
including amino acids Arg355-Ala413. An
alanine scan of all non-conserved serines and threonines within this
region failed to alter the response of hIK1 to ATP, suggesting that
hIK1 itself is not directly phosphorylated. Additionally, substitution
of amino acids Arg355-Met368 of hIK1 into the
corresponding region of rSK2 resulted in an ATP-dependent
activation, which was ~50% of that of hIK1. These results
demonstrate that amino acids Arg355-Ala413
within the C terminus of hIK1 confer sensitivity to ATP. Finally, we
demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca2+.
The human intermediate conductance KCa channel, hIK1,
is required for a variety of physiological processes including
transepithelial ion transport (2-5), vasodilation (6, 7), T cell
activation (8, 9), cell proliferation (9-11), and regulatory volume decrease (9, 10). In addition to demonstrating modulation by
intracellular Ca2+, we and others have demonstrated that
hIK1 activity can be dynamically regulated by phosphorylation (1, 9,
12-14). Several of these studies have demonstrated an
ATP-dependent activation of hIK1 in excised, inside-out
membrane patches that can be reversed by exogenous phosphatases and/or
kinase inhibitors (1, 12, 13). Based on these observations, we
speculated that the phosphorylation-dependent modulation of
hIK1 plays a critical role in modulating the physiological processes in
which hIK1 is involved.
In our previous study we demonstrated, in excised, inside-out patches,
that addition of ATP (1 mM) resulted in, on average, a 3-fold increase in hIK1 activity (1), having an EC50 of
50 µM.1
The stimulatory effect of ATP exhibited a slow onset, requiring several
minutes for the maximal response, was strictly
Mg2+-dependent, and could be mimicked by
neither hydrolyzeable (ADP, GTP, UTP, CTP, ITP) nor non-hydrolyzeable
(AMP-PNP,2 AMP-PCP) ATP
analogs. The ATP-dependent stimulation of hIK1 could be
reversed by both alkaline and acid phosphatases, suggesting that ATP
activates hIK1 via a membrane-delimited kinase. In patches both from
T84 cells, which natively express hIK1, (1, 2, 4) and from the
Xenopus oocyte heterologous expression system, inhibitors of cAMP-dependent protein kinase could
partially reverse that ATP-dependent activation. However,
mutation of the only cAMP-dependent protein kinase
consensus phosphorylation site, serine 334, as well as of all four
protein kinase C consensus phosphorylation sites (Thr101,
Ser178, Thr329, Ser388) resulted in
channels that remained sensitive to phosphorylation. These results
suggested that hIK1 was itself not the target for phosphorylation.
In this article we demonstrate that unlike hIK1, the SKCa
channels, which share both sequence and functional homology to hIK1, fail to respond to ATP in excised, inside-out patches. Therefore, we
used a chimeric hIK1/rSK2 strategy to define an amino acid region,
arginine 355 through alanine 413, within the C terminus of hIK1 that
confers complete ATP dependence. In addition, we demonstrate that the
first 14 amino acids of this region, residues Arg355-Met368, which have been shown to
interact with calmodulin in a Ca2+-dependent
manner, are critical to the ATP-dependent modulation of hIK1.
Xenopus laevis Oocyte Preparation
X. laevis care and handling procedures were in
accordance with University of Pittsburgh guidelines. X. laevis were obtained from either Xenopus 1 (Dexter, MI)
or Nasco (Fort Atkinson, WI). Frogs were anesthetized with
3-aminobenzoic acid ethyl ester, ovaries were surgically removed, and
oocytes were dissected in modified Barth's solution containing the
following: 88 mM NaCl, 2.4 mM
NaHCO3, 1 mM KCl, 0.82 mM
MgSO4, 0.33 mM Ca(NO3), 0.41 mM CaCl2, 10 mM HEPES, and 1%
penicillin-streptomycin. The oocyte follicular cells were removed by
incubation in 5 mg/ml collagenase (Life Technologies, Inc.) plus
0.5 mg/ml trypsin inhibitor in Ca2+-free ND-96 (in
mM concentrations: 96 NaCl, 1 KCl, 1 MgCl2, 5 HEPES; pH adjusted to 7.5 with NaOH) at room temperature for ~60 min.
The oocytes were then incubated in 100 mM
K2HPO4 (pH adjusted to 6.5 with HCl) containing
1 mg/ml bovine serum albumin for 30 min to remove any remaining
follicular cells. Stage 5 and 6 oocytes were pre-sorted and allowed to
incubate overnight in modified Barth's solution at 20 °C prior to
injection of cRNA.
Molecular Biology
In Vitro Transcription--
All cDNAs were subcloned into
the oocyte expression vector pBF containing both 5' and 3' untranslated
regions of the Xenopus Site-directed Mutagenesis and Chimera
Generation--
Site-directed mutants were generated using the
QuikChange site-directed mutagenesis kit (Stratagene) according to the
manufacturer's instructions. Chimeras between hIK1 and rSK2 were
generated by overlap extension polymerase chain reaction using either
Taq (Stratagene) or Pfx (Life Technologies, Inc.)
polymerase. The chimeras were subcloned into the pBF vector using the
EcoRI and XhoI restriction sites. The fidelity of
all mutations and chimeras was confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence
alignment (NCBI Blast 2.0) with hIK1 (GenBankTM
accession number AF022150) and/or rSK2 (GenBankTM accession
number U69882).
To illustrate the chimera nomenclature utilized throughout the
manuscript, we will use the SK-355IK construct as an example. In
SK-355IK the N-terminal portion of the construct is derived from amino
acids Met1-Val466 of rSK2. Based on published
sequence alignments (15), amino acid Val466 of rSK2
corresponds to residue Val354 of hIK1. A chimeric junction
is therefore introduced between amino acid residues Val466
of rSK2 and the next residue of hIK1, Arg355. Thus, in the
SK-355IK construct, amino acids Met1-Val466
are derived from rSK2, at which point the hIK1 residues
Arg355-Lys427 are substituted.
Electrophysiology
The oocyte vitelline membrane was mechanically dissected prior
to patch clamping in a hypertonic solution containing the following (in
mM concentrations): 200 K-gluconate, 20 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES (pH adjusted to 7.4 with
NaOH). Single-channel currents were recorded in the inside-out patch
configuration using an Axon 200B amplifier (Axon Instruments) and
stored on videotape for later analysis. Pipettes were fabricated from
#0010 glass (World Precision Instruments) and heat-polished to
resistances of 3-6 megohms. The pipette solution contained the
following (in mM concentrations): 145 K-gluconate, 5 KCl,
2.5 MgCl2, 10 HEPES, and 1 EGTA (pH adjusted to 7.4 with
KOH). The bath contained the following (in mM
concentrations): 145 K-gluconate, 5 KCl, 2.5 MgCl2, 10 HEPES, and 1 EGTA (pH adjusted to 7.2 with KOH). Sufficient
CaCl2 was added to obtain the desired free
[Ca2+] (program kindly provided by Dr. Dave Dawson,
University of Michigan). For experiments with no added
Ca2+, Ca2+ was excluded from the bath, and EGTA
was maintained at 1 mM (estimated free Ca2+ < 10 nM). Every chimera tested was strictly
Ca2+-dependent, because Ca2+-free
buffer eliminated all channel activity. Constructs that were not
activated by ATP (Roche Molecular Biochemicals) were always tested in
parallel with a chimera known to be modulated by ATP as a positive
control. In addition, constructs not expressing current were tested
minimally on three separate oocyte injections in parallel with
constructs that did express current. All recordings were
maintained at a holding potential of Statistics
All data are presented as means ± S.E., where n
indicates the number of experiments. Statistical analysis was performed
using a Student's t test. A value of p < 0.05 is considered statistically significant and is reported.
ATP-dependent Activation Is Selective for hIK1--
We
previously demonstrated that ATP activates hIK1 via a kinase-mediated
process (1). Because hIK1 shares ~40% sequence homology with the
SKCa family of K+ channels (15, 16), we
determined whether the SKCa channels (rSK2, hSK1, and rSK3)
could similarly be modulated by ATP in excised, inside-out patches from
Xenopus oocytes. As shown for representative experiments in
Fig. 1, following the generation of
stable current, addition of ATP (1 mM) failed to activate
rSK2 (B), hSK1 (C), and rSK3 (D).
However, subsequent addition of the known hIK1 and rSK2 opener, 1-EBIO
(300 µM) (3, 17, 18), induced a significant increase in
current. In contrast to our results with the SKCa channels,
the activity of hIK1 increased in response to ATP addition (Fig.
1A), as previously reported (1). The average current
upon patch excision for the various constructs was as follows: rSK2,
95 ± 16 pA (n = 10); rSK3, 453 ± 132 pA
(n = 6); hSK1, 117 ± 56 pA (n = 6). This current then decreased an average of 42.1 ± 6.0%
(n = 22) to a steady-state level averaging as
follows: rSK2, 35 ± 12 pA; rSK3, 186 ± 53 pA; hSK1,
90 ± 39 pA. Subsequent addition of ATP (1 mM) failed
to activate the SKCa channels (rSK2, 36 ± 12 pA;
rSK3, 171 ± 47 pA; hSK1, 56 ± 15 pA), whereas addition of
1-EBIO (300 µM) resulted in a significant activation
(rSK2, 176 ± 40 pA; rSK3, 1311 ± 298 pA; hSK1, 352 ± 111 pA). In contrast to these results on the SKCa channels,
following patch excision, current due to hIK1 activity decreased from
143 ± 47 to 43 ± 4 pA (n = 6), with the
subsequent addition of ATP increasing current 3.0 ± 0.3-fold to
132 ± 21 pA. These data demonstrate that despite 40% sequence
homology and relatedness in the mechanism of
Ca2+-dependent gating among the
SKCa channels and hIK1, ATP-dependent modulation is specific for hIK1.
The Cytoplasmic C-terminal Tail of hIK1 Confers
ATP-dependent Activation--
Based on the observation
that ATP activates hIK1 but has no effect on the SKCa
family of channels, we used a chimeric strategy between hIK1 and rSK2
to define a motif within hIK1 that confers ATP-dependent
activation. Initially, we generated the chimera IK199-SK (depicted
schematically in Fig. 2A) in
which the initiation codon through amino acid Tyr199 (start
of the putative fifth transmembrane helix) was derived from hIK1, and
the remainder of the construct was derived from rSK2 (amino acids
Met307-Ser580). As shown for one experiment in
Fig. 2A, addition of ATP (1 mM) to an excised,
inside-out patch failed to modulate IK199-SK current flow, whereas the
subsequent addition of 1-EBIO (300 µM) increased current.
In 11 experiments, the base-line current averaged 62 ± 20 pA,
unaffected by ATP (57 ± 17 pA), whereas the subsequent addition
of 1-EBIO (300 µM) resulted in an increase in current to
126 ± 37 pA (p < 0.01). The failure of ATP to
activate the IK199-SK channel suggests that the C-terminal tail of hIK1
is critical for conferring ATP-dependent activation.
To clarify the role of the C-terminal tail in the
ATP-dependent activation of hIK1 we generated the chimeric
channel SK-299IK, in which only amino acids
His299-Lys427 were derived from hIK1 (see Fig.
2B for schematic). ATP robustly activated this chimera, as
shown for a representative experiment in Fig. 2B. On
average, after current rundown, perfusion of ATP stimulated a 2.9 ± 0.5-fold increase in the activity of this chimera (p < 0.05; n = 6) from a steady-state current of 121 ± 55 pA. The time course for this activation was similar to that
observed for wild type hIK1. The fold increase in current of this
chimera in response to ATP did not differ significantly from the
3.0-fold potentiation observed for wild type hIK1 (p = 0.88), suggesting that the region that defines the ATP dependence of
hIK1 is fully contained within the C-terminal cytoplasmic tail.
The ATP-dependent Motif of hIK1 Resides Entirely within
Amino Acids Arg355-Ala413 of the C-terminal
Cytoplasmic Tail--
In an attempt to more narrowly define the domain
of hIK1 responsible for the phosphorylation-dependent
gating observed, we employed a truncation strategy. Unfortunately,
truncation of only 26 amino acids of hIK1
Lys402STOP) resulted in channels that failed to
express current (n = 0 of 19). Only when we truncated
14 amino acids (Leu414STOP) were functional channels
observed. As shown for one experiment in Fig.
3A, L414Stop was robustly
activated by ATP. In five experiments, L414Stop current increased
3.7 ± 0.3-fold upon perfusion of ATP from a steady-state level of
54 ± 16 pA (p < 0.05). These data demonstrate
that hIK1 requires the majority of the distal C-terminal tail for
functional expression, and amino acids
Leu414-Lys427 are not required for
ATP-dependent activation.
Because truncations of hIK1 failed to express functional channels, we
generated three additional chimeras, SK-321IK, SK-342IK, and SK-355IK,
in which progressively smaller portions of the C terminus of hIK1 were
appended onto rSK2. Representative experiments, demonstrating the
ATP-dependent activation of SK-321IK, SK-342IK, and
SK-355IK, are shown in Fig. 3, B, C, and
D, respectively. Each of these chimeras was activated by ATP
(1 mM), averaging 2.7 ± 0.6-fold from a steady-state
activity of 166 ± 76 pA (n = 6; SK-321IK),
2.8 ± 0.5-fold from a basal activity of 53 ± 24 pA
(n = 3; SK-342IK), and 2.5 ± 0.5-fold from a
steady-state current level of 108 ± 61 pA (n = 9;
SK-355IK). The ATP-induced fold increase in current for these three
chimeras did not significantly differ from the fold increase observed
for hIK1 (p = 0.65, 0.77, and 0.29, respectively).
These data indicate that the 59-amino acid region,
Arg355-Ala413 of hIK1 wholly defines the
domain responsible for conferring ATP dependence. The amino acid
sequence of this region is shown in Fig.
4. In contrast to these results, the
complimentary chimera to SK-355IK, i.e. IK354-SK, in which
the distal C-terminal tail of rSK2 (amino acids
Lys467-Ser580) was appended after
Val354 of hIK1, failed to express functional
channels and could not be evaluated. Similar to our truncations, this
result suggests that the distal C terminus of hIK1 is essential to the
functional expression of these channels.
The ATP-dependent Regulation of hIK1 Does Not Depend
upon Direct Phosphorylation of hIK1--
The amino acid sequence
between residues Arg355 and Ala413 of hIK1
lacks consensus phosphorylation residues; however, the domain does
contain 10 non-consensus serines and threonines. Only two of these
residues are conserved between hIK1 and rSK2. Because kinases have been
shown to phosphorylate ion channels at non-consensus sites, we mutated
these non-conserved residues to alanines (shown in bold in
Fig. 4). ATP (1 mM) activated the mutant channel
S367A/S372A, increasing current from an average steady state of 32 ± 10 to 126 ± 13 pA (n = 4, p < 0.01). ATP also activated the triple mutation S386A/S387A/S388A from a
basal activity of 56 ± 17 to 179 ± 47 pA (n = 5, p < 0.05). Finally, introducing the
T407A/S411A/T412A mutations into hIK1 had no effect on the ability of
ATP to activate the channel, because ATP increased current from 44 ± 6 to 137 ± 35 pA (n = 7, p < 0.05). These data demonstrate that ATP-dependent modulation
of hIK1 is independent of non-conserved serines and threonines within
amino acids Arg355-Ala413 of hIK1.
A 14-Amino Acid Region, Arg355-Met368, of
hIK1 Confers Partial ATP Dependence to rSK2--
To further narrow the
domain of hIK1 responsible for ATP-dependent regulation, we
generated additional rSK2/hIK1 chimeras in which smaller segments of
the distal C terminus of hIK1 were appended onto rSK2. These additional
chimeras, SK-383IK and SK-369IK, overlap with the region of hIK1 that
is critical for the Ca2+-dependent interaction
of calmodulin (19) and rSK2 (20, 21). It is potentially for this reason
that these constructs did not express functional channels as
well as the chimeras outlined above. To increase our current signal, we
studied these chimeras at 10 µM Ca2+, because
we have previously shown that ATP robustly activates hIK1 even at this
saturating level of Ca2+ (1). In 10 experiments, SK-383IK
failed to respond to ATP, having an average current of 59 ± 11 pA, which continued to run down to 47 ± 9 pA in the presence of
ATP. As a positive control, 1-EBIO (300 µM) increased the
current to 121 ± 18 pA (p < 0.001). Similar to
SK-383IK, SK-369IK failed to respond to ATP (1 mM), although 1-EBIO (300 µM) produced a significant increase
in current (Fig. 5A). In seven
experiments, the current averaged 113 ± 28 pA, which was not
affected by ATP (108 ± 29 pA), whereas 1-EBIO increased current
to 172 ± 44 pA (p < 0.05). These data suggest that the 14 amino acids of hIK1,
Arg355-Met368, are critically important for
the ATP-dependent regulation of the channel.
To further define the role of this 14-amino acid domain in the
ATP-dependent activation of hIK1, we generated a chimera in which we swapped amino acids Arg355-Met368 of
hIK1 into the homologous region of rSK2 (amino acids
Lys468-Leu481), SK+14IK. This 14-amino acid
region is shown boxed in Fig. 4. As shown for one experiment in Fig.
5B, substituting these 14 amino acids from hIK1 into rSK2
conferred ATP sensitivity to rSK2. In 10 experiments the steady-state
current in 10 µM Ca2+ averaged 105 ± 48 pA, and this increased 1.6 ± 0.1-fold in response to ATP
(p < 0.05). This activation was ~50% of that seen
for hIK1 in response to ATP (3.0-fold). These results demonstrate that, within amino acids Arg355-Ala413 of hIK1, the
14-amino acid region Arg355-Met368 is critical
to conferring ATP-dependent activation.
We also generated chimeras in which we substituted similar small
regions of rSK2 into the distal C terminus of hIK1 in an attempt to
demonstrate loss of ATP-dependent activation in hIK1. Unfortunately, similar to what we observed both with hIK1 truncations and the IK354-SK chimera, these constructs failed to express functional channels.
The ATP-dependent Activation of hIK1 Is
Ca2+-independent--
Previously, we demonstrated that
phosphorylation potentiates hIK1 activity only in the presence of
elevated Ca2+ (i.e. greater than 100 nM). However, these studies did not address the question of
whether the ATP-dependent activation (phosphorylation) of
hIK1 requires Ca2+ to proceed. To test this hypothesis we
employed the following protocol, for which a representative experiment
is shown in Fig. 6. Upon patch excision
into a bath containing 10 µM free Ca2+, hIK1
current decreased to a steady-state level of activity (68 pA).
Subsequently, Ca2+-free buffer was perfused for 1 min to
guarantee complete washout of Ca2+. Note that current
activity decreased to 0 pA, demonstrating that free Ca2+
had decreased to below the threshold for maintaining channel activity
(<100 nM). At this point, Ca2+-free buffer
containing ATP (1 mM) was perfused for 3 min. This amount
of time is sufficient to obtain near maximal activation in the presence
of elevated Ca2+ (see Figs. 1-3 and 5). Note that this
failed to increase current flow, demonstrating that ATP cannot activate
hIK1 in the absence of Ca2+. Following perfusion with ATP,
Ca2+-free buffer without ATP was again perfused for 1 min.
During this phase, 10 units/ml apyrase (tri- and diphosphatase, Sigma) was transiently added to the perfusate to ensure complete depletion of
ATP from the bath. Finally, the initial solution containing 10 µM free Ca2+ was perfused again, resulting in
a large activation of hIK1 to 215 pA. The result of this one experiment
is consistent with an ATP-dependent phosphorylation event
occurring in the absence of Ca2+, because the current flow
observed in 10 µM Ca2+ subsequent to ATP was
3.16-fold greater than the initial steady-state level. In eight
experiments, the initial steady-state current (prior to ATP) averaged
56.8 ± 9.9 pA, and this was completely inhibited upon removal of
Ca2+. Re-addition of 10 µM Ca2+,
following exposure to ATP in Ca2+-free buffer, increased
current 3.0 ± 0.3-fold above the initial steady-state value to
171.0 ± 23.6 pA. This fold increase in activity is not
significantly different from that observed when hIK1 was stimulated by
ATP in the continued presence of Ca2+ (p = 0.96). These data demonstrate that ATP-dependent
phosphorylation proceeds in the absence of Ca2+.
We previously demonstrated that ATP activates hIK1 in a
protein kinase A inhibitor 5-24-dependent manner in
excised, inside-out patches. These results suggested a role for
phosphorylation in the regulation of this KCa channel.
However, mutation of the single consensus cAMP-dependent
protein kinase phosphorylation site (S334A) failed to modify the
response of hIK1 to ATP (1). In the present study, we employed
hIK1/rSK2 chimeras and hIK1 truncations to identify amino acids
Arg355-Ala413 as necessary and sufficient for
the ATP-dependent regulation of hIK1. The
Arg355-Ala413 domain is devoid of consensus
phosphorylation sites, and mutation of all serines and threonines
within this region that are present in hIK1 and not rSK2 resulted in
channels that remained sensitive to ATP. The first 14 amino acids of
this region, Arg355-Met368, play a critical
role in the ATP-dependent modulation of hIK1, because
substitution of these amino acids into rSK2 produced an ATP-dependent increase in current that was ~50% of the
full hIK1 response.
Several mechanisms could account for the necessity of the
Arg355-Ala413 domain in the
ATP-dependent regulation of hIK1. One explanation is that
ATP directly binds this region of the channel. This interaction could
then induce a conformational change in the channel promoting phosphorylation of an hIK1 residue outside of the
Arg355-Ala413 region. This mechanism cannot be
dismissed, but we believe it to be unlikely, because
Arg355-Ala413 lacks consensus ATP binding
sites. As previously reported, hIK1 can be activated by ATP when all
consensus phosphorylation sites are individually mutated (1). Thus,
this mechanism would also require phosphorylation at a novel
non-consensus site or at multiple residues. Alternatively, the
stimulatory effect of ATP could depend upon phosphorylation of multiple
non-consensus residues within the
Arg355-Ala413 region. We believe this
mechanism to be unlikely, because substitution of the smaller
Arg355-Met368 region of hIK1 into rSK2 confers
partial ATP sensitivity. This region does contain a single serine
(Ser367), although mutation of this residue in hIK1 to
alanine does not modulate the sensitivity of the channel to ATP.
Rather, we speculate that the phosphorylation-dependent
regulation of hIK1 is mediated via an alternative ( Interestingly, truncations involving only 25 amino acids of the hIK1 C
terminus failed to express functional channels. In contrast, it has
been reported that C-terminal truncations in excess of 100 amino acids
of rSK2 have no overt functional consequences (20). In our present
study, chimeric swaps in which portions of rSK2 were appended onto, or
swapped into, hIK1 resulted in channels that failed to express
functional channels, whereas chimeras in which portions of hIK1
were either swapped into, or appended onto, rSK2 had no affect on
channel expression. Together, these data suggest that unlike rSK2, the
region that confers phosphorylation dependence upon hIK1 is required
for the functional expression of the channel. As first recognized by
Joiner et al. (16), this region of both hIK1 and rSK2
contains a potential leucine zipper motif (leucines are
italicized and underlined in Fig. 4). Because leucine zippers are known to stabilize a variety of protein-protein interactions (30), it will be important to evaluate the role of this
leucine zipper in the ATP-dependent modulation and
functional expression of hIK1.
We previously demonstrated that phosphorylation does not increase hIK1
activity in the absence of Ca2+ (1). Here we demonstrate
that ATP-dependent phosphorylation occurs in the absence of
Ca2+, although this phosphorylation event is insufficient
to activate the channel until Ca2+ is increased above the
threshold required for channel gating (Fig. 6). These data argue
against a role for Ca2+-dependent kinases, such
as calmodulin-dependent kinase, in the ATP-dependent activation of hIK1. These results are
consistent with our previous report demonstrating that the peptide
inhibitor of Ca2+/calmodulin kinase II,
Ca2+/calmodulin kinase II inhibitor 281-309, failed to alter
the ATP-dependent activation of hIK1. Although the region
we define as critical for phosphorylation-dependent
activation overlaps with the region of hIK1 that binds calmodulin in a
Ca2+-dependent manner, our results demonstrate
that hIK1 need not be in either a
Ca2+/calmodulin-dependent or a conductive state
for phosphorylation to occur.
In summary, using a series of truncations and hIK1/rSK2 chimeras, we
have defined amino acids Arg355-Ala413 as
necessary and sufficient for the ATP-dependent modulation of hIK1. The first 14 amino acids of this region,
Arg355-Met368, are critical because they
impart partial ATP dependence to rSK2. Mutation of all serines and
threonines within the Arg355-Ala413 domain
that are not conserved in rSK2 results in channels that remain
sensitive to ATP-dependent phosphorylation. Thus, the
ATP-dependent activation of hIK1 may be independent of
direct phosphorylation of the hIK1 channel itself.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin gene flanking the
multi-cloning site. The plasmid was linearized using either
PvuI or MluI (Roche Molecular Biochemicals), and
5' capped cRNAs were generated using SP6 polymerase (mMESSAGE mMACHINETM in vitro transcription kit, Ambion). cRNAs were
evaluated both spectrophotometrically and by agarose gel
electrophoresis with ethidium bromide staining. Oocytes were injected
with 10-50 ng of cRNA 2-4 days prior to recording.
100 mV. The voltage was
referenced to the extracellular compartment, as is the standard method
for membrane potentials. Recordings were acquired onto computer using
Pclamp software (version 6.0.2, Axon Instruments) with a low pass
filter frequency of 400 Hz and a sample frequency of 1 KHz. Digitized
recordings were analyzed using Biopatch software (version 3.3, Bio-Logic). Diary plots were constructed by averaging current in pA
over 15-30-s intervals of the experimental record.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The effect of ATP (1 mM) on hIK1
and the homologous SKCa channels. Top,
schematics of hIK1 and rSK2. Representative diary plots are shown for
hIK1 (A), rSK2 (B), hSK1 (C), and rSK3
(D) in response to ATP (1 mM). Each construct
was expressed heterologously in Xenopus oocytes and recorded
in the excised, inside-out patch configuration at a holding potential
of 100 mV in symmetric K+. For constructs not responding
to ATP (rSK2, hSK1, rSK3), the known hIK1 and SK2 opener 1-EBIO (300 µM) was added as a positive control. Each diary plot is
sampled at 30-s intervals.
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Fig. 2.
The C-terminal cytoplasmic tail of hIK1
confers ATP dependence. Schematics of both the IK199-SK
(A) and the SK-299IK chimeras (B) in which
filled circles represent amino acids derived from rSK2, and
open circles represent amino acids from hIK1. Single letter
amino acid nomenclature and arrows denote the hIK1 amino
acid that defines the chimeric junction. Also shown are representative
diary plots from chimeras IK199-SK (A) and SK-299IK
(B) in response to ATP (1 mM); 1-EBIO (300 µM) is used as a positive control for the IK199-SK
chimera that failed to respond to ATP. Chimeras were heterologously
expressed in Xenopus oocytes and recorded in the excised,
inside-out patch configuration at a holding potential of 100 mV in
symmetric K+. The records were sampled at 30-s
intervals.
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Fig. 3.
The C-terminal motif,
Arg355-Ala413, is necessary and sufficient for
the ATP-dependent activation of hIK1. Schematics for
the Leu414STOP (A), SK-321IK
(B), SK-342IK (C), and SK-355IK (D)
constructs. Filled circles represent amino acid residues
derived from rSK2, open circles represent amino acid
residues from hIK1, and a double line break represents the
point of channel truncation. Single letter amino acid nomenclature and
arrows denote the hIK1 amino acids that define the chimeric
junction/truncation. Representative diary plots for each chimera in
response to ATP (1 mM) are shown to the right of each
schematic (A-D). Excised, inside-out patches from
Xenopus oocytes expressing each construct were recorded at a
holding potential of 100 mV in symmetric K+. Each record
was sampled at 30-s intervals.
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Fig. 4.
Amino acid sequence for
Arg355-Ala413. The amino acid sequence is
shown in single-letter code. The boxed region represents the
14-amino acid region, Arg355-Met368, that is
critical for the phosphorylation-dependent activation of
hIK1. The residues in bold type indicate serines and
threonines that were mutated to alanines. The five leucines of the
potential leucine zipper motif are italicized and
underlined.
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Fig. 5.
Amino acids
Arg355-Met368 of hIK1 confer partial ATP
dependence. Schematics of the SK-369IK (A) and the
SK+14IK (B) chimeras are shown. The filled
circles represent amino acid residues derived from rSK2, whereas
the open circles depict residues from hIK1. Single-letter
amino acid nomenclature and arrows denote the hIK1 amino
acids that define the chimeric junctions. A, as shown in the
representative diary plot, SK-369IK failed to respond to ATP (1 mM); 1-EBIO (300 µM) was added as a positive
control. B, a representative diary plot from the SK+14IK
chimera showing activation in response to ATP (1 mM).
Excised, inside-out patches from Xenopus oocytes were
recorded at a holding potential of 100 mV in symmetric
K+. Records were sampled at 30-s intervals.
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Fig. 6.
hIK1 can be phosphorylated in the absence of
Ca2+. Following patch excision into 10 µM free Ca2+, current due to hIK1 declined to
a steady state. Perfusion of Ca2+-free buffer (1 mM EGTA) resulted in the complete loss of channel activity.
Next, Ca2+-free buffer with 1 mM added ATP was
perfused onto the patch for 3 min. Subsequently, Ca2+-free
buffer was perfused for 1 min. During this period, addition of 10 units/ml apyrase was included to ensure complete removal of ATP.
Finally, the initial buffer (10 µM Ca2+) was
re-perfused, and the instantaneous current response was compared with
control, steady-state channel activity. Excised, inside-out patches
were recorded from Xenopus oocytes at a holding potential of
100 mV in symmetric K+. The diary plot was sampled at
15-s intervals
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) subunit that interacts with amino acids Arg355-Ala413. The
role of
-subunits in the kinase-dependent regulation of ion channels is well established, because these accessory molecules confer cell type-specific regulation. For example, the sensitivity of
the hslo KCa channel to cAMP-dependent
protein kinase is dependent upon co-expression with hKCNMB1. In the
absence of the subunit, hslo activity is stimulated by
cAMP-dependent protein kinase, whereas in the presence of
hKCNMB1, hslo activity is inhibited (24). The literature suggests that
a similar paradigm may be true for hIK1. We previously demonstrated a
role for cAMP-dependent protein kinase-mediated
phosphorylation of hIK1 in both endogenous (T84 cells) and heterologous
(Xenopus oocytes) expression systems. However, when hIK1 was
expressed in HEK293 cells, the channel was modulated via
cAMP-dependent protein kinase-independent phosphorylation (1). Khanna et al. (9) also reported cell-specific
phosphorylation-dependent modulation of hIK1. These authors
showed that, when natively expressed in T lymphocytes, hIK1
activity was modulated via calmodulin-dependent kinases. In
contrast, when hIK1 was expressed heterologously in Chinese hamster
ovary cells, calmodulin-dependent kinase inhibitors had
no effect on channel activity. Although our results are suggestive of
the involvement of a
-subunit in the ATP-dependent
regulation of hIK1, this would require that Xenopus oocytes
endogenously express this
-subunit. Currently, we have no direct
evidence that Xenopus oocytes express this proposed subunit.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK54941-02 (to D. C. D.).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.
§ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, S312 Biomedical Science Tower, University of Pittsburgh, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8755; Fax: 412-648-8330; E-mail: dd2+@pitt.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M007716200
1 A. C. Gerlach and D. C. Devor, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
AMP-PNP, adenosine
5'-(,
-imino)triphosphate;
AMP-PCP, adenosine
5'-(
,
-methylenetriphosphate).
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