From the Departments of Cell Physiology & Pharmacology and ¶ Biochemistry, University of Leicester,
P. O. Box 138, Leicester LE1 9HN, United Kingdom
Received for publication, February 14, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Protein kinase A (PKA) is targeted to
discrete subcellular locations close to its intended substrates through
interaction with A kinase-anchoring
proteins (AKAPs). Ion channels represent a diverse
and important group of kinase substrates, and it has been shown that
membrane targeting of PKA through association with AKAPs facilitates
PKA-mediated phosphorylation and regulation of several classes of ion
channel. Here, we investigate the effect of AKAP79, a
membrane-associated multivalent-anchoring protein, upon the function
and modulation of the strong inwardly rectifying potassium channel,
Kir2.1. Functionally, the presence of AKAP79 enhanced the response of
Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a
pool of PKA anchored close to the channel. Antibodies directed against
a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79,
indicating that the two proteins exist together in a complex within
intact cells. In support of this, glutathione S-transferase
fusion proteins of both the intracellular N and C domains of Kir2.1
isolated AKAP79 from cell lysates, while glutathione
S-transferase alone failed to interact with AKAP79.
Together, these findings suggest that AKAP79 associates directly with
the Kir2.1 ion channel and may serve to anchor kinase enzymes in close
proximity to key channel phosphorylation sites.
The phosphorylation state of ion channels is governed by the
activation of protein kinases and phosphatases, which in turn respond
to fluctuations in the local concentration of second messengers such as
calcium and cyclic AMP. It has been shown recently that much of the
specificity seen in intracellular phosphorylation events comes from the
targeting of protein kinases and phosphatases to specific subcellular
structures and organelles through association with anchoring or adaptor
proteins (1, 2). Cyclic AMP-dependent protein kinase
(protein kinase A or PKA),1
for example, is targeted to discrete subcellular locations through interaction with A kinase-anchoring
proteins or AKAPs (3, 4). AKAPs form a family of more than
thirty different functionally related proteins. Each anchoring protein
contains at least two functional motifs: an amphipathic helix that
binds the regulatory (RII) subunits of the PKA holoenzyme (5, 6) and a
specialized anchoring domain that tethers the AKAP·PKA complex to
specific intracellular sites close to their intended substrate (7). The
modulation of ion channel activity through protein phosphorylation is
an important physiological control mechanism, and ion channels represent a diverse group of kinase substrates. It has been shown that
membrane targeting of PKA through association with AKAPs facilitates
PKA-mediated phosphorylation and regulation of AMPA-kainate and NMDA
glutamate ion channels, skeletal and cardiac calcium channels, and
calcium-activated potassium channels (8, 9, 10, 11). Interestingly,
AKAP15/18, the anchoring protein that targets PKA to L-type calcium
channels does so not only by membrane association through a lipid
anchor but also by a possible direct interaction with Ca2+
channel Inwardly rectifying potassium (Kir) channels are another group of ion
channels whose modulation is influenced by the presence of AKAPs (14).
Intrinsic gating and block of the Kir channel pore by intracellular
magnesium and polyamines ensures that Kir channels permit
K+ entry under hyperpolarization more readily that they
permit K+ exit under depolarization (15, 16). This
asymmetry in the current-voltage relation of the channel allows
modification of the electrical properties of cells without excessive
K+ loss. Consequently, Kir channels are involved in setting
and maintaining the resting membrane potential, buffering extracellular K+, and the generation of prolonged action potentials in
the heart and in fertilized egg cells (17). Kir channels are also
regulated by protein phosphorylation and direct G-protein activation
and thus play a role in the fine-tuning of cellular excitability (18, 19, 20). Cloning of members of the Kir family and examination of their
primary sequences suggest the presence of multiple consensus phosphorylation sites (21, 22, 23), although experimental data for the
involvement of kinase enzymes in the regulation of cloned Kir channel
activity are contradictory, with evidence in favor as well as against a
role for protein kinase A and/or protein kinase C (24, 25, 26).
Here, we present evidence that suggests that AKAP79 (recently renamed
AKAP5),2 a
multivalent-anchoring protein that binds PKA, PKC, and the protein
phosphatase calcineurin (PP2B) (27, 28), is not only required for the
reliable modulation of the strong inward rectifying K+
channel, Kir2.1, but further that AKAP79 is targeted to the
intracellular N and C domains of Kir2.1 to anchor kinases close to key
channel phosphorylation sites.
Antibodies, Polyacrylamide Gel Electrophoresis, and
Immunoblotting--
The following primary antibodies were used: rabbit
polyclonal HA-probe (Y-11; Santa Cruz Biotechnologies, Inc.); mouse
monoclonal anti-AKAP79 (clone 22; BD Transduction Laboratories).
Horseradish peroxidase-, fluorescein (FITC)- and Texas Red-conjugated
anti-rabbit and anti-mouse secondary antibodies were from Jackson
ImmunoResearch Laboratories, Inc. Protein extracts were resolved by
SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide-Tris gels
and transferred electrophoretically onto nitrocellulose membranes
(Hybond ECL, Amersham Pharmacia Biotech). Membranes were blocked
overnight at 4 °C in blocking solution containing 5% (w/v) skim
milk powder and 0.1% Tween 20 in Tris-buffered saline. Primary
antibodies were diluted in blocking solution containing 1% skim milk
powder and 0.1% Tween 20 in Tris-buffered saline and incubated with
the membranes for 2-3 h at room temperature. Membranes were washed in
Tris-buffered saline and then incubated with horseradish
peroxidase-conjugated secondary antibodies for a further hour at room
temperature. Labeled bands were visualized using enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech) according to the
manufacturer's protocol.
Cells and Cell Transfection--
Chinese hamster ovary (CHO),
COS-7, and HEK-293 cells were grown in minimal essential medium
(without nucleosides), Dulbecco's modified Eagle's medium, and
minimal essential medium supplemented with 1% non-essential amino
acids, respectively. Medium was supplemented with 10% (v/v) fetal
bovine serum. All medium and reagents were from Life Technologies, Inc.
No antibiotics were used. Cells were transiently transfected using
FuGENE6TM transfection reagent (Roche Molecular Biochemicals)
according to the manufacturer's protocol. Transfections were performed
in six-well culture plates with cells at 50-80% confluence.
Plasmids and DNA Constructs--
The Kir2.1 expression construct
in pcDNA3 has been previously described (29). The AKAP79 expression
construct in pcDNA3 was a generous gift from Dr. John Scott, Vollum
Institute, Portland, OR. The enhanced green fluorescent protein-Kir2.1
fusion (EGFP·Kir2.1) was produced as described previously (30). The
expression vector encoding EGFP (pEGFP-N1) was purchased from
CLONTECH. To confirm the presence of AKAP79 within
cells during electrophysiological recordings, AKAP79 was subcloned as a
SacI-BamHI fragment into the bicistronic
expression vector pIRES2-EGFP (CLONTECH). This permits AKAP79 and EGFP to be translated simultaneously from the same
mRNA transcript and allows visual selection of cells expressing both proteins.
For insertion of the HA epitope into the extracellular M1-H5 loop of
Kir2.1, complimentary oligonucleotides encoding the HA epitope
(YPYDVPDYA) were annealed to form a SphI adapter and ligated into a unique SphI site in Kir2.1 cDNA. The amino acid
sequence of the HA-tagged Kir2.1 reads
117KVSKACYPYDVPDYAACV123 at the site of the
epitope insertion. The HA·Kir2.1 construct was subcloned from the
plasmid vector pBluescript into pcDNA3 as an
EcoRI-XhoI fragment for transfection into COS-7
and HEK-293 cell lines.
Glutathione S-transferase (GST) fusion protein constructs
encoding the N-terminal (1-71) and C-terminal (302) regions were amplified using the polymerase chain reaction with the following sets of primers: N-(1-71), 5'-GGATCCATGGGCAGTGTGAGAACCAACCGCTA-3' and
5'-GAATTCGTCTGCCAGGTACCTCTGTCCCTTCTC-3'; C-(302-428),
5'-GGATCCGAGGCGACTGCCATGACAACTCAATG-3' and
5'-GAATTCTCATATCTCCGATTCTCGCCTTAAGGG-3'.
Products of the reaction were cloned into the pGEM-T vector (Promega),
digested with BamHI and EcoRI, and subcloned into
the pGEX-2T bacterial expression plasmid (Amersham Pharmacia Biotech). The GST fusion protein construct C-(421-428) was constructed by annealing complimentary oligonucleotides encoding the C-terminal eight
amino acids of Kir2.1 to form a BamHI-EcoRI
adapter and ligating this into pGEX-2T.
GST Pull-down Assays--
To identify possible interactions
between Kir2.1 and AKAP79, GST fusion proteins of the intracellular N
(residues 1-71) and C (residues 302-428 and residues 421-428)
domains of Kir2.1 were incubated with lysates of COS-7 cells expressing
AKAP79. The expression plasmid pGEX-2T containing the GST fusion
protein cDNA was transformed into Escherichia coli XL1
Blue competent cells (Stratagene). 15-ml overnight cultures were grown
in Luria Bertani medium containing 100 µg/ml ampicillin (LBA). 200 µl of the overnight culture were then used to inoculate 4 × 12 ml of LBA. Cells were grown by shaking at 37 °C until the
A600 was between 0.5 and 0.8. Expression of the
GST fusion proteins was subsequently induced with 1 mM
isopropyl- Coimmunoprecipitation--
COS-7 cells were plated onto six-well
plates and transiently transfected with cDNA encoding AKAP79 and
HA-tagged Kir2.1. Cell lysates were prepared and cleared as described
for GST pull-down assays. 5 µg of rabbit polyclonal HA-probe (Y-11)
or 5 µg of rabbit non-immune control serum (Santa Cruz
Biotechnologies) was added to 500 µl of cleared lysate and incubated
at 4 °C overnight. 30 µl of prewashed protein A-agarose beads
(Santa Cruz Biotechnologies) were added to the antibody/lysate mix and
incubated with gentle inversion for 90 min at 4 °C. The
protein-coupled agarose beads were then pelleted by gentle
centrifugation and washed five times in PBS. Immunoprecipitated
proteins were recovered in 2× loading buffer, resolved by 10%
SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblotting.
Electrophysiology--
Whole-cell currents were recorded from
either CHO or COS-7 cells typically 24-48 h post-transfection using an
Axopatch 200A amplifier (Axon Instruments). Currents recorded in
response to voltage steps were filtered at 5 kHz ( Immunocytochemistry--
HEK-293 or COS-7 cells were plated onto
polylysine-coated coverslips and transiently transfected with HA-tagged
Kir2.1 and AKAP79. Cells were fixed and permeabilized 48 h
post-transfection in a solution containing 80 mM
Na2HPO4, 20 mM
NaH2PO4, 2% paraformaldehyde, and 0.1% Triton
X-100. Fixed cells were incubated overnight at 4 °C with rabbit
polyclonal anti-HA and mouse monoclonal anti-AKAP79. Both primary
antibodies were diluted 250-fold in PBS with 10% (v/v) goat serum. The
following morning, the coverslips were washed 5× 10 min in PBS, then
incubated in goat anti-rabbit secondary antibody (cross-adsorbed
against mouse) conjugated with Texas Red and goat anti-mouse secondary
antibody (cross-adsorbed against rabbit) conjugated with FITC in PBS + 10% goat serum for 2 h at room temperature. Coverslips were
washed in PBS for 3× 10 min and mounted onto microscope slides using
fluorescent mounting medium (Dako Ltd) before viewing. Confocal images
were obtained using a PerkinElmer UltraViewTM imaging system.
Coexpression of AKAP79 and Kir2.1 Has No Effect upon Channel
Function--
Membrane currents were recorded from CHO or COS-7 cells
using the conventional whole-cell clamp technique typically 24-48 h
after transfection with the cDNAs encoding Kir2.1 and AKAP79, or
with Kir2.1 alone. To confirm the presence of AKAP79 within cells
during electrophysiological recordings, AKAP79 was subcloned into the
bicistronic expression vector pIRES2-EGFP
(CLONTECH). This permits AKAP79 and EGFP to be
translated simultaneously from the same mRNA transcript and allows
visual selection of successfully transfected cells expressing AKAP79.
The presence of Kir2.1 was confirmed by recording whole-cell Kir
currents. For cells expressing Kir2.1 alone, we cotransfected the
cDNA encoding Kir2.1 with the empty (no AKAP79) pIRES2-EGFP
expression vector. Fig. 1A
shows whole-cell currents recorded from a CHO cell transfected with both Kir2.1 and AKAP79. Currents were recorded in response to voltage
steps from a holding potential of AKAP79 Enhances the Response of the Kir2.1 to cAMP but Only in the
Presence of Phosphatase Inhibitors--
Preliminary results indicated
that elevation of intracellular cAMP had little effect upon whole-cell
Kir2.1 currents either in the presence or the absence of AKAP79 (Fig.
2, A and B). In these experiments, whole-cell currents recorded at
In the presence of phosphatase inhibitors and AKAP79, the
increase in whole-cell Kir current in response to elevated
intracellular cAMP was reflected in a 10 mV rightward shift in the
relationship between chord conductance and voltage (Fig.
3). The voltage at which the chord
conductance was half-maximal (V0.5) shifted from V0.5 =
AKAP79 is a multivalent-anchoring protein that also binds protein
kinase C (27, 28). We therefore tested the effect of activators of PKC
on Kir2.1 currents in the presence and absence of AKAP79. The
extracellular application of 1 µM
didecanoyl-rac-glycerol, a cell permeant activator of PKC,
or 1 µM phorbol myristate acetate had no effect upon
Kir2.1 current in either the presence or absence of AKAP79 or
phosphatase inhibitors (not shown).
AKAP79 Coimmunoprecipitates with HA-tagged Kir2.1--
The results
of the functional studies outlined above indicate that AKAP79
facilitates cAMP-induced modulation of Kir2.1, presumably by anchoring
PKA in close proximity to the ion channel. To establish whether any
physical interaction between Kir2.1 and AKAP79 occurs within intact
cells, coimmunoprecipitation experiments were performed. COS-7 cells
were transfected with cDNAs encoding AKAP79 and HA-tagged Kir2.1.
HA-tagged Kir2.1 channels were found to be functionally indistinguishable from untagged wild-type Kir2.1 channels (data not
shown). Antibodies directed against the HA epitope tag on Kir2.1 were
able to immunoprecipitate AKAP79 (Fig.
4), suggesting that Kir2.1 and AKAP79
exist in a complex within cells.
AKAP79 Interacts with the Intracellular N and C Domains of
Kir2.1--
Kir channels have a relatively simple structure compared
with Na+ and Ca2+ channels and members of the
voltage-gated K+ (Kv) channel family. Each subunit in the
Kir tetramer consists of an intracellular N and C terminus and two
membrane-spanning domains (M1 and M2) separated by a short stretch of
amino acids (the H5 or P-region) that dips back into the membrane from
the extracellular side to form the selectivity filter and channel pore
(21, 22). The only intracellular portions of the channel subunit are
found at the N terminus (residues 1-71) before the channel protein
disappears into the membrane to form M1, and at the C terminus when the
protein emerges from M2 (182). It is therefore relatively
straightforward to create GST fusion proteins of the intracellular
portions of the Kir channel and test for direct interaction with AKAP79
in pull-down assays.
To identify possible interactions, GST fusion proteins of the
intracellular N (residues 1-71) and C (302) terminal domains of
Kir2.1 were screened for their ability to bind AKAP79 from lysates of
COS-7 cells transfected with cDNA encoding AKAP79. GST fusion
proteins of both the N- and the C-domains of Kir2.1 were able to
isolate AKAP79 from COS-7 lysates, whereas GST alone failed to interact
with AKAP79 (Fig. 5A).
It has recently been reported that AKAP79 binds to the Src homology 3 (SH3) and guanylate kinase-like (GK) domains of the PDZ protein,
PSD-95/SAP90 (31). The C terminus of Kir2.1 contains the conserved
PDZ-domain recognition motif (X(S/T)X(V/I)) that mediates binding to members of the PSD-95/SAP90 protein family. Whereas
PSD-95 is not present in either COS-7 cells or Escherichia coli, where the GST fusion proteins were originally expressed (see
"Experimental Procedures"), we felt it important to test whether
AKAP79 binds to Kir2.1 via interaction with an unidentified, endogenous
PDZ protein from these cells. For this purpose we constructed a
shortened C-terminal GST fusion protein encoding just the last eight
C-terminal amino acids of Kir2.1 (421). This shortened GST·C fusion protein, GST·C-(421-428), which includes the Kir2.1 PDZ binding motif, failed to isolate AKAP79, suggesting that AKAP79 binds
directly to the intracellular C-domain of Kir2.1 at some point(s)
between residues 302 and 421 and not via a C-terminal PDZ protein (Fig.
5B).
AKAP79 and Kir2.1 Are Both Localized to the Plasma Membrane of
HEK-293 Cells--
Fig. 6 (A
and B) shows confocal images of HEK-293 cells that have been
transfected with HA·Kir2.1 and AKAP79, stained with antibodies
against the HA epitope tag and AKAP79, and visualized with secondary
antibodies conjugated with the non-overlapping fluoroprobes, FITC (Fig.
6A) and Texas Red (Fig. 6B), respectively. The
images show both proteins to be localized to the plasma membrane, demonstrating proximity between Kir2.1 and AKAP79 within intact cells.
Interestingly, we were unable to use EGFP-tagged Kir2.1 channels to
monitor Kir2.1 distribution, as the presence of AKAP79 seemed to quench
the EGFP·Kir2.1 fluorescence. Fig. 6 (C and D) show typical confocal images of a HEK-293 cell transfected with EGFP-tagged Kir2.1 (EGFP attached to the N terminus of Kir2.1) and
AKAP79. The cell has been permeabilized and stained with anti-AKAP79. EGFP fluorescence is almost undetectable (Fig. 6C). AKAP79
distribution within the same cell is shown in Fig. 6D. The
reduction in EGFP fluorescence is not a result of cell permeabilization
because permeabilized HEK-293 cells expressing EGFP·Kir2.1 alone
retain their brightness (Fig. 6E). Nor is the fluorescence
reduction simply because of the coexpression of AKAP79 and EGFP,
because cells transfected with EGFP and AKAP79 were indistinguishable from cells transfected with EGFP alone (data not shown). Rather, the
effect of reduced fluorescence seems to be a side effect of the
association of Kir2.1 and AKAP79. EGFP fluorescence is known to be
sensitive to a number of factors, particularly pH, and it seems
possible that the anchoring of AKAP79 and its associated proteins may
change some factor in the very local environment of the ion channel
that affects the fluorescence of the attached EGFP molecule.
Our findings suggest that the protein kinase A-anchoring protein,
AKAP79, associates directly with intracellular regions of the strong
inwardly rectifying K+ channel, Kir2.1 to anchor PKA close
to channel phosphorylation sites. The primary sequence of Kir2.1
suggests the presence of one putative PKA phosphorylation site at
position Ser-426 on the C terminus and four putative PKC
phosphorylation sites, two on the N terminus (Ser-3, Thr-6) and two on
the C terminus (Ser-357, Thr-383). AKAP79, in common with many adaptor
proteins, binds several different enzymes, in this case PKA, PKC and
the Ca2+ calmodulin-dependent protein
phosphatase-2B, calcineurin (27, 28). Whereas activators of PKC had no
discernable effect upon Kir2.1 currents either in the presence or in
the absence of AKAP79, elevation of intracellular cAMP caused a marked
increase in whole-cell Kir2.1 current in the presence but not in the
absence of AKAP79. Interestingly, the effects of elevated cAMP were
only observed in cells pretreated with the phosphatase inhibitors
okadaic acid and cypermethrin. Phosphatase anchoring via association
with AKAP79 could potentially increase phosphatase activity close to
the channel, although cypermethrin, which specifically inhibits
calcineurin with an IC50 in the subnanomolar range, was
found to be largely ineffective on its own (data not shown).
Alternatively, the reliance on phosphatase inhibitors may simply
reflect a relatively high endogenous phosphatase activity within these
cell systems and suggests that channel phosphorylation and
dephosphorylation may be a fast, dynamic process. There has been a
report of similar findings for cloned L-type Ca2+ channels
expressed in HEK-293 cells where phosphorylation of the The functional effects on channel activity of coexpressing AKAP79 and
Kir2.1 suggest that AKAP79 also facilitates the phosphorylation of
Kir2.1 by anchoring PKA close to the ion channel. The low molecular weight anchoring protein AKAP15/18 has been shown to target PKA to
L-type Ca2+ channels by membrane association through
myristoylation and palmitoylation of residues at its N terminus and,
possibly, by direct interaction with Ca2+ channel subunits
(12, 13). AKAPs contain at least two functional domains: an amphipathic
helix that binds the regulatory (RII) subunits of the PKA holoenzyme
(5, 6) and a specialized targeting region that tethers the AKAP·PKA
complex to specific intracellular sites (7). Early studies suggested
that AKAP79 targeting was mediated through interactions with
cytoskeletal proteins (34, 35), although more recent studies suggest
that AKAP79 is anchored to the plasma membrane through direct binding of basic domains on the protein to acidic phospholipids, including the
phosphoinositide PtdIns(4,5)P2 (36). While the primary
target of AKAP79 is the plasma membrane, there may be a subsidiary
interaction with intracellular regions of channel proteins to create a
channel/AKAP complex that could enhance the speed and specificity of
PKA-induced phosphorylation. Coimmunoprecipitation of AKAP79 with
HA-tagged Kir2.1 certainly suggests that these two proteins exist in a
complex within intact cells. Kir channels have a relatively simple
structure when compared with Na+/Ca2+ channels,
and Kv channels, and this makes it possible to create GST fusion
proteins of the entire intracellular portion of the Kir channel and
test for direct interaction with AKAP79. GST pull-down assays
demonstrate that AKAP79 binds to both the N and C intracellular domains
of Kir2.1. Interaction between AKAPs and their target proteins at
multiple sites is not unprecedented because AKAP79 has recently been
shown to bind to the AKAP79 also binds to the Src homology 3 (SH3) and guanylate kinase-like
(GK) domains of the membrane-associated guanylate kinase (MAGUK)
proteins, PSD-95 and SAP97. This recruits PKA to ionotropic glutamate
receptors because PSD-95 interacts with NR2B subunits of the NMDA
receptor, and SAP97 binds to GluR1 subunits of AMPA receptors (31). The
C terminus of Kir2.1 contains the PDZ-domain recognition motif
(X(S/T)X(V/I)) that mediates binding to the
PSD-95/SAP90 protein family, and Kir2.1 has been shown to interact with
PSD-95 in yeast two-hybrid screens and heterologous expression systems
(38). The C-terminal AKAP binding we detect for Kir2.1 is unlikely to
be via interaction with a PDZ protein because GST·C-(421-428), a
shortened C-terminal fusion protein harboring the PDZ motif, failed to
isolate AKAP79 in pull-down assays. Nevertheless, the ability of AKAP79
to bind to PSD-95 and directly to intracellular regions of Kir2.1 may
offer additional stability to potential protein complexes containing
the ion channel, PDZ protein and AKAP. It is also notable that NMDA
receptors retain the option of recruiting PKA through the simultaneous
association of two separate anchoring proteins: AKAP79, which binds to
the receptor via PSD-95 (31) and yotiao, which binds directly to the
C-terminal C1 exon cassette of the NMDA receptor (9). Because the
native AKAP partner of Kir2.1 has yet to be identified, it is tempting
to speculate that a similar situation may exist for K+ channels.
In conclusion, we present evidence for the direct association of a
protein kinase A-anchoring protein with a K+ channel. This
is potentially important given the unique role of K+
channels in shaping the electrical properties and firing patterns of
cells, and the profound effects that K+ channel modulation
has upon cellular excitability. It seems increasingly likely that
in vivo K+ channels, like Ca2+
channels and ionotropic glutamate receptors, exist in highly organized
complexes made up of AKAPs and associated signaling proteins that
ensure rapid and efficient phosphorylation of the channel protein in
response to localized cell signals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits (12, 13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside and incubation was
continued for a further 4-5 h. Cells were pelleted by centrifugation
and resuspended in 500 µl of phosphate-buffered saline (PBS).
Resuspended cells were lysed by mild sonication with a sonicator probe
for 2 × 5 s on ice. 50 µl of 10% Triton X-100 was then
added to the 500 µl of sonicated suspension and incubated on ice for
5 min. The suspension was centrifuged at 10,000 × g
for 10 min at 4 °C, and the pellets were discarded. The supernatant
was added to 100 µl of washed glutathione-Sepharose beads and
incubated for 30 min at 4 °C with inversion. Extracts from
transfected COS-7 cells were prepared from a six-well plate 24-48 h
post-transfection. Cells were briefly washed in PBS and then 500 µl
of lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, pH 7.6) containing 1%
Triton X-100 and protease inhibitors (1:100 dilution, Sigma protease
inhibitor mixture containing AEBSF, aprotinin, bestatin, leupeptin,
pepstatin A) was added to each well and incubated on ice for 30 min.
The resulting extract was cleared of insoluble debris by centrifugation
at 10,000 × g for 15 min at 4 °C. 100 µl of
Sepharose beads containing the GST fusion proteins were added to 500 µl of the soluble fraction of the lysate and incubated for 1-2 h at
4 °C with gentle inversion. The protein-coupled beads were pelleted
by gentle centrifugation and washed five times in PBS. The beads were
resuspended in 2× loading buffer, and samples were separated by
SDS-polyacrylamide gel electrophoresis with 10% acrylamide gels,
transferred to nitrocellulose membrane, and analyzed by immunoblotting
as described above.
3dB, 8-pole
Bessel), digitized at 10 kHz using a DigiData 1200 interface (Axon
Instruments), and analyzed using software written in AxoBasic (Axon
Instruments) by Dr. N. W. Davies (University of Leicester, UK).
Electrodes were pulled from borosilicate glass (outer diameter 1.5 mm;
Clarke Electromedical, Pangbourne, UK) and fire polished to give a
final resistance of 5 megohm when filled. The pipette-filling solution contained (mM): KCl, 140; MgCl2, 1; EGTA, 10; HEPES, 10;
Na2ATP, 3; pH 7.2. The external solution contained (mM):
KCl, 35; NaCl, 105; MgCl2, 2, CaCl2, 2; HEPES;
10; pH 7.25. 100 µM cAMP (CalBiochem) was added to the
pipette-filling solution in some experiments. The phosphatase
inhibitors, okadaic acid and cypermethrin were purchased from Tocris
Cookson, Inc. Didecanoyl-rac-glycerol, a cell permeant
activator of PKC was purchased from CalBiochem. Phorbol 12-myristate
13-acetate was from Sigma-Aldrich. The junction potential between
pipette and external solutions was sufficiently small (<1.5 mV) to be
neglected. As far as possible, analogue means were used to correct
capacity transients. Up to 90% compensation was routinely used to
correct for series resistance. All experiments were performed at room
temperature (18-22 °C), and the results are expressed as mean ± S.E. Statistical significance was evaluated using the Student's
unpaired t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
35mV (the K+
equilibrium potential, EK) to test potentials ranging from
+55 mV and
105 mV in 10 mV increments. Voltage steps positive to EK elicited only small outward currents, whereas steps
negative to EK produced substantial inward currents. No
significant whole-cell currents were detected in non-transfected cells.
The presence of the kinase-anchoring protein, AKAP79 within the cells
had no significant effect upon whole-cell Kir2.1 current amplitudes
(Fig. 1B) or voltage-dependent properties of Kir
channel currents, such as the relationship between whole-cell
conductance and voltage (Fig. 1C), or the time constant of
Kir2.1 channel gating (Fig. 1D). This suggests that basal
Kir2.1 channel activity is unaffected by coexpression with AKAP79.
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Fig. 1.
Characterization of Kir2.1 currents in the
presence of AKAP79. A, membrane currents recorded from
a single CHO cell expressing AKAP79 and Kir2.1 in response to voltage
steps from a holding potential of 35 mV to test potentials ranging
from +55 to
105 mV in 10 mV increments. Extracellular
[K+] was 35 mM; intracellular
[K+] was 140 mM. Outward currents are defined
as being positive and are shown as upward deflections; inward currents
are defined as being negative. B, mean current-voltage
relation for cells expressing AKAP79 and Kir2.1 (filled
symbols), or Kir2.1 alone (open circles),
n = 6 each. C, relationship between chord
conductance and membrane potential for cells expressing AKAP79 and
Kir2.1 (filled symbols), or Kir2.1 alone (open
symbols). Chord conductance was computed as gK = IK/(V
EK). Its relation to membrane
potential may be fitted by a Boltzmann relation with the relative
conductance g'K = (1 + exp((V
V0.5)/k))
1, where V0.5 (mV) gives
the voltage at which g'K = 0.5 and k (mV) is a
factor affecting the steepness of the relationship. For cells
expressing AKAP79 and Kir2.1, V0.5 =
32.9 ± 0.9 mV;
k = 9.4 ± 1.0 mV (n = 6). For cells
expressing Kir2.1 alone, V0.5 =
33.1 ± 1.1 mV;
k = 10.6 ± 0.6 mV (n = 6). D,
relationship between time constant (t) for activation of
inward current and membrane potential for cells expressing AKAP79 and
Kir2.1 (filled symbols; n = 6) and cells
expressing Kir2.1 alone (open symbols; n = 6). The solid line shows the least-squares fit for cells
expressing AKAP79 and Kir2.1, giving t = 3.9 exp(V/32.3) ms. The best fit to the results for Kir2.1 alone
(dotted line) gave t = 8.1exp(V/24.1)
ms.
80 mV from cells
expressing AKAP79 and Kir2.1 increased by only 13.4 ± 1.9% (mean ± S.E.; n = 6) during a 5-min exposure to
100 µM intracellular cAMP. Cells expressing the channel
alone showed a similar 11.1 ± 2.8% (n = 6)
increase in whole-cell current over the same time interval. It has
previously been reported that cAMP-dependent phosphorylation of L-type Ca2+ channel subunits in HEK-293
can only be detected in the presence of phosphatase inhibitors (10). We
therefore tested whether the phosphatase inhibitors okadaic acid (an
inhibitor of protein phosphatases 1 and 2A) and cypermethrin (a potent
inhibitor of calcineurin, phosphatase 2B) augmented the effect of cAMP
on Kir2.1 currents. Following a 30-40-min pretreatment of the cells in
200 nM okadaic acid and 200 nM cypermethrin,
CHO cells expressing both AKAP79 and Kir2.1 showed a 25.6 ± 3.6%
(n = 5) increase in whole-cell Kir2.1 current over a
5-min period when exposed to 100 µM intracellular cAMP
(Fig. 2, C and D). In contrast, cells transfected
with Kir2.1 alone showed an 8.1 ± 2.5% (n = 5)
increase in Kir2.1 current when exposed to 100 µM cAMP
over the same period.
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Fig. 2.
The presence of AKAP79 enhanced the response
of Kir2.1 to elevated intracellular cAMP but only in the presence of
phosphatase inhibitors. A, relative increase in
whole-cell current recorded at 80 mV ([K]o = 35 mM; [K]i = 140 mM) over time for
cells expressing AKAP79 and Kir2.1 (filled symbols) or
Kir2.1 alone (open symbols). 100 µM cAMP was
included in the pipette-filling solution. Time = 0 represents the point just after the whole-cell recording configuration
has been established. CHO cells expressing both AKAP79 and Kir2.1
showed a 13.4 ± 1.9% (mean ± S.E., n = 6)
increase in whole-cell current over a 5-min period, not significantly
different from the increase in current seen in cells transfected with
Kir2.1 alone, 11.1 ± 2.8% (n = 6) over the same
time period (p = 0.42). B, histogram
summarizing the percentage increase in whole-cell current recorded at
80 mV after a 5-min period with 100 µM cAMP included in
the pipette-filling solution. C, relative increase in
whole-cell currents recorded at
80 mV for cells pretreated for 30-40
min in 200 nM okadaic acid and 200 nM
cypermethrin before recording. 100 µM cAMP, 200 nM okadaic acid, and 200 nM cypermethrin were
included in pipette-filling solution. Cells expressing both AKAP79 and
Kir2.1 show a 25.1 ± 3.9% increase in whole-cell current over a
5-min period (n = 5; filled symbols and
histogram D), significantly greater than the 8.1 ± 2.5% increase seen in cells expressing Kir2.1 alone (open
symbols; n = 5; p < 0.05).
D, histogram summarizing the percentage increase in
whole-cell currents recorded at
80 mV after a 5-min period for cells
pretreated with okadaic acid and cypermethrin.
35.1 ± 1.0 mV in control to V0.5 =
25.0 ± 0.8 mV in cAMP-exposed cells expressing AKAP79 and
Kir2.1 (Fig. 3D). The conductance-voltage relation of cells
expressing Kir2.1 alone shifted from V0.5 =
34.3 ± 1.0 mV in control to V0.5 =
29.0 ± 1.2 mV in
cAMP-exposed cells (Fig. 3B).
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Fig. 3.
cAMP induced a rightward shift in the
conductance-voltage curve for Kir2.1 in the presence of AKAP79 and
phosphatase inhibitors. A, whole-cell currents recorded
in the presence of phosphatase inhibitors from a single CHO cell
expressing Kir2.1 alone in response to a voltage step from the holding
potential of 35 mV to a test potential of
80 mV under control
conditions (immediately following patch break-in) and after 10-min
exposure to 100 µM intracellular cAMP. B, mean
conductance-voltage relationship for 5 cells expressing Kir2.1 alone
under control (filled symbols) and following exposure to 100 µM intracellular cAMP for 10 min (open
symbols). The voltage at which the conductance was half-maximal
shifted from V0.5 =
34.3 ± 1.0 mV in control to
V0.5 =
29.0 ± 1.2 mV in cAMP-exposed cells
(p < 0.05). C, whole-cell currents recorded
in the presence of phosphatase inhibitors from a single CHO cell
expressing AKAP79 and Kir2.1 in response to a voltage step from the
holding potential of
35 mV to
80 mV under control conditions and
after 10-min exposure to 100 µM intracellular cAMP.
D, mean conductance-voltage relationship for 5 cells
expressing AKAP79 and Kir2.1 under control (filled symbols)
and following exposure to 100 µM intracellular cAMP for
10 min (open symbols). The voltage at which the conductance
was half-maximal shifted from V0.5 =
35.1 ± 1.0 mV
in control to V0.5 =
25.0 ± 0.8 mV in cAMP-exposed
cells (p < 0.005)
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Fig. 4.
AKAP79 coimmunoprecipitates with Kir2.1.
COS-7 cells were transfected with cDNAs encoding AKAP79 and
HA-tagged Kir2.1. Immunoprecipitation was performed using antibodies
directed against the HA epitope tag on Kir2.1 or non-immune IgG as
described under "Experimental Procedures." Precipitated proteins
were separated by SDS-polyacrylamide gel electrophoresis, transferred
onto nitrocellulose membrane, and immunoblotted with anti-AKAP79. 10%
of cell lysate was run in the extract lane. AKAP79 runs as a single
band with a molecular size of 79 kDa. Additional bands visible in the
extract lane represent unidentified, endogenous COS-7 proteins.
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Fig. 5.
AKAP79 binds to the N and C regions of
Kir2.1. A, GST fusion proteins of the intracellular N
(residues 1-71) and C (residues 302-428) domains of Kir2.1 were
screened for their ability to isolate AKAP79 from lysates of COS-7
cells transfected with cDNA encoding AKAP79. GST fusion proteins
bound to glutathione-Sepharose beads were incubated with cell lysates.
Bound proteins were resolved by SDS-polyacrylamide gel electrophoresis,
transferred onto nitrocellulose, and immunoblotted with anti-AKAP79.
AKAP79 was specifically retained by GST·N-(1-71) and
GST·C-(302-428) but not control GST. 10% of the cell lysate was run
in the input lane. Membranes were stained for protein to
ensure that equivalent amounts of each GST fusion protein were used in
each pull-down assay. B, GST fusion proteins of the final
eight C-terminal amino acids of Kir2.1 GST·C-(421-428) failed to
retain AKAP79.
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Fig. 6.
AKAP79 and Kir2.1 are localized to the plasma
membrane. A and B,
confocal image of HEK-293 cells transfected with AKAP79 and HA·Kir2.1
and stained with anti-AKAP79 and anti-HA. The subcellular distribution
of HA·Kir2.1 (A) was visualized by addition of a
FITC-conjugated secondary antibody, and AKAP79 by a Texas
Red-conjugated secondary antibody (B). Both proteins are
found predominantly at the plasma membrane. C and
D, confocal image of HEK-293 cells transfected
with EGFP·Kir2.1 and AKAP79. Cells have been permeabilized and
stained with anti-AKAP79. EGFP·Kir2.1 fluorescence (C) is
greatly reduced by coexpression with AKAP79, when compared with
EGFP·Kir2.1 alone (E).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1C channel subunit was only observed in the presence of the
phosphatase inhibitors, okadaic acid and FK506, both being ineffective
on their own (10). This and other studies highlight one of the features
of heterologous expression systems, which while offering the advantage
of a controlled experimental environment, often do not mimic the native
conditions needed for ion channel modulation. For example, the weak
inward rectifying K+ channel Kir1.1 (ROMK1) is
predominantly expressed in the kidney where it plays an important role
in K+ homeostasis (22). The anti-diuretic hormone,
vasopressin is known to enhance the activity of native Kir1.1 channels
in the kidney via a cAMP-dependent pathway (32, 33). When
cloned Kir1.1 channels are expressed in intact Xenopus
oocytes, however, the channels appear insensitive to elevation of
intracellular cAMP, unless coexpressed with the kinase-anchoring
protein, AKAP79 (14). Overlay assays using the AKAP-binding RII subunit
of PKA as a probe show the presence of a 100-120 kDa RII-binding
protein in kidney membranes, which is absent in oocytes. This suggests that cAMP-induced modulation of Kir1.1 relies upon an anchored pool of
PKA, which is not naturally present within oocyte membranes.
2-adrenergic receptor through sites
on both the third intracellular loop and at the C-terminal tail
(37).
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FOOTNOTES |
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* This work was supported by the Royal Society and the Wellcome Trust.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. Tel.: 116 252 3090; Fax: 116 252 5045; E-mail: cd12@le.ac.uk.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101425200
2 The HUGO Gene Nomenclature Committee has recently approved a new nomenclature scheme for the AKAP family.
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ABBREVIATIONS |
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The abbreviations used are:
PKA, cyclic
AMP-dependent protein kinase;
AKAP, cyclic
AMP-dependent protein kinase-anchoring protein;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid;
NMDA, N-methyl-D-aspartate;
PKC, protein
kinase C;
C-terminal, carboxyl-terminal;
N-terminal, amino-terminal;
HA, hemagglutinin;
CHO, Chinese hamster ovary;
COS-7, African Green
monkey kidney cells;
HEK-293, human embryonic kidney 293;
EGFP, enhanced green fluorescent protein;
GST, glutathione
S-transferase;
AEBSF, 4-(2-aminoethyl)benzenesulfonyl
fluoride hydrochloride;
Ptd-Ins(4, 5)P2,
phosphatidylinositol 4,5-bisphosphate;
ROMK1, renal outer medulla
potassium channel;
PBS, phosphate-buffered saline;
FITC, fluorescein
isothiocyanate;
Kir, inwardly rectifying potassium channel.
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