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The Journal of Neuroscience, 1999, 19:RC21:1-6
RAPID COMMUNICATION
Dopaminergic Modulation of Voltage-Gated Na+ Current
in Rat Hippocampal Neurons Requires Anchoring of cAMP-Dependent Protein
Kinase
Angela R.
Cantrell,
Victoria C.
Tibbs,
Ruth E.
Westenbroek,
Todd
Scheuer, and
William A.
Catterall
Department of Pharmacology, University of Washington, Seattle,
Washington 98195-7280
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ABSTRACT |
Activation of D1-like dopamine (DA) receptors reduces peak
Na+ current in acutely isolated hippocampal neurons
via a modulatory mechanism involving phosphorylation of the
Na+ channel  subunit by cAMP-dependent protein
kinase (PKA). Peak Na+ current is reduced 20-50%
in the presence of the D1 agonist SKF 81297 or the PKA activator
Sp-5,6-dichloro-l- -D-ribofuranosyl benzimidazole-3',5'-cyclic monophosphorothionate (cBIMPS).
Co-immunoprecipitation experiments show that Na+
channels are associated with PKA and A-kinase-anchoring protein 15 (AKAP-15), and immunocytochemical labeling reveals their
co-localization in the cell bodies and proximal dendrites of
hippocampal pyramidal neurons. Anchoring of PKA near the channel by an
AKAP, which binds the RII regulatory subunit, is necessary for
Na+ channel modulation in acutely dissociated
hippocampal pyramidal neurons. Intracellular dialysis with the
anchoring inhibitor peptides Ht31 from a human thyroid AKAP and AP2
from AKAP-15 eliminated the modulation of the Na+
channel by the D1-agonist SKF 81297 and the PKA activator cBIMPS. In
contrast, dialysis with the inactive proline-substituted control peptides Ht31-P and AP2-P had little effect on the D1 and PKA modulation. Therefore, we conclude that modulation of the
Na+ channel by activation of D1-like DA receptors
requires targeted localization of PKA near the channel to achieve
phosphorylation of the subunit and to modify the functional
properties of the channel.
Key words:
Na+ current; neuromodulation; cAMP-dependent protein kinase; A-kinase-anchoring protein; hippocampus; dopamine receptors; phosphorylation
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INTRODUCTION |
Voltage-gated
Na+ current is the primary inward current
underlying excitability in the hippocampus and throughout the
CNS. The subunit of the brain voltage-gated
Na+ channel is a target for
phosphorylation by cAMP-dependent protein kinase (PKA) at multiple
consensus sites on the intracellular loop between domains I and II
(Costa et al., 1982 ; Costa and Catterall, 1984; Rossie and Catterall,
1987, 1989; Rossie et al., 1987). PKA activation reduces peak
Na+ current amplitude in cultured rat
brain neurons and in mammalian cells (Li et al., 1992) or
Xenopus oocytes (Gershon et al., 1992; Smith and Goldin,
1996, 1997) expressing rat brain Na+
channels. Similarly, activation of D1-like dopamine (DA) receptors, which couple to the stimulation of adenylyl cyclase, decreases endogenous Na+ current in acutely isolated
striatonigral and hippocampal neurons (Surmeier et al., 1992;
Schiffmann et al., 1995; Cantrell et al., 1997). This modulatory effect
requires direct phosphorylation of the Na+
channel subunit by PKA at Ser-573 on the intracellular loop connecting domains I and II (Cantrell et al., 1997; Smith and Goldin,
1997). This modulation occurs very rapidly, suggesting that PKA may be
concentrated near the channel via a targeting mechanism.
The type II PKA holoenzyme is a heterotetramer consisting of a dimer of
regulatory subunits, each of which binds to and inactivates one
catalytic subunit. After binding of cAMP, the regulatory subunit dimer
reversibly releases activated catalytic subunits, which phosphorylate
target proteins (Krebs and Beavo, 1979; Taylor, 1989). An important
factor in determining the specificity of PKA signaling is localization
and anchoring of the kinase in the vicinity of the protein to be
phosphorylated. Numerous A-kinase anchoring proteins (AKAPs) have been
described, which anchor PKA near target proteins by binding to the RII
regulatory subunits (Rubin, 1994; Dell'Acqua and Scott, 1997; Murphy
and Scott, 1998). AKAPs possess a conserved amphipathic helix that
binds to the RII dimer and an additional unique targeting domain that
mediates localization of the kinase to specific subcellular targets
within cells (Carr et al., 1991, 1992). Recent experiments have
identified a novel AKAP, AKAP-15, which is associated with skeletal
muscle Ca2+ channels (Gray et al., 1997).
AKAP-15 has an N-terminal lipid anchor, which targets it to the plasma
membrane and an amphipathic alpha helix, which binds PKA (Gray et al.,
1998a). It associates with Ca2+ channels
in skeletal muscle fibers and in transfected cells (Gray et al.,
1998a), and it is implicated in cAMP-dependent modulation of calcium
channels in skeletal muscle cells (Gray et al., 1998a) and in
regulation of calcium channels and insulin release from pancreatic cells (Lester et al., 1997; Fraser et al., 1998).
Because AKAPs are abundantly expressed in the CNS, we hypothesized that
anchoring of PKA near the Na+ channel
protein might be required for D1-like DA receptor modulation of the
Na+ current. In agreement with this idea,
AKAP-15 was recently isolated from partially purified preparations of
brain Na+ channels (Tibbs et al., 1998).
PKA activity co-purifies and co-immunoprecipitates with the brain
Na+ channel, suggesting that PKA is
physically associated with the channel (Tibbs et al., 1998). AKAP-15 is
the prominent AKAP in these preparations and is likely to be involved
in targeting PKA to the brain Na+ channel
(Tibbs et al., 1998). In the experiments described here, we provide
further evidence for association and co-localization of the RII
regulatory subunit of PKA and AKAP-15 with brain sodium channels, and
we demonstrate that localization of the kinase near the
Na+ channel is required for D1- and
PKA-dependent modulation of the voltage-gated
Na+ current in rat hippocampal pyramidal neurons.
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MATERIALS AND METHODS |
Co-immunoprecipitation and phosphorylation of the
Na+ channel. Rat brain sodium channels were
purified as described by Hartshorne and Catterall (1984). Approximately
10 pmol of these partially purified sodium channels were incubated at
0°C with 10 µg of affinity-purified anti-RII antibody (kindly
provided by Dr. Stanley McKnight, Department of Pharmacology,
University of Washington) or control antibody for 2 hr at 4°C in a
final volume of 0.5 ml in 50 mM Tris-HCl, pH 7.4, 75 mM NaCl, 2.5 mM EDTA, 0.1% Triton X-100 (bRIA
buffer). In some cases, purified sodium channel was first preincubated with 0.2 mM Ht31 or 0.2 mM Ht31-P peptide (Carr
et al., 1991, 1992) for 30 min at room temperature in 0.5 ml bRIA
buffer containing aprotinin (10 µg/ml), leupeptin (10 µg/ml),
pepstatin A (1 µM), benzamidine (15.7 µg/ml), and
4-(aminoethyl)benzenesulfonyl fluoride (1 mM) before
incubation with 10 µg of affinity-purified anti-RII antibody.
Preswollen protein A-Sepharose (2-5 mg) beads were added to each 0.5 ml reaction and incubated 0.5 hr at 4°C. Protein A-Sepharose beads
containing precipitated complexes were sedimented by centrifugation for
5 sec at 2000 × g and washed three times with 1 ml
bRIA buffer and twice with 50 mM Tris-HCl, pH
7.5, 10 mM magnesium chloride, 1 mM EGTA, and 0.1% Triton X-100 (phosphorylation
buffer). Immunoprecipitated samples were incubated for 15 min at 37°C
in 0.05 ml of phosphorylation buffer containing 0.25 µM PKA and 0.1 mM
[ -32P]ATP (0.005 mCi/mmol). The
precipitated complexes were then washed three times with 1 ml of
phosphorylation buffer, and proteins were eluted by incubation in
SDS-sample buffer at 65°C for 10 min and separated on 6%
Tris-glycine polyacrylamide gels.
-32P-labeled phosphoproteins were
detected by autoradiography.
Immunocytochemical detection of AKAP-15 in the hippocampus.
Single-label immunocytochemical studies in rat brain were performed using the methods described previously (Westenbroek et al., 1998). Anti-AP1 antibodies (Gray et al., 1997) were affinity-purified, diluted
1:20 in a solution containing 0.1% Triton X-100 and 1.0% normal goat
serum in 0.1 M Tris-buffered saline, and used to detect AKAP-15. Bound anti-AP1 antibodies were visualized with biotinylated goat anti-rabbit IgG and fluorescein-labeled avidin, and the sections were examined in a Bio-Rad (Hercules, CA) MRC-600 confocal microscope. Control sections incubated in normal rabbit serum or with no primary antibody showed no specific staining.
Acute dissociation of hippocampal neurons. Hippocampal
neurons from adult (>25 d postnatal) male rats were acutely isolated using procedures previously described (Bargas et al., 1994; Howe and
Surmeier, 1995; Cantrell et al., 1996). In brief, rats were decapitated
under metofane anesthesia. Brains were then quickly removed, iced, and
blocked before slicing. Four hundred to 500 µm slices were cut and
transferred to a low-calcium (100 µM), HEPES-buffered
saline solution containing (in mM): 140 Na isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose, 15 HEPES, pH 7.4 (300-305 mOsm/l). All solutions were bubbled
with 100% O2 before slicing. Slices were then
incubated for 1-6 hr in NaHCO3-buffered Earle's balanced salt solution (Sigma, St. Louis, MO) bubbled with 95% O2 and 5% CO2, pH 7.4 (300-305 mOsm/l). Single slices were removed into the low-calcium
buffer, and with the aid of a dissecting microscope, regions of
hippocampus were removed and placed in a treatment chamber containing
protease type XIV (Sigma; 0.7 mg/ml) in HEPES-buffered HBSS
(Sigma) at 35° C, pH 7.4 (300-305 mOsm/l). After 5-10 min of enzyme
treatment, the tissue was rinsed several times in the low-calcium
buffer and mechanically dissociated using a series of fire-polished
Pasteur pipettes. The cell suspension was then plated into a 35 mm
tissue culture dish (Corning, Corning, NY) mounted on the stage of an
inverted microscope containing 1 ml of HEPES-buffered phosphate-free
HBSS saline. After allowing the cells to settle (~5 min), the
solution bathing the cells was changed to normal external recording solution.
Whole-cell recording. Whole-cell currents were recorded from
pyramidally shaped hippocampal neurons that had at most one or two
short processes (Hamill et al., 1981; Bargas et al., 1994; Howe and
Surmeier, 1995). Electrodes were pulled from 75 µl micropipette glass
(VWR Scientific, West Chester, PA) and fire-polished before use. The
external recording solution consisted of (in mM): 20 NaCl,
10 HEPES, 1 MgCl2, 0.4 CdCl2, 55 CsCl, 5 BaCl2,
and 80 glucose, pH 7.3, with NaOH (300-305 mOsm/l). The internal
recording solution consisted of (in mM): 189 N-methyl D-glucamine, 40 HEPES, 4 MgCl2, 1 NaCl, 0.1 BAPTA, 25 phosphocreatine,
2-4 Na2ATP, 0.2 Na3GTP, and 0.1 leupeptin, pH 7.2, with
H2SO4 (270-275 mOsm/l).
SKF 81297 (Research Biochemicals, Natick, MA) was prepared as a fresh
concentrated stock in water and frozen in aliquots before use.
Sp-5,6-DClcBIMPS (cBIMPS; BioLog, La Jolla, CA) was prepared as
a concentrated stock in DMSO and diluted before use. Appropriate
vehicle controls were performed where necessary.
Electrode resistances were typically 3-6 M in the bath. Final
series resistance values averaged 6-8 M, of which >80% was compensated electronically. Series resistance compensation did not
change significantly during the course of the experiments. Recordings
were obtained using an Axon Instruments (Foster City, CA) 1C patch
clamp. Voltage pulses were delivered and currents recorded using a
personal computer running Basic-FASTLAB software to control an
analog-to-digital/digital-to-analog interface (IDA; Indec
Systems, Capitola, CA).
Drugs were applied using a gravity-fed "sewer pipe" system. The
array of application capillaries (~150 mm inside diameter) was
positioned a few hundred micrometers away from the cell under study.
Solution changes were made by altering the position of the array with a
DC drive system controlled by a microprocessor-based controller
(Newport-Klinger, Inc., Irvine, CA). Complete solution changes were
achieved within <1 sec as judged by TTX block and changes in reversal potential.
Data analysis. Data were collected using standard voltage
step protocols. Least-squares curve fitting and statistical analysis were done using Sigma Plot (Jandel Scientific, Corte Madera, CA). Statistics are presented as means ± SEM.
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RESULTS |
Activation of D1-like DA receptors, which are coupled to the
stimulation of adenylyl cyclase, decreases the
Na+ current in acutely isolated
hippocampal neurons via phosphorylation of the subunit by PKA
(Cantrell et al., 1997). A low molecular weight AKAP, AKAP-15, has been
identified recently and demonstrated to bind to the brain
Na+ channel, suggesting that anchoring of
PKA may be an important element of this signaling pathway (Tibbs et
al., 1998). To further demonstrate a physical association between PKA
and the Na+ channel subunit, the
complex of RII and Na+ channels was
co-immunoprecipitated using anti-RII antibody, and the
immunoprecipitated Na+ channels were then
radiolabeled by phosphorylation in the presence of PKA and
[-32P]ATP and analyzed by SDS-PAGE.
Co-immunoprecipitation of the Na+ channel
was observed with anti-RII antibody but not with preimmune IgG (Fig.
1A).
Co-immunoprecipitation was blocked by preincubating channel
preparations with the Ht31 anchoring inhibitor peptide, whereas
preincubation with a control peptide (Ht31-P) had no effect (Fig.
1A). The phosphoprotein of ~190 kDa present in the
Ht31 and Ht31-P experiments is a proteolytically cleaved fragment of the subunit generated during incubation with the peptides at room
temperature despite the presence of protease inhibitors. These
co-immunoprecipitation experiments provide further evidence for
physical association between the Na+
channel and the RII subunit of PKA.
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Figure 1.
Brain Na+ channel
co-immunoprecipitates with RII, and AKAP-15 is localized in
hippocampal pyramidal neurons. A, RII was
immunoprecipitated from purified sodium channel preparations with
anti-RII antibody or nonimmune IgG, phosphorylated by PKA, and
analyzed by SDS-PAGE and immunoblotting as described in Materials and
Methods. Where indicated, purified sodium channel was first
preincubated with 0.2 mM Ht31 or 0.2 mM Ht31-P
peptide for 30 min at room temperature before immunoprecipitation with
10 µg of affinity-purified anti-RII antibody. A control reaction
using 10 µg of an subunit-specific antibody, anti-SP19, was also
performed. Molecular mass markers are represented as
Mr × 10 3. B,
Localization of AKAP-15 in the cell bodies and proximal dendrites of
pyramidal neurons in the CA3/CA2 regions of the hippocampus.
C, AKAP-15 localization in the CA1 region of the
hippocampus. Scale bar, 100 µm.
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Sodium channels have been shown previously to be localized in low
density in the cell body of hippocampal pyramidal neurons and in higher
density in the mossy fibers and axons of the fimbria, fornix and
perforant path (Westenbroek et al., 1989, 1992). Immunocytochemical studies were performed to determine whether AKAP-15 is also present in
the cell bodies of these neurons, from which we have made
electrophysiological recordings. Using AP1, an antibody that recognizes
AKAP-15 (Gray et al., 1997), we observed labeling of the cell bodies of
pyramidal neurons located in the CA3/CA2 (Fig. 1B)
and the CA1 (Fig. 1C) regions of the hippocampus. Low levels of
labeling were observed in the proximal dendrites of neurons located in
the CA3/CA2 regions (Fig. 1B), but axon tracts were
not labeled. These experiments provide evidence that
Na+ channels and AKAP-15 are localized
together in the cell bodies and proximal dendrites of hippocampal
pyramidal neurons but not in the major axon tracts in the hippocampus.
To determine whether anchoring of PKA near the
Na+ channel is necessary for modulation of
Na+ currents in hippocampal neurons via
the DA/cAMP pathway, we used the peptides Ht31 (Carr et al., 1991,
1992) and AP2 (Gray et al., 1998a). These peptides span the RII binding
domain of human thyroid AKAP Ht-31 and brain AKAP-15, respectively.
Dialysis with these peptides disrupts the interaction between the
kinase and the AKAP by blocking the binding domain of the AKAP,
resulting in disruption of kinase targeting and localization (Carr et
al., 1991). Proline-substituted versions of these peptides (Ht31-P and
AP2-P), which do not bind RII and have no effect on the anchoring
ability of the kinase, are used as negative controls. If D1-like DA
receptor modulation of the Na+ current
requires anchoring of the kinase near the
Na+ channel subunit, the modulation
should be eliminated in neurons dialyzed with Ht31 or AP2 peptides but
not in cells dialyzed with control solution or with the
proline-substituted control peptides.
As illustrated in Figure 2, the ability
of the D1-like DA receptor agonist SKF 81297 to modulate the whole-cell
Na+ current was eliminated by dialysis
with 500 µM Ht31. Whole-cell Na+ current was elicited by a test pulse
to 0 mV from a holding potential of  70 mV. After stabilization of the
current amplitude, SKF 81297 was applied via rapid perfusion, and its
effect on the whole-cell current was recorded. As previously reported
(Cantrell et al., 1997), application of SKF 81297 (10 µM)
resulted in a rapid reduction in the magnitude of the peak current in
cells dialyzed with control internal solution (Fig.
2A,C,E; 21.5 ± 6.7%; n = 4).
This modulation occurred without significant changes in the kinetics or
voltage dependence of the current. In cells dialyzed with 500 µM Ht31, the reduction in peak current in
response to SKF 81297 was greatly attenuated (Fig.
2B,D,E; 4.2 ± 2.3%; n = 4;
p < 0.05). In contrast, cells dialyzed with the
proline-substituted Ht31-P peptide (500 µM)
responded nearly normally to the application of SKF 81297 (Fig.
2E; 16.8 ± 4.0%;n = 4;
p > 0.05). Because 10 µM SKF
81297 is sufficient to maximally activate D1-like DA receptors
(Cantrell et al., 1997), this result indicates that anchoring by AKAPs
is required for modulation of voltage-gated
Na+ channels by the D1-like DA receptor
pathway, even when the DA receptor is fully activated.
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Figure 2.
D1-like DA receptor modulation of the
Na+ channel in the rat hippocampus is prevented by
dialysis with Ht31 peptide. A, B, Plot of
peak, Na+ current elicited by a test pulse to 0 mV
from a holding potential of 70 mV versus time in control and in the
presence of the D1 agonist SKF 81297 (10 µM), as
indicated by the black bar for a neuron dialyzed with
control solution (A) or 500 µM Ht31
peptide (B). C, D,
Representative whole-cell current traces in control extracellular
solution or SKF 81297 (smaller trace) from either a cell dialyzed with
control solution (C) or 500 µM Ht31
(D). E, Statistical summary of the
percentage reduction in peak current for cells dialyzed with control
solution, 500 µM Ht31, or 500 µM Ht31-P.
*Statistical significance; Student's t test,
p < 0.05.
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To directly activate anchored PKA, modulation by the membrane-permeant
activator cBIMPS (50 µM) was also studied (Fig.
3). The reduction in peak
Na+ current caused by direct activation of
PKA was attenuated similarly to modulation by SKF 81297 in the presence
of the Ht31 peptide (Fig. 3). These data indicate that anchoring of the
kinase near the Na+ channel plays an
important role in mediating cAMP-dependent modulation of the
Na+ current in rat hippocampal neurons
after activation of D1-like DA receptors.
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Figure 3.
Modulation of the Na+ channel
by the PKA activator cBIMPS in the rat hippocampus is prevented by
dialysis with Ht31 peptide. A, B, Plot of
peak current elicited by a test pulse to 0 mV from a holding potential
of 70 mV versus time in control and in the presence of the PKA
activator cBIMPS (50 µM), as indicated by the
black bar for a neuron dialyzed with control solution
(A) or 500 µM Ht31 peptide
(B). C, D,
Representative whole-cell current traces in control extracellular
solution or cBIMPS (smaller trace) from a cell dialyzed
with either control solution (C) or 500 µM Ht31 (D). E,
Statistical summary of the percentage reduction in peak current for
cell dialyzed with control solution, 500 µM Ht31, or 500 µM Ht31-P. *Statistical significance; Student's
t test, p < 0.05.
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The novel brain AKAP, AKAP-15, targets PKA to voltage-gated brain
Na+ channels as well as voltage-gated
skeletal muscle calcium channels (Gray et al., 1997, 1998a; Tibbs et
al., 1998). A PKA-binding peptide containing the amphipathic helix
derived from AKAP-15 (AP2) blocks voltage- and PKA-dependent
potentiation of Ca2+ channel activity in
skeletal muscle (Gray et al., 1998a). Because AKAP-15 is abundantly
expressed in the CNS and is associated with Na+ channels, we repeated our experiments
using the AP2 and proline-substituted AP2-P peptides. As shown in
Figure 4, dialysis with the AP2 peptide also attenuated the ability of the D1 agonist to reduce the
Na+ current (6.6 ± 2.4%;
n = 6; p < 0.01), whereas dialysis
with the AP2-P peptide had no effect (21.5 ± 2.8%;
n = 7). These results further substantiate our
conclusion that PKA anchoring near the Na+
channel by an AKAP is necessary for D1-like DA receptor modulation. Because AKAP-15 is co-immunoprecipitated with brain
Na+ channels and is co-localized with them
in hippocampal pyramidal neurons, it is likely that it is the AKAP
responsible for anchoring PKA near the Na+
channel to mediate neuromodulation by DA and other activators of
adenylyl cyclase.
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Figure 4.
D1-like DA receptor modulation of the
Na+ channel in the rat hippocampus is prevented by
dialysis with AP2 peptide. A, B, Plot of
peak current elicited by a test pulse to 0 mV from a holding potential
of 70 mV versus time in control and in the presence of the D1 agonist
SKF 81297 (10 µM), as indicated by the black
bar for a neuron dialyzed with control solution
(A) or 500 µM AP2 peptide
(B). C, D,
Representative whole-cell current traces in control extracellular
solution or SKF 81297 (smaller trace) from a cell
dialyzed with either control solution (C) or 500 µM AP2 (D). E,
Statistical summary of the percentage reduction in peak current for
cells dialyzed with control solution, 500 µM AP2, or 500 µM AP2-P. *Statistical significance; Student's
t test, p < 0.05.
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DISCUSSION |
Our experiments demonstrate the importance of PKA anchoring in
neurotransmitter modulation of voltage-gated brain
Na+ channels by showing that this form of
ion channel modulation is blocked specifically by anchoring inhibitor
peptides. We previously found that D1-like DA receptor activation
resulted in the modulation of the functional properties of the
voltage-gated Na+ current in these neurons
by reducing the peak current amplitude (Cantrell et al., 1997). The
modulation occurred without significant alterations in the voltage
dependence or kinetics of channel activation or inactivation (Li et
al., 1992; Cantrell et al., 1997). We further demonstrated that the
modulation involved the activation of PKA and resulted from
PKA-dependent phosphorylation of the subunit at Ser-573 in the
intracellular loop between domains I and II of the subunit
(Cantrell et al., 1997; Smith and Goldin, 1997). The present work
defines an additional required element in the signaling
pathway-anchoring of PKA to the Na+
channel via an AKAP.
Signal transduction mechanisms that involve PKA-dependent
phosphorylation events often require that the kinase activity be precisely localized within the cell to confer signaling specificity. This is achieved in part by subcellular targeting of the kinase through
association with A-kinase-anchoring proteins or AKAPs (Rubin, 1994;
Dell'Acqua and Scott, 1997; Gray et al., 1998a,b). AKAPs act as
additional regulatory mechanisms allowing for the activation of
compartmentalized pools of PKA. This is an extremely important
mechanism, because PKA has been implicated in the control of a number
of physiological processes in neurons, including the modulation of
multiple classes of ion channels (Brandon et al., 1997). Specific
targeting and anchoring of PKA ensures that the proper substrates
become phosphorylated in response to incoming signals.
Numerous AKAPs have been purified from a variety of tissues in the past
several years (Rubin, 1994; Dell'Acqua and Scott, 1997). Each AKAP
possesses two binding sites, a conserved RII binding domain, which
mediates binding to the kinase, and an additional, unique targeting
domain, which mediates localization of the kinase to particular
subcellular compartments or substrates. The recently discovered plasma
membrane AKAP, AKAP-15 (Gray et al., 1997, 1998a; Tibbs et al., 1998),
is a likely candidate for mediating targeting of PKA to the
Na+ channel. Purified preparations of rat
brain Na+ channels are phosphorylated by
co-purifying PKA (Costa et al., 1982; Tibbs et al., 1998). Analysis of
these preparations using an RII overlay technique to detect proteins
with high affinity for the RII subunit of PKA identified AKAP-15 in
these preparations, suggesting that this AKAP is likely to target PKA
to neuronal Na+ channels (Tibbs et
al., 1998). Co-immunoprecipitation experiments support the
conclusion that PKA is associated with
Na+ channels, and immunocytochemical
labeling shows that AKAP-15 and Na+
channels are co-localized in the cell bodies of hippocampal pyramidal neurons (Fig. 1). Block of PKA modulation by the anchoring inhibitor peptide Ht31 shows that anchored PKA is required for
Na+ channel modulation. Our
electrophysiological results using the AKAP-15-derived peptide AP2
further support the conclusion that this novel AKAP is required for
Na+ channel modulation. However, our
results are not conclusive on this point, because the AP2 peptide is
expected to bind RII and thereby prevent its association with all
endogenous AKAPs. Na+ channels in the
major axon tracts in the hippocampus are not associated with AKAP-15.
Either they are not regulated by PKA or their regulation does not
require anchoring by AKAP-15. Experiments are currently in progress to
define the mechanism of interaction of Na+
channels and AKAP-15 and to determine their subcellular localization in
different classes of neurons. These experiments should further clarify
the role of AKAP-15 in regulating the activity of the Na+ channel in the CNS.
AKAPs have recently been demonstrated to play a role in mediating
PKA-dependent regulation of other voltage-gated ion channels in
skeletal, cardiac, and smooth muscle tissue. For example, in skeletal
muscle transverse tubules and in cardiac muscle regulation of L-type
calcium currents requires anchoring of PKA (Johnson et al., 1994, 1997;
Gao et al., 1997), as does regulation of calcium-activated potassium
channels in tracheal myocytes (Wang and Kotlikoff, 1996). AKAP-15 is
implicated in regulation of L-type calcium channels in skeletal and
cardiac muscle and in pancreatic cells (Fraser et al., 1998; Gray
et al., 1998a). Thus, the anchoring of PKA near ion channels through
AKAP targeting is emerging as an important theme in ion channel
regulation, and AKAP-15 may be involved in this regulation in a wide
range of cell types. Our current work broadens the scope of this
regulatory mechanism to voltage-gated Na+
channels in hippocampal neurons.
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FOOTNOTES |
Received April 12, 1999; revised June 17, 1999; accepted June 21, 1999.
This research was supported by National Institutes of Health Research
Grant NS15751 to W.A.C., an individual postdoctoral National Research
Service Award to A.R.C., and a predoctoral National Research
Service Award to V.C.T. from National Institutes of Health Training
Grant T32 NS07332.
Correspondence should be addressed to Dr. William A. Catterall,
Department of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195-7280.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 1999, 19:RC21 (1-6). The
publication date is the date of posting online at
www.jneurosci.org.
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ABSTRACT
INTRODUCTION
