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Volume 17, Number 19,
Issue of October 1, 1997
pp. 7330-7338
Copyright ©1997 Society for Neuroscience
Dopaminergic Modulation of Sodium Current in Hippocampal Neurons
via cAMP-Dependent Phosphorylation of Specific Sites in the Sodium
Channel  Subunit
Angela R. Cantrell1,
Raymond D. Smith2,
Alan L. Goldin2,
Todd Scheuer1, and
William A. Catterall1
1 Department of Pharmacology, University of Washington,
Seattle, Washington 98195-7280, and 2 Department of
Microbiology and Molecular Genetics, University of California, Irvine,
California 92697
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Phosphorylation of brain Na+ channel  subunits by cAMP-dependent protein kinase (PKA) decreases peak
Na+ current in cultured brain neurons and in
mammalian cells and Xenopus oocytes expressing cloned
brain Na+ channels. We have studied PKA regulation
of Na+ channel function by activation of D1-like
dopamine receptors in acutely isolated hippocampal neurons using
whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a
concentration-dependent manner. No changes in the voltage dependence or
kinetics of activation or inactivation were observed. This effect was
mediated by PKA, as it was mimicked by application of the PKA activator
Sp-5,6-dichloro-1- -D-ribofuranosylbenzimidazole-3 ,5-monophosphorothioate(cBIMPS) and was inhibited by the specific PKA inhibitor peptide
PKAI5-24. cBIMPS had similar effects on type IIA brain
Na+ channel subunits expressed in tsA-201 cells,
but no effect was observed on a mutant Na+ channel
subunit in which serine residues in five PKA phosphorylation sites
in the intracellular loop connecting domains I and II
(LI-II) had been replaced by alanine. A single mutation,
S573A, similarly eliminated cBIMPS modulation. Thus, activation of
D1-like dopamine receptors results in PKA-dependent phosphorylation of
specific sites in LI-II of the Na+
channel subunit, causing a reduction in Na+
current. Such modulation is expected to exert a profound influence on
overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.
Key words:
Na+ current;
neuromodulation;
cAMP-dependent protein kinase;
hippocampus;
dopamine receptors;
phosphorylation
INTRODUCTION
Voltage-dependent
Na+ current is the primary inward current underlying
excitability in the hippocampus and throughout the CNS but is not often
recognized as a target for neuromodulation. The neuronal
Na+ channel subunit is phosphorylated by
cAMP-dependent protein kinase (PKA) (Costa et al., 1982 ; Costa and
Catterall, 1984; Rossie and Catterall, 1987) on a family of sites in
the intracellular loop connecting domains I and II (Rossie et al.,
1987; Rossie and Catterall, 1989; Murphy et al., 1993). Activation of
PKA reduces Na+ flux in synaptosomes (Costa and
Catterall, 1984) and peak Na+ current in cultured
rat brain neurons (Li et al., 1992). PKA also reduces current through
type IIA brain Na+ channels heterologously expressed
in mammalian cells (Li et al., 1992, 1993) or in Xenopus
oocytes (Gershon et al., 1992; Smith and Goldin, 1996). These results
suggest that Na+ channels might be modulated by
neurotransmitters coupled to signaling cascades that alter PKA
activity.
Neuronal release of dopamine is a primary modulator of PKA activity in
the CNS. In particular, the hippocampus receives a rich dopaminergic
innervation from the mesocorticolimbic dopamine system (Meador-Woodruff
et al., 1994). Five dopamine receptor subtypes (D1-D5) have been
identified (Sibley and Monsma, 1992; Civelli et al., 1993; O'Dowd,
1993), and all are expressed in the hippocampus (Meador-Woodruff et
al., 1994). These may be broadly classified as D1-like (D1 and D5) or
D2-like (D2-D4) (Stoof and Kebabian, 1981, 1984; Sibley and Monsma,
1992; O'Dowd, 1993). Both classes of receptor couple to adenylate
cyclase, but with antagonistic effects. D1-like receptors stimulate
adenylate cyclase activity, whereas D2-like receptors inhibit it (Stoof
and Kebabian, 1981). Thus, activation of D1-like dopamine receptors
could modulate Na+ currents by activating PKA and
phosphorylating the Na+ channel subunit in
hippocampal pyramidal neurons. Activation of dopamine receptors reduces
peak Na+ current in rat neostriatal neurons through
a PKA-dependent pathway (Surmeier et al., 1992; Schiffmann et al.,
1995); however, no neurotransmitter receptor has been identified that
activates PKA resulting in modulation of Na+ channel
activity in hippocampal pyramidal neurons.
We have examined modulation of Na+ currents by
D1-like dopamine receptors in acutely isolated rat hippocampal
pyramidal neurons. Activation of D1-like dopamine receptors rapidly and
reversibly reduces peak Na+ current with no effect
on the voltage dependence or kinetics of activation or inactivation.
This effect requires the activation of PKA and is mimicked by the
extracellular application of direct activators of PKA. Furthermore, we
show that the modulation requires phosphorylation of serine 573 in the
intracellular loop connecting homologous domains I and II
(LI-II) of the Na+ channel subunit.
Dopaminergic input to the hippocampus from the mesocorticolimbic system
may activate this pathway in vivo, thus contributing to the
regulation of excitability in the hippocampus. A preliminary report of
these results has been presented (Cantrell et al., 1996b).
MATERIALS AND METHODS
Acute dissociation of hippocampal neurons.
Hippocampal neurons from adult (>25 d postnatal) male rats were
acutely isolated using procedures described previously (Bargas et al.,
1995; Howe and Surmeier, 1995; Cantrell et al., 1996a). In brief, rats
were decapitated under metofane anesthesia. Brains were quickly
removed, iced, and blocked before slicing. Slices (400-500 µm) were
cut and transferred to a low-calcium (100 µM),
HEPES-buffered saline solution containing (in mM): 140 sodium isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose, and 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
transferred to the low-calcium buffer. With the aid of a dissecting
microscope, portions of the hippocampus were dissected 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. After allowing the cells to settle (about 5 min),
the bathing solution was changed to normal external recording
solution.
Mammalian cell transfection. tsA-201 cells, which are human
embryonic kidney cells (HEK293 cells) that have been stably transfected with simian virus 40 large tumor antigen (Robert Dubridge, Cell Genesis, Foster City, CA), were used for transfection experiments. tsA-201 cells were maintained in DMEM/F12 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (HyClone, Logan, UT), 25 U of penicillin/ml, and 25 µg of streptomycin/ml (Sigma). They were cotransfected with cDNA encoding the human CD8
marker protein (EBO-pCD-leu2; American Type Culture Collection, Rockville, MD) and a plasmid encoding either the wild-type (WT) rat
brain type IIA Na+ channel subunit, a mutant
Na+ channel subunit in which serines in each of
the consensus PKA phosphorylation sites in LI-II have been
mutated to alanine (PKATOT; designated PKACOMP-A by
Smith and Goldin, 1996, 1997), or a mutant Na+
channel subunit in which serine 573 has been mutated to alanine (S573A). Cells were transfected using the calcium phosphate
precipitation method as described previously (Margolskee et al., 1993).
Successfully transfected cells were then identified with magnetic
polystyrene microspheres coated with anti-CD8 antibody (Jurman et al.,
1994; M-450 CD8 Dynabeads, Dynal, Great Neck, NY).
Whole-cell voltage-clamp recording. Whole-cell
currents were recorded from pyramidally shaped hippocampal neurons that
had at most one or two short processes or from tsA-201 cells expressing WT or mutant Na+ channel subunits (Hamill et
al., 1981; Bargas et al., 1995; Howe and Surmeier, 1995). Electrodes
were pulled from 75 µl micropipette glass (VWR Scientific,
Westchester, PA) and fire-polished before use. A low
Na+ extracellular recording solution was used for
recording from hippocampal neurons to reduce current magnitude. It
contained (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. For recording from tsA-201 cells, the extracellular solution
contained (in mM): 140 NaCl, 10 HEPES, 1 MgCl2, 0.4 CdCl2, 25 CsCl, and 5 BaCl2, pH 7.3, with NaOH, 300-305 mOsm/l. The
intracellular recording solution contained (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 H2S04, 270-275 mOsm/l. SKF 81297, SKF 38393, and SCH 23390 (Research Biochemicals, Natick, MA) were
prepared as concentrated stocks in water and frozen in aliquots before
use. Tetrodotoxin (TTX; Calbiochem, San Diego, CA), PKA inhibitor
(PKAI) and PKC inhibitor (PKCI) (PKAI5-24 and
PKCI19-36, respectively; Peninsula Labs, Belmont, CA) were
prepared as concentrated stocks in water and diluted immediately before
recording.
Sp-5,6-dichloro-1--D-ribofuranosylbenzimidazole-3,5-monophosphoro-thioate (cBIMPS; BioLog, LaJolla, 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. Recordings
were obtained using an Axon Instruments (Foster City, CA) 1C patch
clamp. Voltage pulses were delivered, and currents were recorded using
a personal computer running Basic-FASTLAB software to control an
analog-to-digital/digital-to-analog interface (IDA, Indec Systems,
Capitola, CA). Series resistance compensation (70-80%) was routinely
used.
Pharmacological compounds were applied using a gravity-fed "sewer
pipe" system. The array of application capillaries (~150 mm inner
diameter) was positioned a few hundred micrometers away from the cell
under study. Solutions were changed by altering the position of the
array with a microprocessor-controlled DC motor (Newport-Klinger, Inc.,
Irvine, CA). Complete solution changes were achieved within 1 sec, as
estimated from the rate of block by TTX or the rate of change in
reversal potential after a solution change.
Pulse protocols and data analysis. Data were collected using
standard voltage step protocols. Conductance
(g) was calculated from peak current measured
during test pulses to varying voltages according to
g(V) = I/(V  Vrev), where I was the
current, V was the test pulse voltage, and
Vrev was the measured reversal potential. The
voltage dependence of inactivation was measured using 20-msec-long conditioning pulses to a varying potential followed by a test pulse to
10 mV. Peak test pulse current was plotted as a function of
conditioning pulse voltage. Both conductance-voltage and
inactivation-voltage curves were fit with a Boltzmann function of the
form:
where Vh was the half-activation or
inactivation voltage, k was a slope factor, and
gmax was the maximum conductance.
Dose-response curves were fit with a Langmuir isotherm of the form:
where Ipeak was the maximum current
elicited by the pulse, Isensitive was the
blockable current, [M] was the agonist concentration, EC50 was the concentration of agonist producing 50% block,
Iresistant was the portion of the current
resistant to modulation, and n was usually set to 1 (varying
n between 0.5 and 1.5 did not significantly enhance the fits
for the data described here). Least squares curve fitting and
statistical analysis were done using Sigma Plot (Jandel Scientific,
Corte Madera, CA). Statistics are presented as mean ± SEM. When
the sample sizes allowed, box plots of the data were drawn. In the box
plot, the median is represented as the central bar of the box, the
edges of the box are the interquartiles (technically fourths), and the
"whiskers" are lines drawn to the most extreme points in the sample
that are not outliers (defined as points beyond interquartile ± 1.5 × interquartile range); outliers are shown as circles or asterisks
(Tukey, 1977).
RESULTS
Na+ current in hippocampal neurons is modulated
by activation of D1-like dopamine receptors
As an initial step in studying dopaminergic modulation of
Na+ channels by phosphorylation, we examined the
effect of agonists of D1-like dopamine receptors. Whole-cell
Na+ currents were recorded in acutely dissociated
hippocampal neurons using solutions that isolated voltage-dependent
Na+ current (see Materials and Methods). Under these
recording conditions, all of the remaining voltage-dependent current
was blocked by the specific Na+ channel blocker TTX
(see Cantrell et al., 1996a). Application of the D1-like dopamine
receptor agonist SKF 81297 (Arnt et al., 1992) rapidly and reversibly
inhibited the whole-cell Na+ current (Fig.
1A,B). Significant
reductions in current were observed in 73.4% of the cells tested
(n = 34). In the responding cells, saturating
concentrations of SKF 81297 (5 µM) reduced current an
average of 22.2 ± 1.7% (n = 25). The magnitude
of the response varied from cell to cell with a range of 7.1 to 36.8%,
as indicated by the box plot in Figure 1B,
inset. Similar effects were observed for a second agonist,
SKF 38393 (Fig. 1C,D). Saturating concentrations of this
agonist (5-10 µM) reduced current an average of
26.8 ± 2.8% (n = 10). These results are in good
agreement with data previously reported for the modulation of
Na+ current by dopamine receptor agonists in the
neostriatum (Surmeier et al., 1992; Schiffmann et al., 1995). To
confirm that these effects were mediated via D1-like dopamine
receptors, the concentration dependence of the effect and its
sensitivity to specific antagonists were tested. The effects of SKF
81297 were concentration-dependent, as shown in Figure
2A,B.
Concentration-response plots for SKF 81297 were fit with a single
Langmuir isotherm having a mean IC50 of 825.4 ± 128.8 nM (Fig. 2A; n = 8 independent experiments). Modulation by 5-10 µM SKF
81297 was prevented in the presence of the D1-like dopamine receptor
antagonist SCH 23390 (10 µM; Fig. 2C,D). It
was recovered after SCH 23390 removal, confirming that the tested cell
was responsive to SKF 81297. These results demonstrate that the
observed effects were mediated by the activation of D1-like dopamine
receptors. Similar effects of SCH 23390 were observed in four other
experiments. Taken together, these data confirm that SKF 81297 acts at
D1-like dopamine receptors on hippocampal pyramidal neurons to produce
a dose-dependent reduction in tetrodotoxin-sensitive, voltage-dependent
Na+ current.
Fig. 1.
D1-like dopamine receptor agonists reduce
whole-cell Na+ current. A, The
absolute value of peak inward Na+ current is plotted
as a function of time during the experiment. The cell was exposed to
SKF 81297 (5 µM) by rapid perfusion during the period
indicated by the solid bar. Test pulses to 20 mV (40 msec long) were applied once every 2 sec from a holding potential of
75 mV. The same pulse protocol was used in subsequent figures unless
otherwise indicated. B, Representative current traces
obtained under control conditions (1) and in the
presence of SKF 81297 (2) recorded at the times
indicated in A. Inset, Box plot showing the extent of current reduction in cells that responded to SKF 81297. C, Effect of SKF 38393 (10 µM) on peak
inward Na+ current as described in A.
D, Representative current traces obtained under control
conditions (1) and in the presence of SKF 38393 (2) at the times indicated in C.
Inset, Box plot showing the extent of reduction in cells
that responded to SKF 38393.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Involvement of a D1-like dopamine receptor in the
signaling pathway. A, Dose-response curve for SKF 81297 reduction in current. The solid line is a least squares
fit of a Langmuir isotherm to the data with
KI = 745 nM.
Inset, Box plot summarizing similar data from eight
independent experiments. B, Representative current traces from the cell in A obtained in control and in the
presence of the indicated SKF 81297 concentrations. C,
Peak Na+ current as a function of time as described
for Figure 1A. The cell was first exposed to 10 µM SCH 23390, then to a mixture of SCH 23390 and 10 µM SKF 81297, and finally to SKF 81297 alone, as
indicated by the bars. D, Representative
current traces from the experiment shown in C.
[View Larger Version of this Image (30K GIF file)]
Characterization of effects of D1-like dopamine receptor activation
on Na+ current properties
The catalytic subunit of PKA was previously found to reduce peak
Na+ current in excised patches from cultured neurons
with no discernible effects on the voltage dependence or kinetics of
activation or inactivation (Li et al., 1992). As a comparison with
those results, we tested whether D1-like dopamine receptor agonists
altered these properties of the current in hippocampal pyramidal
neurons. The time course of Na+ currents was similar
before and after application of 5 µM SKF 81297 (Figs.
1B, 3A-D),
and the reduction in peak Na+ current was comparable
at all test potentials (Fig. 3A-E). This indicates that the
voltage dependence of activation of the Na+ current
was unchanged, as illustrated by the superimposition of
current-voltage curves before and after agonist application (Fig.
3E). The half-activation voltage and slope factor in control conditions were 44.2 ± 5.3 mV (n = 8) and
5.3 ± 0.3 mV (n = 8), respectively, and
44.9 ± 5.4 mV (n = 8) and 5.5 ± 0.3 mV
(n = 8) in the presence of 5 µM SKF 81297 (Fig. 3F,G). Likewise, the voltage dependence of
inactivation during 50 msec prepulses was also unaffected, having a
half-inactivation voltage of 51.8 ± 1.5 mV (n = 13) and slope factor of 9.2 ± 0.7 mV (n = 13) in
control and a half-inactivation voltage of 54.7 ± 1.6 mV and
slope factor of 10.8 ± 0.9 mV (n = 13) in the
presence of SKF 81297 (n = 13) (Fig. 3F,G).
Thus, the effects of D1-like receptor activation occur without dramatic
alterations in the voltage-dependent properties of the
Na+ channel, consistent with the characteristics of
Na+ channel modulation by PKA (Li et al., 1992,
1993).
Fig. 3.
Voltage dependence of D1-like dopamine receptor
effect on Na+ currents. A-D,
Whole-cell Na+ current elicited by test pulses to
the indicated potentials from a holding potential of 75 mV in the
absence and presence of 5 µM SKF 81297. In each panel,
the smaller current was obtained in the presence of SKF 81297. E, Current-voltage relationship for peak
Na+ current in control (closed
squares) and in the presence of SKF 81297 (closed
circles). The SKF 81297 points have been scaled to facilitate
comparison of the voltage dependence of the Na+
current in the absence and presence of the compound (shaded
triangle). F, Boltzmann fits of the activation
and inactivation curves for the same cell in control (closed
symbols) and in the presence of SKF 81297 (open
symbols). G, Box plot showing the range of half-activation and half-inactivation voltages obtained in control and
in the presence of SKF 81297.
[View Larger Version of this Image (23K GIF file)]
In previous experiments, the effects of PKA activation on brain
Na+ channels were studied by applying the purified
catalytic subunit of PKA to the cytoplasmic surface of excised
inside-out patches (Li et al., 1992, 1993). To demonstrate that direct
activation of PKA produced the same effects as activation of D1-like
dopamine receptors in hippocampal neurons, the modulation of
Na+ current by the potent and membrane-permeant
activator of PKA cBIMPS (Sandberg et al., 1991) was studied.
Application of cBIMPS (20-50 µM) rapidly and reversibly
reduced Na+ currents in rat hippocampal neurons
(Fig. 4A,B). Currents
were reduced in all seven cells treated with 50 µM
cBIMPS, with a mean reduction of 21.7 ± 4.5%. Consistent with
the preceding studies, the reduction in current attributable to
activation of PKA occurred without changes in the voltage-dependent
properties or kinetics in rat hippocampal neurons.
Fig. 4.
Reduction of Na+ current by PKA
activation. A, Peak Na+ currents
elicited in control and in the presence of 50 µM cBIMPS
plotted as a function of time during the experiment. B,
Representative current traces in control (1) and
in the presence of cBIMPS (2) recorded at the
times indicated in A. Inset, Box plot
showing the extent of current reduction in response to cBIMPS.
C, Peak Na+ currents plotted as a
function of time. The cell was first exposed to 5 µM SKF
81297, then 50 µM cBIMPS, and finally to a mixture of SKF
81297 and cBIMPS as indicated by the bars. The time
course was corrected for a 20% rundown in the currents by subtraction of an exponential fit to the currents during control periods. D, Representative current traces in cBIMPS
(2) and in the presence of cBIMPS plus SKF 81297 (3) recorded at the times indicated in C.
[View Larger Version of this Image (32K GIF file)]
The physiological effects of D1-like dopamine receptor activation
require PKA
To test directly for the involvement of PKA in the signaling
pathway mediating the effects of D1-like dopamine receptor activation in hippocampal pyramidal neurons, we have used the specific peptide inhibitor of PKA, PKAI5-24 (Cheng et al., 1986). When
PKAI5-24 was included in the pipette solution at a
concentration of 10 µM, and 5-7 min were allowed for
intracellular dialysis, SKF 81297 no longer modulated whole-cell
Na+ current in hippocampal neurons (Fig.
5B). Although a slight
increase in Na+ current is observed for the cell in
Figure 5B, an average of results on six cells showed that
SKF 81297 reduced peak current by 5.5 ± 0.7%
(p < 0.006; n = 6). During the
same series of experiments, cells dialyzed with control internal
solutions responded to SKF 81297 treatment with an average
Na+ current reduction of 22.1 ± 4.2%
(n = 8; Fig. 5A). The loss of SKF 81297 modulation was not attributable to loss of critical cellular components
during the dialysis period, because a similar dialysis period was
included in the control experiments. As an additional negative
control for the effects of intracellular dialysis with a peptide
fragment, we repeated the above experiment using the PKC-specific
inhibitor peptide PKCI19-36 (House and Kemp, 1987).
When PKCI19-36 was added to the pipette solution at a
concentration of 2 µM, and 5-7 min were allowed for
intracellular dialysis, a protocol sufficient to block
Na+ channel modulation by PKC (Cantrell et al.,
1996a), 5 µM SKF 81297 still reduced whole-cell
Na+ current an average of 28.2 ± 10.5%
(n = 6; Fig. 5C). These data demonstrate
that the block of SKF 81297 modulation by PKAI5-24 is
attributable to the inhibition of PKA and not to nonspecific effects of
the peptide itself. Consistent with this result, pre-exposure to 50 µM cBIMPS to activate PKA occluded any further modulation by SKF 81297 (Fig. 4C,D; n = 5). Thus,
activation of PKA is required for D1-like dopamine receptor-mediated
reduction of Na+ current in hippocampal neurons.
Fig. 5.
Block of D1-like dopaminergic
Na+ current modulation by an inhibitor of PKA.
Modulation of peak Na+ current by 10 µM SKF 81297 with a control pipette solution
(A) and using pipette solutions containing the
PKA inhibitor PKAI5-24 (B) or the
PKC inhibitor PKCI19-36 (C).
Experimental procedures were as described in Figure
1A.
[View Larger Version of this Image (23K GIF file)]
Modulation of Na+ current by PKA requires
phosphorylation of the Na+ channel subunit in
one or more identified PKA sites in LI-II
The next set of experiments examined the molecular substrate
for the PKA phosphorylation initiated by activation of the D1-like dopamine receptor. The subunit of the neuronal
Na+ channel is phosphorylated at up to four sites by
PKA in intact neurons (Rossie and Catterall, 1987). Serine residues
573, 610, 623, and 687 are phosphorylated by PKA both in
vitro and in intact cells (Rossie et al., 1987; Rossie and
Catterall, 1989; Murphy et al., 1993). A mutant channel in which each
of these serines plus nearby serine 554 had been mutated to alanine was
resistant to the effects of PKA activation when expressed in
Xenopus oocytes (Smith and Goldin, 1996). Because there may
be significant differences between PKA modulation in Xenopus
oocytes and mammalian cells, it was important to determine that these
same sites were responsible for the modulatory effects of PKA in
mammalian cells under the conditions we had used to activate PKA in
hippocampal neurons. For these experiments, we used the same mutant
type IIA rat brain Na+ channel subunit in which
the serine residues in each of the identified PKA sites had been
mutated to alanine (PKATOT; designated PKACOMP-A by
Smith and Goldin, 1996). Plasmids containing cDNA encoding either WT or
PKATOT mutant rat brain type IIA Na+
channel subunits were transiently expressed in the tsA-201 cell
subclone of HEK293 cells. Application of 20 and 50 µM
cBIMPS to tsA-201 cells expressing WT type IIA Na+
channel subunits reduced the expressed Na+
current in a concentration-dependent manner (Fig.
6A,B). The mean
reduction by 50 µM cBIMPS in these cells was 21.3 ± 2.3% (n = 25; Fig. 6F). In contrast,
the mutant Na+ channel PKATOT was not
modulated by cBIMPS application (Fig. 6C,D,F). The
mean reduction was 2.7 ± 0.9% (50 µM;
n = 16; p < 0.0005, Student's
t test) (Fig. 6F). Recent work has
indicated that the PKA site centered at serine 573 is required to
mediate the effects of PKA on rat brain type IIA Na+
channels expressed in Xenopus oocytes (Smith and Goldin,
1997). To test whether the same site is required for PKA modulation
when the channel is expressed in mammalian cells, we used a mutant type
IIA rat brain Na+ channel subunit in which
serine 573 had been mutated to alanine (S573A). When transiently
expressed in tsA-201 cells, mutant S573A channels were also not
modulated significantly by cBIMPS application (Fig.
6E,F). The mean reduction was 3.9 ± 2.1% (50 µM; n = 11; p < 0.0005, Student's t test) (Fig. 6F).
These experiments demonstrate that PKA-dependent modulation of neuronal
Na+ channels expressed in mammalian cells requires
phosphorylation of serine 573 of the Na+ channel subunit.
Fig. 6.
Modulation by PKA activation requires direct
phosphorylation of the Na+ channel subunit.
A, tsA-201 cell expressing WT rat brain type IIA subunits was exposed to cBIMPS (20-50 µM) during the
periods indicated by the solid bars. B,
Representative current traces obtained under control conditions
(1) and in the presence of cBIMPS (2,
3) at the times indicated in A. The absolute
value of peak inward Na+ current is plotted as a
function of time during the experiment. C, A tsA-201
cell expressing expressing mutant PKATOT was exposed to
cBIMPS (20-50 µM) during the periods indicated by the
solid bars. The absolute value of peak inward
Na+ current is plotted as a function of time during
the experiment. D, Representative current traces
obtained under control conditions (1) and in the
presence of cBIMPS (2, 3) at the times indicated in
C. E, A tsA-201 cell expressing
expressing mutant S573A was exposed to cBIMPS (50 µM)
during the periods indicated by the solid bar. The
absolute value of peak inward Na+ current is plotted
as a function of time during the experiment. F, Bar
graphs showing the response to 50 µM cBIMPS in tsA-201 cells expressing WT, PKATOT, or S573A mutant
Na+ channels.
[View Larger Version of this Image (33K GIF file)]
DISCUSSION
Activation of D1-like dopamine receptors in hippocampal pyramidal
neurons reduces Na+ current via phosphorylation of
the Na+ channel by PKA
This study describes a complete signaling pathway linking
activation of a neurotransmitter receptor on the cell surface of a
neuron to PKA-dependent phosphorylation of a specific site on the
Na+ channel subunit for the first time. Earlier
work had shown that Na+ currents in cultured rat
brain neurons and in mammalian cell lines expressing the subunit of
the rat brain type IIa Na+ channel were reduced by
application of the catalytic subunit of PKA to the cytoplasmic surface
of excised patches of membrane (Li et al., 1992). That reduction
occurred without significantly altering the voltage-dependent
properties of the channel or its rate of inactivation. The present work
demonstrates that D1-like dopamine receptor activation in acutely
isolated hippocampal neurons produces a similar modulation of
Na+ current and that the modulation requires
activation of PKA. Furthermore, we show that PKA-dependent reduction of
Na+ current is abolished in a mutant
Na+ channel subunit in which each of the serines
in PKA consensus sites that have been shown to be phosphorylated by PKA
in biochemical experiments (Rossie et al., 1987; Rossie and Catterall,
1989; Murphy et al., 1993) has been mutated to alanine (Smith and
Goldin, 1996, 1997), as well as in the single serine mutant S573A.
Thus, the reduction in Na+ current by activation of
D1-like dopamine receptors requires phosphorylation of serine 573. Experiments with other single-site mutants will be required to
determine whether the other phosphorylated serine residues may also
have significant effects in mammalian cells. In Xenopus
oocytes, phosphorylation of serines 610 and 623 has a small effect,
whereas phosphorylation of serines 554 and 687 has none (Smith and
Goldin, 1997).
There have been few reports of receptor-mediated modulation of
Na+ channel activity in neurons.
Na+ currents in Aplysia neurons are
modulated by the peptide FMRFamide (Brussaard et al., 1991), but the
mechanism of receptor coupling to Na+ channel
modulation is unknown. Consistent with the present results in
hippocampal neurons, activation of dopaminergic receptors modulates whole-cell Na+ current in neostriatal neurons via a
PKA-dependent signaling pathway (Surmeier et al., 1992; Schiffmann et
al., 1995). Because striatal neurons are specialized for response to
dopaminergic neurotransmission, and dopamine has regulatory effects in
them that are not observed in other neurons, extension of this
regulatory mechanism for Na+ channels to hippocampal
neurons suggests that it may be a widely distributed component of the
response to dopamine and other neurotransmitters that activate
adenylate cyclase.
In hippocampal neurons work from our laboratory has previously shown
that activation of muscarinic receptors modulates whole-cell Na+ current (Cantrell et al., 1996a). Those effects
occur by a pathway requiring activation of PKC but not activation of
PKA. The dopaminergic pathway described here is a distinct pathway for
Na+ channel modulation that is distinguished by its
dependence on PKA phosphorylation. Thus, two biochemically distinct
pathways for Na+ current modulation by
neurotransmitters are present in hippocampal neurons.
The effects of PKA activation on Na+ currents
demonstrated in the present paper are similar to previous results using
cultured rat brain cells or expressed rat brain type IIA
Na+ channel subunits (Li et al., 1992, 1993). A
notable exception is that phosphorylation of Ser1506 in the
intracellular loop connecting homologous domains III and IV by PKC was
required to observe measurable PKA-dependent modulation in a mammalian
cell line in the previous study (Li et al., 1993). In the present work,
PKA-dependent modulation was not blocked by inclusion of the peptide
inhibitor of PKC, PKCI19-36, in the recording pipette. We
have found that this difference in the requirement for PKC activity to
observe inhibition of Na+ currents by activation of
PKA is caused by differences in the holding potential in the two
studies (Cantrell et al., 1997). At a holding potential of 110 mV, as
used in the studies of Li et al. (1993), little effect of PKA on
Na+ current is observed unless PKC is also
activated. At more positive holding potentials, the requirement for PKC
activation is progressively reduced and is insignificant at the holding
potential of 75 mV used here. These results show that the convergent
regulation of Na+ channels by the PKA and PKC
pathways is voltage-dependent. The voltage dependence of these effects
of phosphorylation provides an additional element of specificity and
sensitivity to modulation of brain Na+ channels.
Dopaminergic receptor subtypes responsible for modulation
All five of the known dopamine receptor subtypes are expressed in
the hippocampus (Meador-Woodruff et al., 1994). The D1-like (D1 and D5)
dopamine receptors stimulate adenylate cyclase and increase PKA
activity (Stoof and Kebabian, 1981, 1984; Sibley and Monsma, 1992;
O'Dowd, 1993). Although we have not yet determined which receptor
subtype is responsible for mediating the effects we observe, the D1 and
D5 dopamine receptor subtypes are good candidates for mediating the
PKA-dependent reduction of Na+ current that we have
characterized in hippocampal pyramidal neurons. A more detailed
determination, however, must await the development and availability of
better subtype-specific blockers.
Functional implications of Na+ channel
modulation through D1-like dopamine receptors in the hippocampus
The hippocampus receives a rich dopaminergic innervation from the
mesocorticolimbic dopamine system, and multiple dopamine receptor
subtypes are expressed on the cell surface of hippocampal pyramidal
neurons (Civelli et al., 1993; Meador-Woodruff et al., 1994). The
effects of dopamine in the hippocampus are primarily inhibitory,
resulting in elevation of spike threshold, slower firing rates, and in
some cases membrane hyperpolarization (Biscoe and Straughan, 1966;
Herrling, 1981; Stanzione et al., 1984; Pockett, 1985; Malenka and
Nicoll, 1986; Berretta et al., 1990). Although it is currently
postulated that these effects are primarily a consequence of
enhancement of a slow Ca2+-dependent
K+ conductance (Bernardo and Prince, 1982; Hass and
Konnerth, 1983; Pockett, 1985; Malenka and Nicoll, 1986), it is likely
that these effects are mediated by a concerted action of the
transmitter on several conductances including the
Na+ channel. Reduction of neuronal
Na+ currents would be expected to influence the
excitability of the target neurons strongly. For example, reduction of
peak Na+ current would be expected to shift the
voltage threshold for action potential generation toward more
depolarized potentials. Thus, a stronger depolarization would be
required to elicit a response. The frequency at which the cell is
capable of generating action potentials might also be reduced.
Additionally, reduction in peak Na+ current by PKA
could serve as a neuroprotective safeguard, preventing excitotoxicity
resulting from prolonged hyperexcitability. The effects of activation
of D1-like receptors are consistent with a role for modulation of
Na+ currents as an important contributing factor to
the inhibitory effects of dopamine in the hippocampus.
FOOTNOTES
Received June 26, 1997; accepted July 23, 1997.
This research was supported by National Institutes of Health Grant
NS15751 to W.A.C., a National Institutes of Health postdoctoral fellowship from Training Grant T32 DA07278 and Individual Postdoctoral Fellowship NS10147 to A.R.C., and National Institutes of Health Grant
NS26729 and an Established Investigator Award from the American Heart
Association to A.L.G.
Correspondence should be addressed to Dr. William A. Catterall,
Department of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195-7280.
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