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 Previous Article | Next Article 
The Journal of Neuroscience, July 1, 1999, 19(13):5301-5310
Voltage-Dependent Neuromodulation of Na+ Channels by
D1-Like Dopamine Receptors in Rat Hippocampal Neurons
Angela R.
Cantrell,
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
through phosphorylation of the  subunit of the
Na+ channel by cAMP-dependent protein kinase (PKA).
Here we report that neuromodulation of Na+ currents
by DA receptors via PKA is voltage-dependent in the range of  110 to
70 mV and is also sensitive to concurrent activation of protein
kinase C (PKC). Depolarization enhanced the ability of D1-like DA
receptors to reduce peak Na+ currents via the PKA
pathway. Similar voltage-dependent modulation was observed when PKA was
activated directly with the membrane-permeant PKA activator DCl-cBIMPS
(cBIMPS; 20 µM), indicating that the membrane potential
dependence occurs downstream of PKA. PKA activation caused only a small
(2.9 mV) shift in the voltage dependence of steady-state inactivation
and had no effect on slow inactivation or on the rates of entry into
the fast or slow inactivated states, suggesting that another mechanism
is responsible for coupling of membrane potential changes to PKA
modulation. Activation of PKC with a low concentration of the
membrane-permeant diacylglycerol analog oleylacetyl glycerol also
potentiated modulation by SKF 81297 or cBIMPS, and these effects were
most striking at hyperpolarized membrane potentials where PKA
modulation was not stimulated by membrane depolarization. Thus,
activation of D1-like DA receptors causes a strong reduction in
Na+ current via the PKA pathway, but it is effective
primarily when it is combined with depolarization or activation of PKC.
The convergence of these three distinct signaling modalities on the
Na+ channel provides an intriguing mechanism for
integration of information from multiple signaling pathways in the
hippocampus and CNS.
Key words:
Na+ current; neuromodulation; cAMP-dependent protein kinase; protein kinase C; 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 neuronal
voltage-gated Na+ channel is a primary target for
phosphorylation by both cAMP-dependent protein kinase (PKA) (Costa et
al., 1982 ; Costa and Catterall, 1984; Rossie and Catterall, 1987;
Murphy et al., 1993) and protein kinase C (PKC) (Costa and Catterall,
1984; Murphy et al., 1993). The neurotransmitters dopamine (DA) and
acetylcholine modulate Na+ currents via these
signaling mechanisms in hippocampal neurons (Cantrell et al., 1996,
1997). Application of oleylacetyl glycerol (OAG), a membrane-permeant
activator of PKC, reduces peak Na+ current and slows
the rate of inactivation of Na+ channels expressed
in Xenopus oocytes or cultured mammalian cells (Sigel and
Baur, 1988; Dascal and Lotan, 1991; Numann et al., 1991; West et al.,
1991). Activation of muscarinic acetylcholine receptors coupled to
phospholipase C exerts a similar PKC-dependent inhibitory effect on
Na+ current in acutely isolated hippocampal
pyramidal neurons (Cantrell et al., 1996).
The subunit of brain Na+ channels is
phosphorylated by PKA at multiple consensus sites on the intracellular
loop between domains I and II (Rossie et al., 1987; Rossie and
Catterall, 1989; Murphy et al., 1993). PKA reduces peak
Na+ current amplitude in cultured rat brain neurons
(Li et al., 1992) and in mammalian cells (Li et al., 1992) or
Xenopus oocytes (Gershon et al., 1992; Smith and Goldin,
1996, 1997) expressing brain Na+ channels.
Similarly, activation of D1-like DA receptors, which couple to
activation 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 (Cantrell et al., 1997; Smith and Goldin, 1997).
Small changes in resting membrane potential can have dramatic effects
on the integrative properties and on the threshold and frequency of
action potential firing in central neurons (Jahnsen and Llinas, 1984;
Hultborn and Kiehn, 1992; McCormick and Von Krosigk, 1992; Gorelova and
Reiner, 1996; Kiehn et al., 1996; McCormick and Bal, 1997; Surmeier and
Kitai, 1997). In the experiments reported here, we have examined the
role of membrane potential and PKC activation in modulation of
whole-cell Na+ current by D1-like DA receptors in
acutely isolated rat hippocampal pyramidal neurons. Our results show
that membrane potential is a crucial determinant of the neuromodulation
of Na+ channels via the PKA pathway. Depolarization
strongly enhances the effect of dopaminergic agonists and PKA
activation. This voltage dependence does not reflect a simple shift in
the steady-state inactivation of Na+ channels after
phosphorylation. Concurrent activation of PKC also enhances the ability
of D1-like DA receptor activation to modulate the functional properties
of the neuronal voltage-gated sodium current via a parallel pathway.
These results provide the first evidence, to our knowledge, for
voltage-dependent neuromodulation and raise the possibility that
membrane voltage and PKC phosphorylation alter the extent or pattern of
phosphorylation of Na+ channels by PKA and thereby
alter neuromodulation of channel activity.
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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., 1994; Howe and Surmeier, 1995; Cantrell et al., 1996). 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 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, 5%
CO2, pH 7.4, 300-305 mOsm/l. Single slices were
then 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 the 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 the cells were allowed to
settle (~5 min), the solution bathing the cells was changed to normal
external recording solution.
Mammalian cell transfection
tsA-201 cells, an embryonic kidney cell line 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/RBL) supplemented with 10% fetal calf serum (Hyclone), 25 U/ml penicillin, and 25 µg/ml streptomycin (Sigma). They were
cotransfected with cDNA encoding the human CD8 marker protein
(EBO-pCD-leu2; American Type Culture Collection) and a plasmid encoding
wild-type or mutant rat brain type IIa Na+ channel
subunit. Cells were transfected using the calcium phosphate precipitation method as described previously (Margolskee et al., 1993).
Successfully transfected cells were then identified by labeling with
magnetic polystyrene microspheres coated with anti-CD8 antibody (Jurman
et al., 1994) (Dynabeads M-450 CD8, Dynal, Great Neck, NY).
Whole-cell recording
Hippocampal neurons. Whole-cell currents were
recorded from pyramidally shaped hippocampal neurons that had at most
one to 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, 80 glucose, pH 7.3 with NaOH, 300-305 mOsm/l. The internal recording
solution consisted of (in mM): 188.9 N-methyl
D-glucamine, 40 HEPES, 4 MgCl2, 1 NaCl,
0.1 BAPTA, 25 phosphocreatine, 2-4 Na2ATP, 0.2 Na3GTP, 0.1 leupeptin, pH 7.2 with
H2SO4, 270-275 mOsm/l. SKF 81297 (RBI,
Natick, MA) and PKCI19-36 (Peninsula Labs, Belmont, CA)
were prepared as fresh concentrated stocks in water and frozen in
aliquots before use. Sp-5,6-DCl-cBIMPS (cBIMPS; BioLog, LaJolla, CA)
and OAG (Alexis Biochemicals, San Diego, CA) were prepared as
concentrated stocks 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. The series resistance compensation did not
change significantly during a typical experiment. Recordings were
obtained using an Axon Instruments 1C patch clamp (Axon Instruments, Foster City, CA). Voltage-pulses were delivered and currents were recorded using a personal computer running Basic-FASTLAB software to
control an AD/DA interface (IDA, Indec Systems, Sunnyvale, CA).
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. Solution changes were made by altering the position of the array
with a DC drive system controlled by a microprocessor-based controller
(Newport-Klinger, Irvine, CA). Complete solution changes were achieved
within <1 sec as judged by the rate of TTX block of
Na+ current and changes in reversal potential in
response to a change in Na+ concentration.
tsA-201 cells. Whole-cell currents were recorded from
successfully transfected cells identified using DynaBeads. The external recording solution consisted of (in mM): 140 NaCl, 10 HEPES, 1 MgCl2, 0.4 CdCl2, 25 CsCl, 5 BaCl2, pH 7.3 with NaOH, 300-305 mOsm/l.
The internal recording solution was identical to that described for
hippocampal neurons. Other recording parameters were as described above.
Data analysis
Data were collected using standard voltage step
protocols. Least-squares curve fitting and statistical analysis were
performed using Sigma Plot (Jandel Scientific). Statistics are
presented as means ± SEM.
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RESULTS |
Modulation of Na+ current by the D1 dopamine
receptor agonist SKF 81297 is enhanced by membrane
depolarization
Activation of D1-like DA receptors, which are coupled to
the stimulation of adenylyl cyclase, decreases endogenous
Na+ current in acutely isolated hippocampal neurons
via phosphorylation by PKA (Cantrell et al., 1997). To determine
whether membrane potential could affect physiological modulation of
Na+ currents by DA receptor activation, hippocampal
neurons were studied under whole-cell voltage clamp using a holding
potential of either 70 or 85 mV. From a holding potential of 70
mV, a 40 msec test pulse to 20 mV was applied once every 2 sec for 1-2 min in control solution until the Na+ current
magnitude stabilized. The D1 agonist, SKF 81297 (5 µM), was then applied. Reduction in peak Na+ current was
measured as the difference in the magnitude of the current elicited by
the test pulse in control conditions and in the presence of the
agonist. The agonist was then washed out of the bath and the current
amplitude was allowed to recover. This control-drug-wash sequence was
then repeated at the hyperpolarized potential. At a holding potential
of 70 mV, application of SKF 81297 reduced peak
Na+ current by 21.2 ± 2.4% (n = 20) (Fig. 1A,D), but
only by 7.5 ± 1.4% when the holding potential was 85 mV
(n = 23) (Fig. 1C,D). Similar results were
obtained when the control-drug-wash sequence was performed at the
hyperpolarized potential and then repeated at the depolarized
potential. Together, these results indicate that the effect of DA
receptor activation on Na+ current is voltage
dependent.
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Figure 1.
Modulation of whole-cell Na+
current by D1-like dopamine receptor activation is enhanced at
depolarized holding potentials in rat hippocampal neurons.
A-C, Representative current traces elicited by a test
pulse to 20 mV from the indicated holding potential in control and in
the presence of 5 µM SKF 81297 (smaller
trace). D, Bar graph summarizing the effect of
membrane depolarization on the magnitude of SKF 81297 modulation in a
population of neurons (n  20 for each group). The
mean of each population is indicated on the graph. An
asterisk indicates statistical significance
(p  0.05; Student's t
test).
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Direct activation of PKA with the membrane-permeant activator cBIMPS
(20 µM) revealed similar dependence on membrane potential (Fig. 2). Depolarization from 85 to
75 mV or 65 mV caused an increase in the percentage reduction of
sodium current by treatment with cBIMPS from 15 to 49% (Fig.
2A-C). Application of cBIMPS at a holding potential
of 85 mV reduced peak Na+ current only 10.1 ± 1.6% (n = 14), but the reduction was 28.1 ± 4.1% (n = 11) at a holding potential of 70 mV (Fig.
2D). This experiment activates PKA downstream of
adenylyl cyclase, placing the membrane potential-sensitive component of
this regulatory pathway downstream of cAMP production. The most likely
voltage-sensitive component in the pathway is the
Na+ channel itself. In contrast, PKC-dependent
modulation of the Na+ channel via muscarinic
receptor activation as described previously (Cantrell et al., 1996) was
not sensitive to membrane potential (data not shown).
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Figure 2.
Membrane potential-dependent modulation of
Na+ current by cBIMPS in rat hippocampal neurons.
A-C, Representative current traces elicited by a test
pulse to 20 mV from the indicated holding potential in control and in
the presence of 50 µM cBIMPS (smaller
trace). D, Bar graph summarizing the effect of
membrane depolarization on the magnitude of cBIMPS modulation in a
population of neurons. An asterisk indicates statistical
significance (p 0.05; Student's
t test).
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Similar effects of membrane potential on PKA modulation of
brain Na+ channel subunits expressed in tsA-201
cells
PKA modulation of Na+ channels via
PKA-dependent phosphorylation at Ser 573 is reproduced when type IIA
Na+ channel subunits are heterologously
expressed in human embryonic kidney tsA-201 cells (Cantrell et al.,
1997). To determine whether depolarization of the membrane exerts
similar effects on PKA-dependent regulation of Na+
current in this heterologous expression system, we compared the reduction of peak Na+ current in response to PKA
stimulation by cBIMPS at holding potentials of 110 mV, 85 mV, and
70 mV. As shown in Figure 3, the
magnitude of modulation was greatly increased by membrane
depolarization, with an average reduction in peak current of 0.2 ± 0.2% (n = 16) at 110 mV and 19.6 ± 1.2%
(n = 64) at 70 mV. Thus, voltage-dependent enhancement of Na+ channel modulation by PKA is
reconstituted when the Na+ channel subunit is
expressed alone in a non-neuronal mammalian cell expression system.
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Figure 3.
Membrane potential-dependent modulation
of Na+ current by cBIMPS in tsA-201 cells
transiently transfected with type IIA Na+ channel
subunits. A-C, Representative current traces
elicited by a test pulse to 0 mV from the indicated holding potential
in control and in the presence of 50 µM cBIMPS
(smaller trace). D, Bar graph
summarizing the effect of membrane depolarization on the
magnitude of cBIMPS modulation in a population of tsA-201 cells
expressing type IIA Na+ channel subunits. An
asterisk indicates statistical significance
(p < 0.05; Student's t
test).
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We repeated the experiments shown in Figures 1 and 2 with 20 mM BAPTA in the intracellular solution to determine whether
elevations in the intracellular calcium concentration might be
responsible for the observed voltage dependence of the modulation. This
concentration of BAPTA should be sufficient to rapidly buffer
intracellular calcium to the low nanomole range. We found that the
voltage-dependence of D1/PKA modulation was similar for cells dialyzed
with 0.1 mM or 20 mM BAPTA in the intracellular
solution, indicating that the intracellular calcium concentration is
not a determining factor for this aspect of Na+
channel modulation (data not shown).
The voltage dependence of D1/PKA modulation is not caused
by a shift in voltage dependence of steady-state inactivation
The effects of membrane potential on D1/PKA modulation
could result from a simple hyperpolarizing shift in the steady-state inactivation curve after phosphorylation, because this would cause a
net increase in the inhibitory effect of phosphorylation as the cell is
depolarized. We therefore tested whether PKA-dependent phosphorylation
of the sodium channel subunit had any effects on the voltage
dependence of inactivation.
We first examined steady-state fast inactivation with a standard
prepulse protocol. Na+ currents were measured during
test pulses to 0 mV after a series of 50 msec depolarizing prepulses
between 110 and 50 mV. The current amplitude was plotted as a
function of prepulse voltage and fit with a Boltzmann function to
determine the half-inactivation voltage and slope factor. Inactivation
curves were compared in control solution and in the presence of the D1
agonist SKF 81297 (10 µM). As shown in Figure
4A, there was only a
small negative shift in the half-inactivation voltage in the presence
of the D1 agonist. The mean values for half-inactivation voltage and slope factor in control conditions were 51.8 ± 1.5 mV
(n = 13) and 9.2 ± 0.7 mV (n = 13), respectively, and 54.7 ± 1.6 mV (n = 13)
and 10.8 ± 0.9 mV (n = 13) in the presence of SKF
81297 (Fig. 4B). This small shift (2.9 mV,
p < 0.05), although statistically significant, would
cause only a 4.7% reduction in peak current at 85 mV and a 9.2%
reduction at 70 mV. Thus, the effects of D1 receptor activation and
subsequent PKA phosphorylation are not caused primarily by a shift in
steady-state fast inactivation.
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Figure 4.
Effects of PKA phosphorylation on the
voltage dependence of steady-state inactivation. A, From
a holding potential of 70 mV, hippocampal neurons were depolarized to
the indicated membrane potentials for 50 msec and then further
depolarized to 0 mV to record peak Na+ currents.
Steady-state fast inactivation curves are presented for control
conditions ( ) and in the presence of 10 µM SKF 81297 ( ). B, Box plot summarizing the effects of PKA
phosphorylation on the half-inactivation voltage and the slope factor.
An asterisk indicates statistical significance
(p 0.05; Student's t
test). C, The peak Na+ current in
response to test pulses to OmV is plotted as a function of time in
control () or 50 µM cBIMPS () in response to
depolarization of the membrane to the indicated holding potential.
D, Voltage dependence of the sum of fast and slow
inactivation of control and in the presence of cBIMPS derived from the
data in C.
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Na+ channels are also inactivated by a
mechanistically distinct slow inactivation process (Narahashi, 1964;
Adelman and Palti, 1968; Rudy, 1978; Ogata and Tatebayashi,
1992). Therefore, we next examined the effects of PKA phosphorylation
on steady-state slow inactivation because a hyperpolarizing shift
in slow inactivation could also account for our results. To test slow
inactivation, cells were held at 110 mV for 100 sec. Current
amplitude was determined by applying a 40 msec test pulse to 0 mV once
every 2 sec. After 100 sec at 110 mV, the holding potential was
changed to 80 mV for 100 sec, 70 mV for 100 sec, 60 mV for 100 sec, and 50 mV for 100 sec (Fig. 4C). This protocol
allowed time for the channels to enter both the fast and slow
inactivated states. The peak current elicited by the test pulse at each
membrane potential was then plotted as a function of voltage and fit
with a Boltzmann equation to determine the half-inactivation voltage
and slope factor (Fig. 4D). The protocol was then
repeated in the same cell in the presence of 50 µM
cBIMPS. As shown in Figure 4C,D, there was little shift in
the half-inactivation voltage or slope factor in the presence of the
PKA activator cBIMPS (50 µM). The half-inactivation voltage and slope factors in control conditions were 60.0 ± 1.8 mV (n = 12) and 6.4 ± 0.6 mV (n = 12), respectively, and 62.6 ± 1.8 mV (n = 12)
and 7.1 ± 0.2 mV (n = 12) in the presence of cBIMPS (Fig. 4D). This small shift (2.6 mV,
p < 0.05), although statistically significant, is
comparable to the effect on fast inactivation alone and would account
for only a 8.6% reduction at 70 mV. Thus, we can conclude that the
effects of D1 receptor activation and subsequent PKA phosphorylation of
the channel are not caused primarily by changes in the voltage
dependence of steady-state slow or fast inactivation.
We next examined the effects of PKA phosphorylation on the rate of
inactivation from the closed state, because an increase in the rate of
closed-state inactivation would cause more channels to inactivate
before opening and thereby reduce peak Na+ current.
Cells were held at 100 mV and depolarized to 70 mV for varying
intervals of time from 1 to 200 msec to induce channel inactivation
without channel opening. The extent of inactivation was then assessed
by depolarization to 0 mV for 5 msec and recording the remaining
current. This protocol was performed for cells in control conditions
and in the presence of cBIMPS. Comparison of the results gave a direct
measure of the effects of PKA phosphorylation on closed-state
inactivation. We found that cells treated with cBIMPS inactivated more
during the second application of the long pulse protocol than under
control conditions during the first application of the pulse protocol
(Fig. 5A, circles)
(n = 6). However, this was an effect of the pulse
protocol or time of the experiment because the same small increase in
inactivation was observed when the pulse protocol was repeated twice in
the absence of cBIMPS (Fig. 5A, squares)
(n = 6). Furthermore, fitting these data to an
exponential function gave rate constants of 71.7 and 67.6 msec for
control and cBIMPS, respectively. This change (4.1 msec) was not
significantly different from that observed in control solution during a
similar recording protocol. Thus, we conclude that the effects of D1
receptor activation and subsequent PKA phosphorylation of the channel
occur without alterations in the rate of steady-state inactivation at
70 mV.
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Figure 5.
Effects of PKA phosphorylation on the rate of
inactivation. A, Hippocampal neurons were depolarized to
70 mV for the indicated times and then peak currents were recorded at
0 mV. A plot of normalized peak current versus prepulse duration at
70 mV in control conditions () and cBIMPS (). The solid
lines are single exponential fits of the data to determine the
inactivation rate constant. Note that the small increase in
inactivation in the presence of cBIMPS was also observed when the
pulse protocol was repeated twice in the absence of cBIMPS ( vs
 ). B, Plot of normalized peak current versus prepulse
duration at 40 mV in control and in the presence of 50 µM cBIMPS demonstrating the rate inactivation from the
final closed states. C, Plot of normalized peak current
versus prepulse duration at 0 mV in control and in the presence of 50 µM cBIMPS demonstrating the rate of inactivation from the
open state.
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Finally, we compared the effects of PKA phosphorylation on the rate of
onset of inactivation from closed state(s) further along the activation
pathway and from the open state. The current amplitude in response to a
test pulse to 0 mV was measured after a prepulse to 40 mV (to study
inactivation from the final closed states) or 0 mV (to study open-state
inactivation). Prepulse duration was increased in 1 msec increments
from 1 to 50 msec to study closed-state inactivation and from 1 to 20 msec to study open-state inactivation. These experiments were conducted
in control solution and then repeated in the presence of 50 µM cBIMPS. As shown in Figure 5B,C, no
difference in the onset of inactivation was observed in the presence of
cBIMPS from either the closed state or the open state. Thus, we can
conclude that the effects of D1 receptor activation and subsequent PKA
phosphorylation of the channel occur without substantial alterations in
the rate of onset of inactivation from either the closed states or the
open state. Altogether, our results indicate that the observed effects
of membrane potential on D1/PKA modulation are not caused primarily by
changes in the rate or voltage dependence of inactivation from closed
or open states. This rules out a simple negative shift in gating
parameters as the mechanism of interaction between membrane potential
and PKA regulation. The possibility that membrane potential changes alter the extent of phosphorylation or the pattern of phosphorylation of the PKA sites in the Na+ channel is considered in Discussion.
Modulation of Na+ current by PKA is also
enhanced by the activation of PKC
Previous work in this laboratory showed that concurrent
activation of PKC potentiated the effects of PKA activation on
Na+ currents in cultures of embryonic neurons and
transfected cells studied at a negative holding potential of 110 mV
(Li et al., 1992, 1993). To determine whether PKC potentiates D1/PKA
regulation of Na+ current at physiologically
relevant resting membrane potentials (70 to 85 mV) in hippocampal
pyramidal neurons, we used the membrane-permeant activator of PKC, OAG.
The magnitude of the modulation was assessed as the difference in the
magnitude of the Na+ current elicited by a test
pulse to 20 mV in control and in the presence of 50 µM
cBIMPS before and after the application of OAG (20 µM)
(Fig. 6A). At a holding
potential of 85 mV, a 40 msec test pulse to 20 mV was applied once
every 2 sec for 1-2 min in control solution until the current
magnitude stabilized, and 50 µM cBIMPS was then applied.
A reduction in peak Na+ current of 6.5% was
observed in this experiment in the presence of cBIMPS. The cBIMPS was
then washed out of the bath, and the current amplitude was allowed to
recover. A low concentration of OAG was applied, which itself caused a
small reduction in peak Na+ current, and the
control-drug-wash sequence was then repeated in the continued
presence of the PKC activator. The magnitude of the cBIMPS effect was
significantly increased to 22.5% reduction of Na+
current in this experiment after activation of PKC (Fig.
6A). In 13 similar experiments at 85 mV, activation
of PKC increased modulation by cBIMPS from 8.9 ± 2.0%
(n = 13) in control to 16.8 ± 2.9%
(n = 13) in the presence of OAG.
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Figure 6.
Modulation by SKF 81297 and cBIMPS is enhanced by
activation of PKC at a hyperpolarized holding potential in rat
hippocampal neurons. A, Time course of modulation by
cBIMPS in the presence and absence of OAG. The peak current is plotted
as a function of time in the presence of various neuromodulators as
indicated by the bars. B, C,
Representative current traces elicited by a test pulse to 20 mV from
a holding potential of 85 mV demonstrating modulation by SKF
81297 with or without previous activation of PKC by OAG.
D, Bar graph summarizing the facilitatory effect of
previous exposure to a low dose (20 µM) of OAG on the
magnitude of SKF 81297 modulation in a population of neurons. An
asterisk indicates statistical significance
(p 0.05; Student's t
test).
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We performed similar experiments with the D1-like agonist SKF 81297 (Fig. 6B-D). As for cBIMPS, concurrent activation of
PKC with OAG significantly increased the extent of neuromodulation by
SKF 81297 (Fig. 6B,C). At a holding potential of 85
mV, the mean peak Na+ current was decreased 6.7 ± 1.2% (n = 17) by SKF 81297 in control and 14.1 ± 1.9% (n = 11) in the presence of OAG. These results show that PKA-dependent modulation of Na+ currents
is enhanced by both membrane depolarization and PKC activation.
Similar effects of OAG were observed on PKA modulation of
Na+ channel subunits expressed in tsA-201 cells
(Fig. 7A-C). At 110 mV,
essentially no modulation was observed under control conditions, but
significant modulation was recovered after application of OAG. Larger
effects of cBIMPS were observed at 85 and 70 mV, as expected from
the voltage dependence of PKA modulation, but these effects were
significantly increased by concurrent activation of PKC at holding
potentials of 85 and 70 mV as well. The relative effect of
PKC activation is greatest at 110 mV, where activation of PKA has no
detectable effect alone.
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Figure 7.
Modulation by cBIMPS is also potentiated
by PKC activation in transiently transfected tsA-201 cells expressing
type IIA Na+ channel subunits. A,
B, Representative current traces elicited by a test pulse to 0 mV from the indicated holding potential demonstrating modulation by
cBIMPS without (A) or with
(B) previous activation of PKC by OAG.
C, Bar graph summarizing the facilitatory effect of
previous exposure to a low dose (20 µM) of OAG on the
magnitude of cBIMPS modulation in a population of tsA-201 cells. An
asterisk indicates statistical significance
(p 0.05; Student's t
test).
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We repeated the experiments shown in Figures 6 and 7 in the presence of
20 mM BAPTA in the intracellular solution to rapidly buffer
intracellular calcium to the low nanomole range. The increase in D1/PKA
modulation in response to concurrent PKC activation was similar for
cells dialyzed with 0.1 mM or 20 mM BAPTA (data not shown). These results indicate that the intracellular calcium concentration is not a determining factor for enhancement of modulation of Na+ channels by PKC.
Membrane potential-dependent enhancement of PKA modulation
is independent of phosphorylation by PKC
To determine whether basal activation of endogenous PKC
was responsible for the effect of membrane depolarization, we tested the magnitude of the PKA-dependent modulation of Na+
currents at a holding potential of 70 mV in hippocampal neurons dialyzed with the specific PKC inhibitor PKCI(19-36)
(House and Kemp, 1987). When PKCI(19-36) was added to the
recording pipette at a concentration of 20 µM and 5-10
min were allowed for dialysis of the peptide, no significant
differences in the magnitude of the modulation by cBIMPS were observed
between the peptide-containing neurons and control neurons (Fig.
8A-C). In comparable
control experiments, this treatment is sufficient to completely prevent
the modulation of currents by OAG or by activation of muscarinic
acetylcholine receptors in hippocampal neurons (Cantrell et al., 1996),
consistent with the conclusion that membrane potential-dependent enhancement of PKA modulation does not require phosphorylation by
PKC.
View larger version (23K):
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|
Figure 8.
Membrane potential-dependent enhancement
of PKA modulation does not require phosphorylation by PKC. A,
B, Peak current elicited by a test pulse to 20 mV from a
holding potential of 70 mV plotted as a function of time in control
solution and in the presence of 50 µM cBIMPS for a
control cell and for a cell dialyzed with 20 µM PKCI.
C, Bar graph summarizing the magnitude of PKA modulation
in control and in the presence of 20 µM PKCI for a
population of neurons. An asterisk indicates statistical
significance (p 0.05; Student's
t test).
|
|
To further address this question, we used a mutant
Na+ channel in which the conserved serine residue
1506 in the loop connecting domains III and IV of the
Na+ channel subunit was mutated to alanine
(S1506A). Previous studies have demonstrated that phosphorylation of
this serine residue is required to observe PKC-dependent modulation of
the Na+ current (West et al., 1991) and
PKC-dependent enhancement of PKA modulation of the
Na+ current (Li et al., 1993). If phosphorylation of
the channel by PKC is necessary for the voltage dependence of the
D1/PKA effect, mutation of serine 1506 should prevent voltage-dependent
PKA modulation. As illustrated in Figure
9, we observed similar voltage-dependent PKA modulation of this mutant channel. Application of cBIMPS at a
holding potential of 110 mV reduced peak sodium current only 2.8 ± 1.2% (Fig. 9B,C) (n = 4), but the
reduction was 17.8 + 5.6% (Fig. 9A,C) (n = 4) at a holding potential of 70 mV. The magnitude of the
voltage-dependent increase in PKA modulation is similar to that
observed for wild-type Na+ channels, confirming that
phosphorylation of serine 1506 by PKC is not needed for
voltage-dependent enhancement of PKA modulation.
View larger version (18K):
[in this window]
[in a new window]
|
Figure 9.
D1/PKA modulation is voltage dependent in tsA-201
cells expressing mutant S1506A Na+ channel subunits. A, B, Representative current traces elicited
by a test pulse to 0 mV from the indicated holding potential in control
and in the presence of 5 µM SKF 81297 (smaller
trace). C, Bar graph summarizing the effect of
membrane depolarization on the magnitude of SKF 81297 modulation in a
population of tsA-201 cells (n 20 for each
group). The mean of each population is indicated on the graph. An
asterisk indicates statistical significance
(p 0.05; Student's t
test). These results indicate that PKC phosphorylation and membrane
depolarization affect PKA modulation of Na+ current
by parallel mechanisms that both enhance the PKA-dependent reduction in
peak Na+ current.
|
|
| |
DISCUSSION |
Membrane depolarization enhances the modulation of
Na+ channels by PKA
Phosphorylation of the subunit of neuronal
Na+ channels serves an important regulatory function
in the CNS. Na+ current in striatonigral and
hippocampal neurons is modulated by activation of D1-like DA receptors,
which act through PKA to reduce peak Na+ current
(Surmeier et al., 1992; Schiffmann et al., 1995; Cantrell et al.,
1997). Our results provide the first evidence for an effect of membrane
potential on modulation of sodium channels by the D1/PKA pathway and,
to our knowledge, the first evidence for voltage-dependent neuromodulation of any ion channel. Depolarization in the range from
110 to 70 mV substantially enhances the reduction of peak Na+ current by PKA activation. At 110 mV, no
detectable reduction in peak Na+ current is caused
by activation of PKA. In contrast, the reduction in peak
Na+ current grows progressively with depolarization,
approaching a 30% reduction in peak current at 70 mV. Thus, over the
range of voltages near the normal resting membrane potential, small depolarizations caused by synaptic input or by subthreshold
inward current through voltage-gated channels would enhance the
effects of DA on Na+ channels, thereby reducing peak
Na+ current and increasing the threshold for action
potential generation.
Voltage-dependent neuromodulation may be particularly important in
light of recent reports that the response properties of some
populations of neurons are state-dependent (Jahnsen and Llinas, 1984;
Hultborn and Kiehn, 1992; Gorelova and Reiner, 1996; Kiehn et al.,
1996; McCormick and Bal, 1997; Surmeier and Kitai, 1997). For example,
neostriatal neurons can exist in either an "up-state," in which the
membrane is depolarized, or a "down-state," in which the membrane
is hyperpolarized (Surmeier and Kitai, 1997). This voltage-dependent
shift in the function of striatonigral neurons has profound effects on
their integrative and action potential firing properties. In light of
our observations, Na+ channels in a neuron residing
in the up-state would be expected to be responsive to an incoming DA
signal, whereas neurons in the down-state would not. The integration of
PKA-dependent and membrane potential-dependent regulation of
Na+ channels may contribute in an important way to
the regulation of neuronal spiking activity by activation of
dopaminergic neurotransmitter pathways and other regulatory pathways
using cAMP as second messenger.
Mechanism of voltage-dependent neuromodulation of
Na+ channels
Previous studies of modulation of single
Na+ channels in cultured brain neurons have shown
that the reduction of peak Na+ current caused by
phosphorylation by PKA results from a shift of channels to a null
gating mode in which channels do not open in response to depolarization
(Li et al., 1992). This effect of PKA might acquire voltage dependence
in two ways. First, changes in membrane potential may alter the
extent of PKA modulation via an effect of the voltage-dependent
gating processes of Na+ channels on phosphorylation
or dephosphorylation of the channel. Second, phosphorylation might
cause a shift in steady-state inactivation of channels, which would
impart a voltage dependence to the functional effects of
phosphorylation. We tested the possibility that PKA may alter
steady-state inactivation using protocols designed to measure the rate
and voltage dependence of both fast and slow inactivation. We observed
only a small shift in the voltage dependence of steady-state fast
inactivation and no shift in the voltage dependence of slow
inactivation or in the rates of fast or slow inactivation in the
presence of a PKA activator. Because phosphorylation does not strongly
affect the gating parameters of Na+ channels, the
alternative hypothesis that voltage directly alters the extent or
pattern of PKA phosphorylation in response to dopaminergic stimulation
deserves further study. In this case, the prolonged changes in membrane
potential in the range of 70 to 110 mV imposed in our experiments
may cause slow conformational changes in the sodium channel
phosphorylation sites that lead to differences in their rates of
phosphorylation or dephosphorylation. Development of new methods will
be required to test this idea because the extent of
Na+ channel phosphorylation must be measured in
single cells whose membrane potential is altered in the range of 110
to 70 mV under voltage-clamp conditions.
Synergistic interaction between membrane depolarization and
PKC in enhancing regulation of Na+ channels by
PKA
Previous work on brain Na+ channels
expressed in transfected cells showed that PKA and PKC act in a
convergent manner to regulate Na+ channels and cause
a reduction in peak Na+ current (Li et al., 1993).
In hippocampal pyramidal neurons, activation of muscarinic
acetylcholine receptors modulates Na+ channels,
causing a slowing of inactivation and a reduction of peak
Na+ current (Cantrell et al., 1996). Those effects
required activation of PKC but not activation of PKA. In transfected
cells at a holding potential of 110 mV, little effect of PKA on
Na+ current is observed unless PKC is also
activated, as reported previously (Li et al., 1992). We were interested
in determining whether PKC-dependent enhancement of the effects of PKA
could be demonstrated in hippocampal neurons under physiological
conditions. In the experiments described here, we observed significant
modulation of Na+ currents by the PKA pathway in the
absence of PKC activation at 85 or 70 mV, but that modulation was
enhanced by concomitant activation of PKC at both membrane potentials.
Evidently, membrane potential and PKC act in a parallel manner to
enhance PKA modulation of peak Na+ current in brain
neurons. A recent report (Kondratyuk and Rossie, 1997) has suggested
that phosphorylation of purified sodium channels by PKC decreases
dephosphorylation of cAMP-dependent phosphorylation sites by
calcineurin or protein phosphatase 2A. This may be the mechanism by
which PKC enhances the ability of PKA to regulate channel function. In
striatonigral neurons residing in the hyperpolarized down-state
(Surmeier and Kitai, 1997), this mechanism would enhance responsiveness
to an incoming DA signal in the presence of an additional signal that
activates PKC, such as activation of muscarinic receptors coupled to
PKC stimulation as described previously (Cantrell et al., 1996).
Functional implications of convergent regulation of
Na+ channels in hippocampal neurons by PKA, PKC, and
membrane depolarization
The hippocampus receives both cholinergic innervation from
neurons of the basal forebrain (Frotscher and Leranth, 1985; Price et
al., 1993; Wainer et al., 1993) and dopaminergic innervation from the
mesocorticolimbic dopamine system (Civelli et al., 1993). PKC-dependent
slowing of inactivation of Na+ currents would
increase the duration of the action potential and possibly alter the
pattern of action potential firing. Reduction of neuronal
Na+ currents attributable to phosphorylation by PKC
and/or PKA would be expected to strongly influence the functional
properties of the target neurons, and these effects would be
strengthened by small depolarizations of the resting membrane
potential. 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. The
convergent regulation of Na+ current by membrane
potential, PKC, and PKA allows signals mediated by voltage-gated ion
channels, neurotransmitter receptors that directly affect membrane
conductance, and neurotransmitter receptors that activate either PKA or
PKC to be integrated at the level of the Na+
channel, which is the final common pathway for signal output from the
cell body in the form of action potentials.
| |
FOOTNOTES |
Received Dec. 8, 1998; revised March 29, 1999; accepted April 20, 1999.
This research was supported by National Institutes of Health Grant
NS15751 to W.A.C. and National Research Service Award postdoctoral fellowship NS10147 to A.R.C. We thank Jacob Brown for excellent technical assistance.
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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85(1):
374 - 383.
[Abstract]
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N. A. Gorelova and C. R. Yang
Dopamine D1/D5 Receptor Activation Modulates a Persistent Sodium Current in Rat Prefrontal Cortical Neurons In Vitro
J Neurophysiol,
July 1, 2000;
84(1):
75 - 87.
[Abstract]
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S. K. Park, S. A. Sedore, C. Cronmiller, and J. Hirsh
Type II cAMP-dependent Protein Kinase-deficient Drosophila Are Viable but Show Developmental, Circadian, and Drug Response Phenotypes
J. Biol. Chem.,
June 30, 2000;
275(27):
20588 - 20596.
[Abstract]
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C.-j. Liu, S. D. Dib-Hajj, and S. G. Waxman
Fibroblast Growth Factor Homologous Factor 1B Binds to the C Terminus of the Tetrodotoxin-resistant Sodium Channel rNav1.9a (NaN)
J. Biol. Chem.,
May 25, 2001;
276(22):
18925 - 18933.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
