Department of Physiology, Kurume University School of Medicine, Kurume 830-0011, Japan
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ABSTRACT |
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Yamada, Kei,
Hiroshi Hasuo,
Masaru Ishimatsu, and
Takashi Akasu.
Characterization of Outward Currents Induced by 5-HT in Neurons
of Rat Dorsolateral Septal Nucleus.
J. Neurophysiol. 85: 1453-1460, 2001.
Properties of the
5-hydroxytryptamine (5-HT)-induced current
(I5-HT) were examined in neurons of
rat dorsolateral septal nucleus (DLSN) by using whole cell patch-clamp
techniques. I5-HT was associated with
an increase in the membrane conductance of DLSN neurons. The reversal
potential of I5-HT was 93 ± 6 (SE) mV (n = 7) in the artificial cerebrospinal
fluid (ACSF) and was changed by 54 mV per decade change in the external
K+ concentration, indicating that
I5-HT is carried exclusively by K+. Voltage dependency of the
K+ conductance underlying
I5-HT was investigated by using
current-voltage relationship. I5-HT
showed a linear I-V relation in 63%, inward rectification
in 21%, and outward rectification in 16% of DLSN neurons.
(±)-8-Hydroxy-dipropylaminotetralin hydrobromide (30 µM), a
selective 5-HT1A receptor agonist, also produced
outward currents with three types of voltage dependency.
Ba2+ (100 µM) blocked the inward rectifier
I5-HT but not the outward rectifier
I5-HT. In
I5-HT with linear I-V
relation, blockade of the inward rectifier K+
current by Ba2+ (100 µM) unmasked the outward
rectifier current in DLSN neurons. These results suggest that
I5-HT with linear I-V
relation is the sum of inward rectifier and outward rectifier
K+ currents in DLSN neurons. Intracellular
application of guanosine-5'-O-(3-thiotriphosphate) (300 µM) and guanosine-5'-O-(2-thiodiphosphate) (5 mM),
blockers of G protein, irreversibly depressed
I5-HT. Protein kinase C (PKC) 19-36 (20 µM), a specific PKC inhibitor, depressed the outward rectifier
I5-HT but not the inward rectifier
I5-HT.
I5-HT was depressed by
N-ethylmaleimide, which uncouples the G-protein-coupled receptor from pertussis-toxin-sensitive G proteins. H-89 (10 µM) and
adenosine 3',5'-cyclic monophosphothioate Rp-isomer (300 µM), protein
kinase A inhibitors, did not depress
I5-HT. Phorbol 12-myristate 13-acetate
(10 µM), an activator of PKC, produced an outward rectifying K+ current. These results suggest that both
5-HT-induced inward and outward rectifying currents are mediated by a G
protein and that PKC is probably involved in the transduction pathway
of the outward rectifying I5-HT in
DLSN neurons.
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INTRODUCTION |
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Lateral septal neurons have
been known to receive afferents containing 5-hydroxytryptamine (5-HT:
serotonin) from the dorsal and medial raphe nuclei (Gall and
Moore 1984; Gallagher et al. 1995
; Jakab
and Leranth 1995
; Köhler et al. 1982
).
5-HT receptors, especially those of the 5-HT1A
subtype, have been demonstrated by in vitro autoradiography with the
highest levels in the lateral septum (Biegon et al.
1982
; Hensler et al. 1991
; Marcinkiewicz et al. 1984
; Pazos and Palacios 1985
;
Vergé et al. 1986
). Molecular cloning has
established that 5-HT1A receptors belong to the
superfamily of the GTP binding protein (G-protein)-coupled receptor
(Albert et al. 1990
; Fargin et al. 1989
).
In the lateral septum, Sim et al. (1997)
have
demonstrated that stimulation of 5-HT1A receptors increases guanosine-5'-O-(3-thiotriphosphate) (GTP
S)
binding. In situ hybridization revealed a substantial distribution of
messenger RNA for the G-protein-coupled inward rectifier
K+ channel (GIRK1-4) in the lateral septum of the
rat brain (Karschin et al. 1996
).
Electrophysiological studies have shown that 5-HT produces a
hyperpolarizing response associated with an increase in
K+ conductance in neurons of the rat dorsolateral
septal nucleus (DLSN) (Goto et al. 1997;
Joëls and Gallagher 1988
; Joëls et al. 1986
, 1987
) through 5-HT1A receptors
(Joël and Gallagher 1988
; Yamada et al.
2000
). Much evidence has accumulated suggesting that 5-HT
produces an inward rectifier K+ current by
directly activating G protein in neurons of the dorsal raphe nucleus
and the hippocampus (Bayliss et al. 1997
;
Katayama et al. 1997
; Pan et al. 1993
;
Penington et al. 1993a
,b
). In the DLSN, however, the
5-HT-induced hyperpolarization seems to be voltage independent in
either normal artificial cerebrospinal fluid (ACSF) or high
K+ ACSF (Joëls and Gallagher
1988
; Joëls et al. 1986
, 1987
). Our preliminary data showed that the 5-HT-induced K+
current was accompanied by inward rectification in DLSN neurons (Yamada et al. 2000
). However,
Ba2+, a blocker for GIRK channels (North
1989
), produced only partial depression of the outward current
mediated by 5-HT1A receptors in DLSN neurons
(Yamada et al. 2000
). The purpose of the present study
is to characterize, in detail, the K+ conductance
underlying the 5-HT-induced outward current in DLSN neurons by using
whole cell patch-clamp methods.
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METHODS |
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Brain slices containing the septal nucleus were obtained from
rats in a manner described previously (Stevens et al.
1984). Male Wistar rats, 80-150 g, were killed by
decapitation, and their brains were rapidly removed and immersed for
8-10 s in cooled ACSF (4-6°C) that was prebubbled with 95%
O2-5% CO2. Transverse slices (400 µm in thickness) were cut with a Vibroslice (Campden Instruments) and left to recover for 1 h in oxygenated ACSF at room temperature (22-24°C). The slice was then transferred to a
recording chamber and submerged in ACSF at 32-33°C. The composition of the ACSF was as follows (in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaHPO4, and 11 D-glucose (pH 7.4 and 295-305 mOsm). Whole cell tight-seal
recordings were made from DLSN neurons using the slice patch technique
(Blanton et al. 1989
; Coleman and Miller
1989
). Patch pipettes were filled with the following internal
solution (mM): 122 K-gluconate, 5 NaCl, 0.3 CaCl2, 2 MgCl2, 1 ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA), 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 5 Na2ATP, and 2 GTP-Na (pH 7.2 adjusted by KOH, 280 mOsm). The tip resistance of the whole cell
patch-pipette was 4-5 M
. In some experiments, GTP was substituted
with 300 µM GTP
S and 5 mM
guanosine-5'-O-(2-thiodiphosphate) (GDP
S). Membrane
potential and current were recorded with an Axoclamp-2B amplifier.
During the whole cell voltage-clamping, sample frequencies were between
5 and 6 kHz and the amplifier gain was 0.8-2.5 nA/mV. Voltage and
current were monitored continuously with a memory oscilloscope
(Nihon-Kohden, RTA-1100). The pClamp system (Axon Instruments)
operating on an IBM-AX computer (Gateway 2000) was used to analyze the
membrane potentials and currents.
Of the drugs used, GTP, GTPS, GDP
S, N-ethylmaleimide
(NEM), tetrodotoxin (TTX), adenosine triphosphate (ATP) disodium salt, forskolin, phorbol 12-myristate 13-acetate (PMA), EGTA,
N6,2'-O-dibutyryladenosine
3':5'-cyclic monophosphate (db-cAMP), adenosine 3',5'-cyclic
monophosphothioate Rp-isomer (Rp-cAMPS), and glibenclamide were
purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO).
Tetraethylammonium (TEA) chloride was purchased from Tokyo Kasei.
(±)-8-Hydroxy-dipropylaminotetralin hydrobromide (8-OH-DPAT) was
purchased from RBI (Natick, MA). 5-HT creatinine sulfate complex was
from Wako Pure Chemical Ind. (Osaka, Japan). N-[2-((
-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89) was purchased from Calbiochem-Novabiochem (La Jolla, CA).
Protein kinase C (PKC) 19-36 was from Peninsula laboratories (Belmont,
CA). Glibenclamide was dissolved in dimethyl sulfoxide (DMSO) and added
to the ACSF, where the final concentration of DMSO (0.1%) had no
direct effect on DLSN neurons. Other drugs were directly dissolved in
the ACSF. Each experimental value was presented as the mean ± SE.
Differences between means were analyzed by Student's
t-test.
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RESULTS |
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5-HT causes an outward current in DLSN neurons
Neurons in the rat DLSN had resting membrane potential of
61 ± 4 mV (n = 62) and input resistance of
132 ± 11 M
(n = 62). DLSN neurons were
voltage-clamped with a whole cell configuration at
60 mV.
Bath-application of 5-HT (10 µM) caused an outward current
(I5-HT) in DLSN neurons (Fig.
1A). Membrane currents
produced by applying either ramp potentials or step command potentials with duration of 300 ms were increased by 5-HT, suggesting an increased
membrane conductance of DLSN neurons (Fig. 1A). Figure 1B shows the reversal potential of
I5-HT examined by changing the holding
membrane potential in a DLSN neuron (Fig. 1B).
I5-HT increased in amplitude at
depolarized membrane potentials, while it decreased when the membrane
was hyperpolarized and reversed its polarity at
86 mV (Fig.
1C,
). In this particular neuron, I5-HT appeared to be voltage
independent in the normal ACSF. In the same cell, the reversal
potential of I5-HT shifted to a
hyperpolarizing membrane potential in 1 mM K+
solution (Fig. 1C,
). An increase in the concentration of
external K+ to 9.7 and 20 mM shifted the reversal
potential of I5-HT to depolarized membrane potentials (Fig. 1C,
and
, respectively).
I5-HT exhibited weak inward
rectification in these high K+ solutions. Pooled
data showed that the reversal potential of I5-HT, measured in ACSF (containing
4.7 mM K+), was
93 ± 6 mV
(n = 8; Fig. 1D), which is close to the
equilibrium potential of K+ in DLSN neurons. In
ACSF containing 1, 9.7, and 20 mM K+, the
reversal potentials of I5-HT were
123 ± 5 mV (n = 6),
73 ± 4 mV
(n = 4), and
53 ± 4 mV (n = 6),
respectively. The relationship between the reversal potential of
I5-HT and the concentration of
extracellular K+ had a slope of 54 mV for 10-fold
change in external K+ concentration (Fig.
1D). This is identical to the expected value of the
equilibrium potential for K+ by the Nernst
equation.
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Properties of the outward current induced by 5-HT
The voltage dependency of the K+ conductance
underlying I5-HT was investigated, in
detail, in DLSN neurons. Figure
2A shows a sample record of
I5-HT obtained from a DLSN neuron.
The current-voltage relationship (I-V curve) constructed
by step command potentials increased its slope in the presence of 5-HT
(Fig. 2B). The component of current activated by 5-HT (net
I5-HT) was obtained by digital subtraction of the control I-V curve from that recorded in
the presence of 5-HT (10 µM). The net
I5-HT showed no obvious rectification in this particular neuron (Fig. 2C).
I5-HT with linear I-V
relation was seen in 61 (63%) of 97 DLSN neurons, where the amplitude
of I5-HT was 108 ± 5 pA
(n = 40) at the holding membrane potential of 60 mV.
Ba2+, at a micromolar concentration, has been
reported to block selectively the inward rectifier
K+ current in various central neurons
(North 1989
). Figure 2D shows the effect of
Ba2+ (100 µM) on
I5-HT with linear I-V
relation in a DLSN neuron. In this neuron, Ba2+
(100 µM) markedly depressed I5-HT at
potentials more negative than
100 mV. However, the depression was
less marked at depolarized membrane potentials.
I5-HT that remained in the presence of
Ba2+ (100 µM) exhibited outward rectification
(Fig. 2Da,
). In contrast, the
Ba2+-sensitive
I5-HT exhibited inward rectification
(Fig. 2Db). The pooled data show that
Ba2+ (100 µM) decreased the amplitude of
I5-HT from 135 ± 8 pA
(n = 8) to 32 ± 4 pA (n = 8) at
130 mV (Fig. 2E, P < 0.01). At
50 mV,
the amplitudes of I5-HT were 121 ± 6 pA (n = 8) and 112 ± 6 pA (n = 8) in the absence and the presence of Ba2+ (100 µM), respectively. This difference was statistically not significant.
These results suggest that I5-HT with
linear I-V relation is composed of a
Ba2+-sensitive, inward rectifier and a
Ba2+-insensitive, outward rectifier
K+ currents in DLSN neurons.
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Effects of Ba2+ on outward and inward rectifier currents
In 16 (16%) of 97 neurons, the application of 5-HT increased the
membrane conductance at potentials more positive than 70 mV (Fig.
3Aa).
I5-HT reversed polarity at
83 mV in
this neuron (Fig. 3Aa). The net
I5-HT obtained by subtraction of the
control I-V curve from that recorded in the presence of 5-HT
(10 µM) showed clear outward rectification (Fig. 3Ab).
Figure 3B shows the effect of Ba2+
(100 µM) on the outward rectifier
I5-HT in a DLSN neuron.
Ba2+ (100 µM) caused no obvious depression of
the I5-HT. At
50 mV, the amplitudes
of I5-HT were 91 ± 7 pA
(n = 6) and 80 ± 6 pA (n = 6) in
the absence and the presence of Ba2+ (100 µM),
respectively. At
130 mV, the amplitudes of
I5-HT were
29 ± 4 pA
(n = 5) and
24 ± 3 pA (n = 5)
in the absence and the presence of Ba2+ (100 µM), respectively. I5-HT having
characteristic inward rectification was seen in 20 (21%) cells out of
a total of 97 neurons in the rat DLSN (Fig. 3C). The
amplitude of the inward rectifier
I5-HT was 42 ± 5 pA
(n = 8) at
60 mV. Ba2+ (100 µM) preferentially depressed I5-HT
at hyperpolarized membrane potentials. Pooled data showed that the
amplitude of I5-HT was depressed from
52 ± 6 pA (n = 5) to 44 ± 8 pA
(n = 5) by Ba2+ (100 µM) at
50 mV. There was no statistically significant difference between
these two data. When recorded at
130 mV, the amplitude of
I5-HT was depressed from
151 ± 7 pA (n = 5) to
23 ± 5 pA (n = 5) in the presence of Ba2+ (100 µM; Fig.
3D). Ba2+-sensitive inward rectifier
I5-HT has been shown previously in LSN
neurons (Yamada et al. 2000
).
|
We examined the effects of other K+ channel
blockers on I5-HT in DLSN neurons.
5-HT (100 µM) produced outward current with amplitude of 121 ± 5 pA (n = 6) at a potential of 60 mV in ACSF containing 1 µM TTX, 0 mM Ca2+ (with 2 mM
EGTA), Cs+ (2 mM), and 20 mM TEA. Glibenclamide
(100 µM), a blocker of the ATP-regulated K+
channel, and extracellular Cs+ (1 mM), a blocker
of nonselective cation channels (IQ)
(Halliwell and Adams 1982
) and
Ih (Bobker and Williams
1989
), did not depress the
I5-HT in DLSN neurons
(n = 4). It has been reported that the M current is an
outward rectifier K+ current that is controlled
by muscarinic receptors in central and peripheral neurons (Brown
1990
). However, hyperpolarizing command potentials (from
40
to
80 mV) did not produce the time-dependent current relaxation that
is the characteristic feature of the M current in either the presence
or the absence of 5-HT (100 µM).
Effects of 8-OH-DPAT on the membrane current in DLSN neurons
8-OH-DPAT (30 µM), a selective agonist for the
5-HT1A receptor (Cervo and Samanin
1987; Kennett et al. 1987
), also produced an
outward current in DLSN neurons (Fig.
4A). The onset and recovery of
the 8-OH-DPAT-induced outward current were slower than those of
I5-HT. The mean amplitude of 8-OH-DPAT
(30 µM)-induced outward current was 58 ± 8 pA
(n = 20) at
60 mV. The outward current induced by
8-OH-DPAT (30 µM) also showed different voltage dependency in
individual neurons (Fig. 4B). 8-OH-DPAT-induced current was associated with inward rectification in 6 neurons, outward
rectification in 4 neurons, and linear I-V relation in 14 neurons of a total of 24 neurons. The proportion of the three types was
similar as in the case of 5-HT.
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G protein mediates I5-HT in DLSN neurons
To inhibit the activity of G-protein in DLSN neurons, the
patch-pipette solution contained GTPS (300 µM) or GDP
S (5 mM) instead of GTP (300 µM). First, we confirmed that 5-HT consistently produced the outward current for at least 80 min under the whole cell
patch-clamp recording in intact neurons (Fig.
5B). The first application of
5-HT (50 µM) produced a typical outward current with amplitude of 120 pA at
60 mV in a DLSN neuron treated with GTP
S (300 µM) for 5 min (Fig. 5Aa). When 5-HT was applied again 10 min after
first application, I5-HT did not
completely recover but gradually shifted in the outward direction even
after removal of 5-HT from the ACSF (Fig. 5Ab) probably
because of continuous diffusion of GTP
S into the intracellular
space. I5-HT decreased in amplitude by
more than 85% of control 30 min after the first application of 5-HT
(Fig. 5Ad). Intracellular application of GTP
S for 40 min
irreversibly depressed I5-HT (Fig.
5B). Figure 5B also shows the effect of GDP
S
on the I5-HT in DLSN neurons.
Intracellular application of GDP
S (5 mM) for 60 min via the patch
pipette strongly depressed the amplitude of
I5-HT. No recovery of
I5-HT was seen as long as whole cell
recording was continued. It has been reported that NEM, a sulfhydryl
alkylating agent, uncouples the G-protein-coupled receptor from the
pertussis toxin (PTX)-sensitive G proteins (Gi and/or Go)
(Nakajima et al. 1990
; Shapiro et al.
1994
). Bath-application of NEM (100 µM) for 5 min depressed
I5-HT by 81 ± 5%
(n = 4) at a holding potential of
60 mV (not shown).
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Effect of protein kinase inhibitors on I5-HT in DLSN neurons
To study the contribution of PKC to
I5-HT, DLSN neurons were dialyzed with
an internal solution containing PKC 19-36 (20 µM), a specific peptide
inhibitor for PKC that is a pseudosubstrate peptide in the regulatory
domain of PKC (House and Kemp 1987). Figure
6A shows
I5-HT taken 5 min after dialysis of a
DLSN neuron with a pipette solution containing PKC 19-36 (20 µM).
5-HT (10 µM) increased the membrane conductance at all membrane
potentials tested. The net I5-HT
obtained by subtraction of the control I-V curve from that
taken in the presence of 5-HT (10 µM) showed a linear I-V
relation in this neuron (Fig. 6Ab). The net
I5-HT was also recorded in the same
neuron internally dialyzed with PKC 19-36 for 20 min (Fig.
6B). 5-HT (10 µM) produced an inward rectifier K+ current in this neuron at this time. The
PKC-sensitive I5-HT, obtained by
subtraction of the control I5-HT from
that recorded after 20 min of dialysis with PKC 19-36, exhibited
outward rectification (Fig. 6C). Statistical data showed
that PKC 19-36 (20 µM) reduced the amplitude of
I5-HT with linear I-V
relation from 148 ± 7 (n = 5) to 61 ± 4 pA
(n = 5) at
40 mV (Fig.
6D, P < 0.01). In contrast, the amplitudes of linear
I5-HT were
163 ± 8 (n = 5) and
158 ± 11 pA (n = 5)
in the absence and the presence of PKC 19-36 (20 µM), respectively,
at
130 mV (Fig. 6D); there was no statistically significance between these two sets of data.
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|
Figure 7A shows the effects of PKC 19-36 (20 µM) on
the outward rectifier I5-HT in a DLSN
neuron. When 5-HT (10 µM) was applied to the bath solution 5 min
after the beginning of whole cell recording, a typical outward current
with amplitude of 132 ± 6 pA (n = 5) at 40 mV
(Fig. 7Aa,
). However, in LSN neurons internally treated with PKC 19-36 (20 µM) for 20 min, 5-HT (10 µM) produced only a
23 ± 2 pA (n = 5) at
40 mV (Fig.
7Aa,
). Thus PKC 19-36 (20 µM) in the pipette solution
depressed I5-HT by about 81%
(n = 5) at a potential
40 mV. The PKC 19-36-sensitive
current of I5-HT showed outward
rectification (Fig. 7Ab). In contrast, at the holding potential of
130 mV, amplitudes of
I5-HT were
41 ± 6 pA
(n = 5) and
36 ± 8 pA (n = 5)
when recorded 5 and 20 min after the application of PKC 19-36, respectively. The difference between these two sets of data were
statistically not significant. The effect of PKC 19-36 on the inward
rectifier I5-HT was also examined in a
DLSN neuron (Fig. 7B). PKC 19-36 did not significantly
depress the inward rectifier I5-HT
(Fig. 7B, a and b). Pooled data from five neurons
showed that amplitudes of I5-HT at
40 and
130 mV were 194 ± 11 and 175 ± 13 pA in the
presence and the absence of PKC 19-36 (20 µM), respectively. At
130
mV, they were 50 ± 4 (n = 5) and 47 ± 5 pA
(n = 5) in the absence and the presence of PKC 19-36 (20 µM). There were statistically no significance between two sets of
data taken at potentials of either
40 or
130 mV (Fig.
7Bc). These results suggest that PKC 19-36 preferentially depresses the outward rectifier I5-HT.
The effects of protein kinase A (PKA) inhibitors on
I5-HT were examined in DLSN neurons.
H-89 (10 µM), a membrane permeable and selective inhibitor of PKA
(Chijiwa et al. 1990), was applied to the ACSF for
10-20 min. H-89 (10 µM) did not significantly depress the
5-HT-induced outward current in DLSN neurons (n = 4).
Rp-cAMPS (300 µM), a membrane permeable cAMP analogue that is known
to be a PKA inhibitor, also produced no significant depression of the
amplitude of I5-HT (n = 4). Both inward and outward rectifier I5-HT were not changed by H-89 and
Rp-cAMPS (n = 5).
Effects of protein kinase activators on the membrane current in DLSN neurons
The results obtained with PKC 19-36 strongly suggest the PKC
mediated the activation of the outward rectifier by 5-HT. It may be
expected, therefore that a PKC activator would turn on a K current with
similar voltage dependence. Therefore the effect of PMA, an activator
of PKC, on the membrane current was examined in DLSN neurons (Fig.
8). PMA (10 µM) was applied to the
intracellular space of DLSN neurons through a patch pipette.
Immediately after completion of the whole cell patch-clamp recording,
bath-application of 5-HT (10 µM) produced an outward current with
linear I-V relation (Fig. 8Ab). In the same cell,
intracellular application of PMA (10 µM) for 20 min produced an
outward current with amplitude of 75 ± 5 pA (n = 6) at 60 mV. The PMA-induced current reversed polarity at
84 ± 5 mV (n = 5; Fig. 8Bb). Amplitudes of the
PMA-induced current recorded at
40 and
130 mV were 134 ± 6 (n = 6) and
23 ± 7 pA (n = 6),
respectively (Fig. 8C). These results indicate that PKC
produces outward rectifier K+ current in DLSN
neurons. By contrast, forskolin (10 µM), an activator of PKA, and
db-cAMP (200 µM) did not produce visible outward current in DLSN
neurons (n = 8).
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DISCUSSION |
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Properties of K+ conductance underlying I5-HT
The present study showed that 5-HT produced an outward current
(I5-HT) associated with an increase in
the membrane conductance of DLSN neurons.
I5-HT reversed polarity at a holding
potential of 93 ± 6 mV, which is close to the equilibrium
potential for K+ in ACSF containing 4.7 mM
K+. The reversal potential of
I5-HT changed by 54 mV per decade change in the external K+ concentration as
predicted by the Nernst equation. These results indicate that
I5-HT is carried exclusively by
K+. It has been shown that the 5-HT-induced
hyperpolarization was not obviously voltage dependent in either normal
ACSF or high K+ solution in DLSN neurons
(Joëls and Gallagher 1988
; Joëls et al. 1986
, 1987
). Our preliminary report showed that although
5-HT produced an inward rectifier K+ current in
some DLSN neurons, Ba2+ (100 µM) did not
completely block the I5-HT
(Yamada et al. 2000
). The present study showed three
types of I5-HT in terms of their voltage dependencies. In 63% of neurons,
I5-HT was associated with linear
I-V relation. I5-HT with
characteristic inward rectification was seen in 21% of the neurons
tested. In remaining 16% of neurons, I5-HT exhibited outward rectification.
Ba2+ (100 µM) almost completely depress
I5-HT of the inward rectifier type.
Such a Ba2+-sensitive inward rectifier
I5-HT is probably identical to that described in raphe nuclei neurons (Bayliss et al. 1997
;
Katayama et al. 1997
; Pan et al. 1993
;
Penington et al. 1983a
,b
). In contrast, Ba2+ (100 µM) did not significantly affect the
outward rectifier I5-HT. In
I5-HT with linear I-V
relation, Ba2+ preferentially depressed the 5-HT
current at hyperpolarizing membrane potentials. As the results, the
Ba2+-resistant component of linear type
I5-HT clearly showed outward rectification activated at potentials more positive than
80 mV. These
results suggest that the I5-HT with
linear I-V relation is the sum of these two component
currents. Previously, we have reported that
5-HT1A receptors are responsible for the outward current produced by 5-HT in DLSN neurons (Yamada et al.
2000
). In the present study, 8-OH-DPAT, a
5-HT1A receptor agonist, produced outward
currents associated with inward rectification, outward rectification,
and linear I-V relation in DLSN neurons. However, further
studies are needed to clarify the subtype of 5-HT receptors that
mediate to both inward and outward rectifying K+
currents in DLSN neurons.
The delayed rectifier K+ current and the
Ca2+-activated K+ current
do not seem to be involved in I5-HT
because 5-HT caused the outward current in an external solution
containing 20 mM TEA and 0 mM Ca2+.
Joëls et al. (1987) have reported that the
5-HT-induced hyperpolarization is not sensitive to
Ca2+ in DLSN neurons.
I5-HT was not blocked by glibenclamide
(100 µM), indicating that the ATP-sensitive K+
current (Schmid-Antomarchi et al. 1987
; Sturgess
et al. 1985
) is not involved in
I5-HT. It has been shown that the M
current is a time-dependent outward rectifier K+
current activated by acetylcholine via muscarinic receptors in autonomic and central neurons (Brown 1990
). The M
channel may not be involved in the outward rectifier current produced
by 5-HT in DLSN neurons because I5-HT
was not associated with the time-dependent relaxation that is the
characteristic feature of the M current at depolarized membrane potentials.
Signal transduction of I5-HT
Molecular cloning has established that
5-HT1A receptors belong to the superfamily of
G-protein-coupled receptors (Albert et al. 1990).
5-HT1A receptors couple to inward rectifier
K+ channels via a PTX-sensitive G protein in
neurons of the dorsal raphe nucleus and the hippocampus (Bayliss
et al. 1997
; Bobker and Williams 1989
;
Katayama et al. 1997
; Penington et al.
1993a
; Sim et al. 1997
). The present study
showed that intracellular application of GTP
S and GDP
S
irreversibly and almost completely suppressed
I5-HT in DLSN neurons. NEM, an
uncoupler of receptors from the PTX-sensitive G protein (Asano
and Ogasawara 1986
; Nakajima et al. 1990
;
Shapiro et al. 1994
), also depressed
I5-HT in DLSN neurons. These results
suggest that a PTX-sensitive G protein mediates
I5-HT in DLSN neurons. Previous
studies have shown that a soluble second messenger is not required for
the G-protein-mediated effect of 5-HT in activating inwardly rectifying
K+ channels in dorsal raphe neurons
(Katayama et al. 1997
; Penington et al.
1993b
). 5-HT activation of inward rectifier
K+ channels in hippocampal neurons in inside-out
patches appeared to be directly mediated by G
(Oh et al.
1995
).
Biochemical assays have demonstrated that the rat
5-HT1A receptor inhibits both basal and
stimulated cyclic AMP accumulation by forskolin (Fargin et al.
1989; Raymond et al. 1989
). In DLSN neurons,
however, db-cAMP and forskolin produced no obvious effect on the
membrane current in DLSN neurons. The PKA inhibitors H-89 and Rp-cAMPS
did not significantly reduce I5-HT
with properties of either inward or outward rectification. These
results suggest that PKA is not involved in the pathway that mediates
I5-HT in DLSN neurons. In addition to
the inhibitory effect on adenylate cyclase, 5-HT, at micromolar
concentration, stimulates phospholipase C activity in Hela cells with
permanently expressed 5-HT1A receptors. This
effect did not appear to be secondary to an inhibition of adenylate
cyclase, and 5-HT1A receptors can stimulate
phosphatidylinositol hydrolysis, resulting in the activation of PKC in
Hela cell (Fargin et al. 1989
; Raymond et al.
1989
). 5-HT1A receptors may be capable of
coupling to multiple G-protein-associated effector systems in a single
cell. The present study showed that PKC 19-36 preferentially depressed
I5-HT with properties of the outward
rectifier K+ conductance, while it did not
significantly affect the inward rectifier
I5-HT in DLSN neurons. PMA, an
activator of PKC, produced outward rectifier K+
current in DLSN neurons. We concluded that a PTX-sensitive G protein
directly mediates the 5-HT-induced inward rectifier
K+ current, while a soluble second messenger,
such as PKC, may mediate the 5-HT1A
receptor-activated outward rectifier K+ current
in DLSN neurons.
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ACKNOWLEDGMENTS |
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Most of this study was supported by The Ishibashi Research Fund and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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FOOTNOTES |
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Address for reprint requests: T. Akasu, Dept. of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan (E-mail: akasut{at}med.kurume-u.ac.jp).
Received 24 August 2000; accepted in final form 18 December 2000.
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REFERENCES |
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