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The Journal of Neuroscience, May 15, 1999, 19(10):3739-3751
Roles of G-Protein 
, Arachidonic Acid, and Phosphorylation
in Convergent Activation of an S-Like Potassium Conductance by
Dopamine, Ala-Pro-Gly-Trp-NH2, and
Phe-Met-Arg-Phe-NH2
Hind
van Tol-Steye1, 2,
Johannes C.
Lodder1,
Huibert D.
Mansvelder1,
Rudi J.
Planta2,
Harm
van Heerikhuizen2, and
Karel S.
Kits1
Departments of 1 Neurophysiology, Research Institute
Neurosciences, and 2 Biochemistry and Molecular Biology,
Faculty of Chemistry, Vrije Universiteit, 1081 HV Amsterdam, The
Netherlands
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ABSTRACT |
Dopamine and the neuropeptides Ala-Pro-Gly-Trp-NH2
(APGWamide or APGWa) and Phe-Met-Arg-Phe-NH2 (FMRFamide or
FMRFa) all activate an S-like potassium channel in the light
green cells of the mollusc Lymnaea stagnalis,
neuroendocrine cells that release insulin-related peptides. We studied
the signaling pathways underlying the responses, the role of the
G-protein 
subunit, and the interference by phosphorylation
pathways. All responses are blocked by an inhibitor of arachidonic acid
(AA) release, 4-bromophenacylbromide, and by inhibitors of
lipoxygenases (nordihydroguaiaretic acid and AA-861) but not by
indomethacin, a cyclooxygenase inhibitor. AA and phospholipase
A2 (PLA2) induced currents with similar
I-V characteristics and potassium selectivity as
dopamine, APGWa, and FMRFa. PLA2 occluded the response to
FMRFa. We conclude that convergence of the actions of dopamine, APGWa,
and FMRFa onto the S-like channel occurs at or upstream of the level of
AA and that formation of lipoxygenase metabolites of AA is necessary to
activate the channel. Injection of a synthetic peptide, which interferes with G-protein subunits, inhibited the
agonist-induced potassium current. This suggests that subunits
mediate the response, possibly by directly coupling to a phospholipase.
Finally, the responses to dopamine, APGWa, and FMRFa were inhibited by activation of PKA and PKC, suggesting that the responses are
counteracted by PKA- and PKC-dependent phosphorylation. The
PLA2-activated potassium current was inhibited by
8-chlorophenylthio-cAMP but not by 12-O-tetradecanoylphorbol 13-acetate
(TPA). However, TPA did inhibit the potassium current induced by
irreversible activation of the G-protein using GTP--S. Thus, it
appears that PKA targets a site downstream of AA formation, e.g., the
potassium channel, whereas PKC acts at the active G-protein or the phospholipase.
Key words:
FMRFamide; dopamine; neuropeptide; K+-current; S-current; molluscs; arachidonic acid; G-protein subunits; neuron; signal transduction; convergence
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INTRODUCTION |
G-protein-mediated activation of
K+ channels involves either direct interaction of
G-proteins with the channels [so-called G-protein-gated inward
rectifier K+ channels (GIRKs); Wickman and Clapham,
1995
] or intervention of an enzyme system and second messengers. The
latter route has been well documented in molluscan systems. Thus,
Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa)-induced
activation of S-channels in sensory cells of Aplysia
(Piomelli et al., 1987a; Belardetti et al., 1989; Buttner et al.,
1989), an S-like current in neuron B5 of Helisoma (Bahls et
al., 1992) and an S-like but voltage-dependent
K+-current in the caudodorsal cells of
Lymnaea (Kits et al., 1997), involves lipoxygenase
metabolites of arachidonic acid (AA). However, other studies suggested
AA-independent activation of K+ channels. Examples
are the S-current activated by the peptide myomodulin in
Aplysia (Critz et al., 1991) and a
K+-conductance activated by dopamine, glutamate, and
a muscarinic agonist in Planorbarius (Bolshakov et al.,
1993).
Previously, we described the convergent activation of an S-like
K+ channel by the classical transmitter dopamine and
the neuropeptides Ala-Pro-Gly-Trp-NH2 (APGWamide or
APGWa) and FMRFa in the light green cells (LGCs) of
Lymnaea stagnalis (van Tol-Steye et al., 1997), a set of
growth-regulating neurons that produce insulin-related peptides
(Geraerts, 1976; Geraerts et al., 1991; Smit et al., 1988). The
responses are G-protein-dependent, but the signaling pathway has not
been elucidated. Here, we focus on three main questions. (1) What is
the signal transduction pathway underlying the convergent activation of
the K+ channel by dopamine and APGWa and FMRFa?
Convergence may occur at any site between the receptors and the
channel, implying that the agonists may use completely different or
identical pathways. To solve this, we used pharmacological tools to
interfere with signal transduction. (2) Which G-protein subunit, 
or
, is the active mediator in these responses? Unlike for GIRKs in
which G subunits induce the response (Clapham and Neer, 1997;
Isomoto et al., 1997), for receptor-operated K+
channels in molluscs (Sasaki and Sato, 1987; Volterra and Siegelbaum, 1988; Bolshakov et al., 1993), it is unclear whether G or subunits are the active mediators. To address this problem, we used a
peptide that specifically interacts with -mediated signaling (Koch et al., 1993; Macrez et al., 1997). (3) Do modulatory pathways interact with the K+ current response? In LGCs,
responses to dopamine are inhibited by cAMP. This resembles the
situation in S channels in which protein kinase A (PKA)-dependent
phosphorylation shuts down the channels (Shuster et al., 1985).
Therefore, we studied for all three messengers the role of
phosphorylation by PKA and protein kinase C (PKC) in modulation of the
current responses. Also, we asked which sites in the signaling route
are targeted by PKA and PKC by studying the effect of PKA and PKC
activation on responses activated downstream of the receptors.
We provide evidence that channel activation by all three agonists
involves lipoxygenase metabolism of AA, implying that not only FMRFa
but also dopamine and APGWa, couple to the AA pathway. G subunits
appear to mediate the response and presumably activate the
phospholipase catalyzing AA formation. The AA pathway is modulated by
phosphorylation through PKA and PKC, with each kinase apparently targeting a separate site in the signaling route.
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MATERIALS AND METHODS |
Animals and cells. Adult pond snails (Lymnaea
stagnalis), bred in the laboratory under standard conditions (van
der Steen et al., 1969), were used. LGCs were dissociated mechanically
from the CNS after 45 min incubation at 37°C in HEPES-buffered
saline (HBS) (see below) supplemented with 0.2% trypsin (type III;
Sigma, St. Louis, MO). Dissociated cells were plated in culture dishes (Costar, Cambridge, MA) containing HBS and were left undisturbed for at
least 1 hr to allow the cells to attach to the bottom of the dish. In
all experiments, isolated cells (diameter of ~60 µm) without
neurites were used. Cells were always used within 7 hr after isolation.
Solutions and chemicals. CNSs and isolated cells were bathed
in HBS containing (in mM): NaCl 30, NaCH3SO4 10, NaHCO3 5, KCl 1.7, CaCl2 4, MgCl2 1.5, and HEPES 10, pH 7.8 adjusted with NaOH. The same buffer was used as extracellular solution
during electrophysiological recordings, unless otherwise specified.
Extracellular high-K+ solution contained 20 mM KCl and 10 mM NaCl. For the remainder, it
was identical to HBS. Internal pipette solution contained (in mM): KCl 29, ATPMg 2, GTP (Tris-salt) 0.1, HEPES 10, EGTA
11, and CaCl2 2.3, pH 7.4 adjusted with KOH.
Solutions of agonists and all other drugs were freshly prepared before
use by dilution from stock solutions into extracellular or
intracellular medium. The following agents were prepared as stocks in
DMSO: 4-bromophenacylbromide (4-BPB), nordihydroguaiaretic acid (NDGA),
indomethacin, and 8-chlorophenylthio-cAMP (8-cpt-cAMP) (all obtained
from Sigma); 12-O-tetradecanoylphorbol 13-acetate [(TPA) also referred
to as phorbol 12-myristate 13-acetate (PMA)], 4--phorbol
12-myristate 13-acetate (4-PMA), staurosporin, and okadaic
acid (all obtained from Research Biochemicals, Natick, MA); AA (Sigma
and Research Biochemicals); and 1-(2-nitrophenyl)ethyl cAMP (NPE-caged
cAMP) (Molecular Probes, Eugene, OR). AA-861 stock solutions (BIOMOL">Biomol,
Plymouth Meeting, PA) were prepared in ethanol. Stocks of
3,4-dihydroxyphenylethylamine hydrochloride (dopamine) (Sigma),
APGWamide (American Peptide Co., Sunnyvale, CA), FMRFamide (Peninsula
Laboratories Inc., Belmont, CA), phospholipase A2
(PLA2) from bee venom (Sigma), and
-interacting peptide (peptide G) (Isogen Bioscience BV, Maarssen,
The Netherlands) were prepared in distilled water. Stocks of cAMP
(Boehringer Mannheim, Mannheim, Germany) were prepared in distilled
water containing 2 mM KOH. cAMP-dependent protein kinase
inhibitor 5-24 amide (wiptide) (Peninsula Laboratories Inc.)
was directly dissolved in pipette medium. Peptide G is an N-acylated
and C-amidated 28-mer: WKKELRDAYREAQQLVQRVPKMKNKPRS, corresponding to
amino acids 643-670 from the -binding sequence of -adrenergic
receptor kinase 1 (ARK1) (Koch et al., 1993). AA-containing
solutions were always prepared under N2 and sonicated before use. Dopamine, APGWa, FMRFa, 8-cpt-cAMP, staurosporin, AA, and
PLA2 were applied to the cell by means of a gravity-driven Y-tube system described previously (Kits et al., 1997) or by means of a
Picospritzer (General Valve, Fairfield, NJ). The vital dye amaranth
(0.01%) (Merck, Darmstadt, Germany) was added to visualize the
application. Because of the continuous perfusion of the culture dish with extracellular solution, applied agents were washed away within seconds after application was stopped. Okadaic acid and NPE-caged cAMP were applied intracellularly via the patch electrode. Peptide G was administered by microinjection. All other solutions were
administered via the pump-driven bath perfusion system, unless stated
otherwise. Bath medium was completely replaced within 5 min.
Transmitters were dissolved in the same solution as used for perfusion.
Recordings were always started in standard extracellular solution. When
high potassium or drugs were bath-perfused during an experiment,
subsequent experiments were performed on a fresh dish of cells.
Electrophysiological recordings and microinjections.
Whole-cell voltage-clamp recordings were made with an Axoclamp-2B or Axopatch-200B amplifier (Axon Instruments, Foster City, CA).
Voltage-clamp electrodes (2-4 M
) were pulled from Clark GC-150T or
GC-150 glass (Clark Electromedical Instruments, Reading, UK). Seal
resistance was 
1 G; after disruption of the patch membrane, series
resistance (<5 M) was compensated for 70%. Sharp injection
electrodes were pulled from Clark GC-150F glass; resistance was 15-20
M when filled with standard pipette medium. After impalement of the
cell, which was confirmed by monitoring the membrane potential using a
custom-built microelectrode amplifier, injection (
1% of the cell
volume) was accomplished under visual control, using a Picospritzer.
Data-acquisition was controlled by a CED 1401 (Cambridge Electronics
Design, Cambridge, UK) or a Digidata 1200 (Axon Instruments) analog-to-digital converter connected to a personal computer. Voltage-clamp software developed in our laboratory, as well as pClamp
(Axon Instruments), was used. Current recordings were filtered at 100 Hz, sampled at >200 Hz, and stored on disk. This system allowed
simultaneous control of applied voltage protocols, data acquisition,
and drug application.
Cells were kept at a holding potential of 
80 mV. Before application
of agonist, a voltage step to 40 mV was made, unless stated
otherwise, to enhance the driving force for potassium. I-V
relationships were determined using ramp protocols, imposing linear
voltage ramps (slope of 34 mV/sec; duration of 5 sec). Current
responses to ramps under control conditions (no agonist present) were
subtracted from those measured after application (10-40 sec) of
agonist to obtain the current-voltage relationship of the
agonist-induced current.
Error bars indicate SEM.
Flash experiments. Uncaging of NPE-caged cAMP was
accomplished using a 35 S mercury arch Flashtube and Strobex model 238 power pack (Chadwick-Helmuth, El Monte, CA), delivering 200 J per
flash. For these experiments, the bottoms of the Costar culture dishes, normally used to plate the cells, were replaced by glass coverslips, and an Axiovert 35 microscope with 40× plan-neofluar objective (both
from Zeiss, Jena, Germany) was used.
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RESULTS |
Arachidonic acid pathway
Previously, we have shown that the transmitter dopamine and the
neuropeptides APGWa and FMRFa activate a single type of potassium channel in the neuroendocrine LGCs of the pond snail Lymnaea
stagnalis (van Tol-Steye et al., 1997). To assess whether AA is
involved in activation of this potassium channel, we tested the effect of 4-BPB. 4-BPB irreversibly blocks the formation of AA by
inhibiting phospholipase A2 activity or by inhibiting the
combined action of phospholipase C (PLC) and diacylglycerol
lipase (Blackwell and Flower, 1983; Piomelli et al., 1987a). The
current responses to all three transmitters were blocked for 90% by
10 µM 4-BPB (Fig.
1). The inhibition was obtained
within 10-20 min.
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Figure 1.
4-BPB inhibits the outward current induced by
dopamine, APGWa, and FMRFa. For each agonist, the control response and
the strongly reduced response in the presence of 4-BPB are shown.
Responses were evoked by 2 sec applications of agonist by means of
Y-tube. Graphs are plots of current amplitudes
(I), normalized to the current response at
t = 0 (I0), against time.
Arrows indicate the start of bath perfusion with 0.01%
DMSO in standard medium (control, open diamonds;
n = 3 for each agonist) or with 10 µM
4-BPB (filled diamonds; n = 3 for each agonist; n indicates number of cells tested per
agonist). SEMs are indicated only by upward error bars. Concentrations
used were as follows: dopamine, 1 µM; APGWa, 7 µM; and FMRFa, 0.1 µM.
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Whereas 4-BPB inhibited the responses, AA (5-50 µM)
mimicked the agonist-induced effects on the LGCs (Figs.
2A,
3).
However, the AA-evoked responses were rather variable in intensity and stability after repeated applications (2-20 sec applications, once per
minute). In some cases, AA application failed to induce a response.
Although we prepared all AA-containing solutions under N2
and sonicated solutions before use, these problems could well be
attributable to the fact that AA is poorly soluble in aqueous solutions, tends to stick to glassware and tubing (Yamada et al., 1994), and is prone to oxidation. In line with this explanation, we
found that a given solution was either effective or ineffective in all
cells tested. To consolidate our observation that AA is capable of
mimicking the effect of dopamine, APGWa, and FMRFa on the LGCs, we
stimulated AA formation in the cell by application of PLA2.
Extracellular application of PLA2 (50-122.5 U/ml,
corresponding to micromolar range) consistently induced a slow
outward current similar to that evoked by the three agonists (Figs.
2A, 3). Figure 2A shows examples of
the AA- and PLA2-induced responses and the dopamine-induced
potassium current.
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Figure 2.
PLA2 and AA are involved in the
signaling route activated by the agonists. A, AA and
PLA2 induce an outward current, similar to that induced by
dopamine. Bars indicate extracellular applications of AA (5 sec, 50 µM), PLA2 (20 sec, 122.5 U/ml), and dopamine
(1 sec, 1 µM). The cells were voltage clamped at 40 mV.
Responses are from different cells. B, C,
FMRFa occludes the response to PLA2. B,
Combined application of FMRFa and PLA2 fails to show
summation of the responses to FMRFa and PLA2. Average
amplitudes (right) of the responses to a maximal dose of
FMRFa (10 µM) alone and to the subsequently applied
combination of FMRFa and PLA2 (50 U/ml)
(n = 4). C, Application of FMRFa
during a response to PLA2 increases the current amplitude
up to the level of the response to FMRFa alone. The right
panel shows the average current amplitudes to FMRFa alone and
to PLA2 alone compared with the response amplitude obtained
when FMRFa was applied during a PLA2 response
(n = 4).
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Figure 3.
AA and PLA2 induce a current with
similar I-V characteristics as the current induced by
dopamine, both in standard and high extracellular
[K+]o. A,
I-V relationships of the current induced by AA (50 µM), PLA2 (50-122.5 U/ml), or dopamine (1 µM) were determined with the use of linear voltage ramp
protocols (34 mV/sec) as described in Materials and Methods.
Left panels, Standard extracellular potassium (1.7 mM; n = 3, n = 4, n = 7 for AA, PLA2, and
dopamine, respectively). Right panels, High
extracellular potassium (20 mM; n = 2, n = 4, n = 2 for AA,
PLA2, and dopamine, respectively). B,
Curves from A were normalized with respect to the
current at 40 mV (1.7 mM K+) or to the
peak inward current amplitude (20 mM
K+).
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Figure 4.
Inhibitors of phospholipase and lipoxygenase
reduce the responses to dopamine, APGWa, and FMRFa. A,
Percentage reduction of the potassium current elicited by dopamine,
APGWa, and FMRFa (top, middle, and
bottom, respectively) after perfusion of the cells with
standard medium (control), 0.01% DMSO-containing
medium (DMSO), the inhib itor of AA-release 4-BPB (10 µM), the
lipoxygenase inhibitor NDGA (15 µM), and the
cyclooxygenase inhibitor indomethacin (Indom; 5 µM). Asterisks indicate significant
difference with respect to the DMSO control (unpaired t
test; p < 0.01). Apart from controls in which
n = 6, experimental groups were
n = 3 for each agonist. B, NDGA also
blocks the current induced by bee venom-derived
PLA2, indicating that exogenous PLA2
acts specifically to produce AA. Shown are the average response
amplitudes to applications of PLA2 (50 U/ml) in the absence
and presence of 15 µM NDGA (n = 11).
NDGA was applied for 10 min before PLA2 was tested.
Inset, Superimposed traces showing a control response to
PLA2 and the strong reduction of this response by NDGA
(responses from the same cell). *p < 0.01;
paired t test.
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If direct application of AA or PLA2 activates the same
channels as the agonists, there should be occlusion of the agonist responses and the PLA2-evoked response. To test this, we
used a saturating dose of FMRFa (10 µM) to evoke the
agonist-induced response because it was shown previously that FMRFa
maximally activates the population of K+ channels
involved and completely occludes the responses to the other agonists
(van Tol-Steye et al., 1997). This response was compared with the
response to combined application of PLA2 (50 U/ml) and
FMRFa (n = 4). Figure 2B shows there
is no summation of the responses, i.e., that FMRFa completely occludes
the response to PLA2. If FMRFa was applied during the
PLA2 current response, an increase in current amplitude was
observed up to the level of the response to FMRFa alone
(n = 4) (Fig. 2C). This indicates that
PLA2 activates the same population of channels as FMRFa but does so less effectively.
As a next test to establish that PLA2 and AA evoke the same
current as dopamine, APGWa, and FMRFa, we determined the reversal potential and voltage dependence of the responses using voltage ramp
protocols (Fig. 3). In standard extracellular potassium, the current
induced by dopamine reversed at 82.0 ± 0.4 mV (± SEM;
n = 4) and that induced by AA and PLA2 at
81.9 ± 2.3 mV (n = 3) and 83.6 ± 0.4 mV
(n = 7), respectively. At high extracellular potassium,
the reversal potential shifted to 23.1 ± 0.1 mV for dopamine
(n = 4), 23.1 ± 1.3 mV for AA
(n = 2), and 21.2 ± 1.1 mV for PLA2
(n = 2). Thus, for the three agents, the shift in reversal potential caused by the change in extracellular potassium concentration amounts to ~60 mV. This corresponds well with the value
calculated by the Nernst equation for potassium (61.6 mV), indicating
that the current induced by AA and PLA2, like that induced by dopamine, APGWa, and FMRFa, is carried by potassium.
The I-V relationship of the K+ channels
activated by dopamine, FMRFa, and APGWa has a characteristic shape that
is a result of outward rectification, accounted for by asymmetry in
potassium concentrations across the membrane and a slight voltage
dependence (van Tol-Steye et al., 1997). The I-V
relationships of the AA- and PLA2-induced currents also
display these properties (Fig. 3A). Outward rectification is
especially evident at standard extracellular potassium. Voltage
dependence is apparent at voltages below approximately 100 mV, where
all conductances decreased despite the increasing driving force. This
is best appreciated at 20 mM
[K+]o. To permit a better comparison,
normalized I-V curves are shown Figure 3B. In
low [K+]o, the I-V
curves of the dopamine-, AA-, and PLA2-induced responses overlap completely at all voltages negative to approximately 20 mV.
Also in high [K+]o, the overall
shape of the I-V relationships is very similar. The
differences between the dopamine- and PLA2-evoked currents at voltages positive to 20 mV reflect effects of the agonist on high
voltage-gated calcium channels that are not mediated by activation of
PLA2 (H. van Tol-Steye and K. Kits, unpublished observations). The much stronger deviation of the AA-induced current at
positive potentials is unexplained.
The data presented suggest that AA, whether applied exogenously or
formed endogenously by application of PLA2,
activates the same type of potassium channel as dopamine, APGWa, and
FMRFa. Together with the fact that an inhibitor of AA formation
potently inhibits the responses to the three transmitters, this
strongly suggests that formation of AA underlies the generation of
these responses.
To test whether AA itself, or one of its metabolites, is responsible
for the generation of the potassium current, we tested several
inhibitors of AA metabolism. NDGA, an inhibitor of the lipoxygenase
pathway (Needleman et al., 1986; Yamada et al., 1994), is active in
Aplysia nervous tissue, with an IC50 of 3 µM (Piomelli et al., 1987a,b). In the LGCs, 15 µM NDGA inhibited the responses evoked by dopamine,
APGWa, and FMRFa for ~90% or more (n = 3 in all
cases) (Fig. 4A) within 20-30 min. A similar amount of
inhibition was observed with 5 µM NDGA within 40-60 min
in a first set of experiments with FMRFa (data not shown).
Indomethacin is an inhibitor of the cyclooxygenase pathway. In
Aplysia ganglia, indomethacin inhibits the formation of
prostaglandines, with an IC50 of 0.5 µM. The
lipoxygenase pathway is only affected at higher concentrations
(IC50 of >10 µM) (Piomelli et al., 1987a). Perfusion of the LGCs during ~25 min with 5 µM
indomethacin did not affect the current responses to dopamine,
APGWa, and FMRFa compared with the control situation in which the cells
were perfused with standard medium containing 0.01% DMSO (Fig.
4A). At this concentration, DMSO did not affect the
responses. As a final additional test of the involvement of the
lipoxygenase pathway, we used the lipoxygenase blocker AA-861 (Yamada
et al., 1994) on dopamine and APGWa responses, both of which were
significantly reduced (dopamine, 64.8 ± 4.1% block with 4 µM AA-861; n = 3; APGWa, 61.6 ± 2.1 and 84.1 ± 0.1% block with 4 and 10 µM AA-861,
respectively; n = 3 for both concentrations; unpaired
t test; p 0.01, tested against 0.05%
ethanol controls; n = 3).
To demonstrate that the exogenous bee venom-derived PLA2
that we used specifically forms AA and starts the same cascade as the
agonists, we tested whether NDGA blocks the PLA2-induced
response. Figure 4B shows that NDGA (15 µM) blocks the response to PLA2 (50 U/ml) by
80 ± 4% (n = 11). In three of these experiments, responses to PLA2 were evoked repeatedly before NDGA was
applied, which confirmed that control responses to PLA2
were stable.
We conclude from these data that (1) AA metabolites of the lipoxygenase
pathway, formed after activation of the receptors for dopamine, APGWa,
and FMRFa, are responsible for generation of the potassium current, and
(2) convergence onto the potassium channel occurs at an early step in
the signal transduction path, i.e., at or even upstream of the level of AA.
Involvement of G-protein subunits
We next asked which G-protein subunit mediates the responses. In
several cases, subunits of heterotrimeric G-proteins have been
implicated in the activation of PLA2 (Jelsema and Axelrod, 1987; Murayama et al., 1990). Because the responses to dopamine, APGWa,
and FMRFa are all G-protein-dependent (van Tol-Steye et al., 1997) and
mediated by AA, as demonstrated above, our strategy was to confine the
action of the subunits. To this end, we investigated the effect
of a synthetic peptide, peptide G, which was demonstrated previously to
interfere with -mediated signaling (Koch et al., 1993; Macrez et
al., 1997). Peptide G consists of 28 amino acids and is derived from
the -binding sequence of ARK1 (Koch et al., 1993).
After three control dopamine applications, peptide G was injected into
the cell (final concentration estimated to be 50 µM),
after which dopamine was applied again. Injection of peptide G caused
an irreversible reduction of the dopamine response by 79 ± 4%
(n = 5), whereas control injections with carrier had
hardly any effect (8 ± 1% reduction of the dopamine response; n = 3) (Fig.
5A).
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Figure 5.
-interacting peptide (peptide G) inhibits
the potassium current response. A, Reduction of the
response to dopamine after injection of peptide G (79 ± 4%;
n = 5) or injection of carrier (8 ± 1%;
n = 3). *p < 0.001; unpaired
t test. B, Induction of the sustained
K+ current as a result of irreversible activation of
the G-protein. Repeated application of agonist with 200 µM GTP--S in the pipette induces a sustained current
(b), which is the irreversible part of the
response. In the right panel, it is also seen as the
increased holding current before the response. Shown are the first and
last response to the agonist, in this example FMRFa;
arrows indicate application of agonist during the step
to 40 mV. The sustained current is only detected at 40 mV, when the
driving force for potassium is large. Notice that the holding current
at 80 mV (a) remains constant.
C1, Effect of peptide G on the sustained potassium
current, induced as in B, using dopamine as agonist.
Sustained current amplitude was measured once every 2 min. Injection of
peptide G (indicated by arrow) is done after the last
dopamine application. (Concentration of peptide G in injection
electrode was 5 mM, and final concentration is estimated to
be 50 µM.) C2, Example trace of the
immediate effect of peptide G on the sustained current at 40 mV.
Arrow marks injection of peptide G. C3,
Like B2 but with injection of carrier instead of peptide G. Obviously,
the carrier has no effect.
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The -interacting peptide in principle might, apart from competing
with effector molecules for binding free , also compete with G
for binding to and thus reduce the amount of functional heterotrimeric G-protein available for activation by receptors (but see
Koch et al., 1994; Macrez et al., 1997). To rule out this option, we
induced sustained and irreversible activation of the G-protein by
repeated applications (approximately six) of agonist, either dopamine
or FMRFa, with the nonhydrolyzable GTP-analog GTP--S (200 µM) in the pipette medium (Fig. 5B). This procedure results in the development of a sustained outward current that only decays over a period of several minutes after washout of the
agonist (cf. van Tol-Steye et al., 1997). Figure 5, C1 and
C2, illustrates the effect of peptide G on this sustained current. Injection of the peptide caused a rapid reduction of the
current (>50% within 1 min). Injection of carrier solution, however,
hardly affected the sustained current (Fig. 5C3). The results obtained for dopamine and FMRFa were highly similar (both n = 4). Thus, also when the G-protein is irreversibly
activated, the peptide has an inhibitory effect on the potassium
current re- sponse. This suggests that this inhibitory effect is
a result of interference with -induced activation of effector
(PLA2) and not of reduction of the amount of
functional heterotrimeric G-protein.
Effects of cAMP on agonist-induced responses
Previous work in our laboratory demonstrated that the
dopamine-induced hyperpolarization of the LGCs is inhibited by the
cAMP-analog 8-cpt-cAMP and by forskolin (which activates adenylyl
cyclase) and IBMX (an inhibitor of phosphodiesterase) (De Vlieger et
al., 1986). To test whether the outward current responses induced by dopamine, APGWa, and FMRFa are all inhibited by cAMP, we used caged
cAMP. NPE-caged cAMP (209 µM) was included in the
pipette medium and allowed to diffuse into the cell after establishing the whole-cell configuration. Caged cAMP was photolyzed by a flash of
UV light (360 nm), as was confirmed in similar experiments on
voltage-gated calcium currents known to be increased by cAMP-analogs in
other cells of Lymnaea (Dreijer and Kits, 1995). When a
flash was given just before application of agonist (at a membrane
potential of 40 mV), the potassium current evoked by the agonist was
greatly reduced [83.8 ± 1.4% (n = 2), 87.5 ± 1.9% (n = 2), and 66.5 ± 4.0%
(n = 5) for dopamine, APGWa, and FMRFa, respectively]
(Figure 6A). The flash
itself had virtually no effect (data not shown). If the flash was given
when the agonist-induced current was at its maximum, the outward
current declined rapidly (within 5 sec), as shown in Figure
6B (dopamine, n = 3; APGWa,
n = 2; FMRFa, n = 6). Thus, cAMP not
only prevents the response but also inhibits an ongoing response. In
the continuous presence of agonist, the current recovered within 30-50
sec to the level before the flash. Probably the recovery reflects
phosphodiesterase activity in the cell rather than desensitization of
the cAMP effect because the current was reinhibited after a second
flash (n = 8) (Fig. 6C, APGWa). The
experiments presented here indicate that the responses induced by
dopamine, APGWa, and FMRFa in the LGCs are all inhibited by cAMP. This
conclusion was further substantiated in experiments using either
extracellularly applied 8-cpt-cAMP, a membrane-permeable nonhydrolyzable cAMP-analog, or intracellularly applied cAMP (Fig. 6D, dopamine; other data not shown).
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Figure 6.
cAMP-dependent phosphorylation inhibits the
responses to dopamine, APGWa, and FMRFa. A1, Peak
current amplitudes as a function of pulse number. a-c
denote traces depicted in corresponding panels in A.
A2, Superimposed K+ current responses
to dopamine, APGWa, or FMRFa as indicated, before
(a) and directly after (b)
flash-photolysis of NPE-caged cAMP and after 60 sec recovery
(c). Arrows indicate timing of the
flashes 2-3 sec before agonist application. NPE-caged cAMP was added
to the pipette solution at 209 µM. All responses were
evoked at a potential of 40 mV. Uncaged cAMP strongly reduces the
responses, although it has no effect on the holding current at 40 mV.
B, Superimposed traces of a response interrupted by
flash-photolysis of caged cAMP at the peak of the potassium current and
the response after 60 sec recovery. Inhibition of current responses
by cAMP is fast and reversible. Shown is an example with dopamine.
C, Effect of repeated flashes on the current response in
the continuous presence of agonist. After the first decrease
attributable to uncaging of cAMP, the response recovers to a level
similar to that before the flash, whereas a second flash induces a
similar inhibition of the response. Shown is an example with APGWa.
D, Percentage reduction of the dopamine response after 3 min perfusion with 1 mM 8-cpt-cAMP, with and without the
PKA inhibitor wiptide (100 µM) in the pipette medium. The
inhibitory effect of 8-cpt-cAMP is strongly reduced by wiptide.
n = 3 for both groups. *p < 0.005; unpaired t test.
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To study whether cAMP acts by stimulation of PKA, we tested the
effect of 8-cpt-cAMP on the dopamine response in the presence of the
PKA inhibitor wiptide. Figure 6D shows that, when
wiptide (100 µM) was included in the electrode, the
inhibitory effect of 8-cpt-cAMP (1 mM) on the dopamine
response was significantly smaller than in the absence of wiptide
(31 ± 5% vs 89 ± 2%; n = 3 for both
treatments; unpaired t test; p < 0.005).
Wiptide had no effect on the dopamine response. We conclude that the
inhibition of the agonist-induced potassium current by cAMP and
analogs is mediated by PKA-dependent phosphorylation.
Effects of phorbol ester on agonist-induced responses
To investigate whether PKC modulates the responses to the three
agonists under study also, we tested the effects of TPA, a phorbol
ester activator of PKC. Bath perfusion of TPA (50 nM) caused a significant reduction of the current induced by all three agonists (Fig. 7A). After
~16 min, the currents induced by dopamine, APGWa, and FMRFa were
reduced by 71.3 ± 4.0% (n = 5), 65.9 ± 5.7% (n = 3), and 88.5 ± 1.3%
(n = 3), respectively. The PKC inhibitor staurosporin
(1 µM) significantly reduced the inhibition by TPA of the
response to FMRFa to 16.2 ± 3.1 (unpaired t test;
p < 0.001; n = 3) (Fig.
7B). Staurosporin did not affect the response to FMRFa.
These data suggest that the responses induced by dopamine, APGWa,
and FMRFa are all inhibited by PKC.
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Figure 7.
Phosphorylation by protein kinase C inhibits the
responses to dopamine, APGWa, and FMRFa. A, Repetitively
induced responses to dopamine, APGWa, and FMRFa as indicated, in the
absence and presence of TPA. Current amplitudes
(I) are normalized to the current response
at t = 0 (I0).
Arrows indicate the start of bath perfusion with 50 nM TPA (black diamonds;
n = 3-5 for each agonist). Control current
responses (contr, open diamonds;
n = 5-6 for each agonist) were obtained from
experiments performed without change of medium. SEMs are indi cated only by upward error bars. TPA strongly reduces the
responses to the agonists. B, Percentage reduction of
the FMRFa response after ~16 min perfusion with 50 nM TPA
or with a combination of TPA and the PKC inhibitor staurosporin (1 µM). Staurosporin strongly reduces the inhibitory effect
of TPA. *p < 0.005; unpaired t
test; n = 3 for both groups.
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Effects of phosphatase inhibitor
Because the K+ current responses are
inhibited by PKA- and PKC-dependent phosphorylation, it is to be
expected that inhibition of phosphatase activity will have similar
effects. To test this, we studied the effect of the phosphatase
inhibitor okadaic acid (Cohen et al., 1990) on the dopamine-induced
response. When okadaic acid was included in the patch electrode (1-2
µM), the dopamine-induced response decreased within ~30
min to 48.2 ± 6.7% of the initial value, measured directly after
rupture of the seal (n = 5; data not shown). Controls
with 1% DMSO in the patch electrode did not show a decrease of the
dopamine-induced current within the same period of time
(n = 2; data not shown).
The results with cAMP, TPA, and okadaic acid suggest that
phosphorylation of the channel or a component in the AA pathway inhibits the responses, or alternatively, that the AA pathway involves
a dephosphorylation step.
The AA pathway and the
phosphorylation-dephosphorylation cycle
To test at what site PKA and PKC interact with the signaling
cascades activated by dopamine, APGWa, and FMRFa, we examined the
effect of PKA and PKC stimulation on the signal transduction pathway
downstream of AA formation. Thus, the potassium current response to
application of exogenous PLA2 (50 U/ml) was measured before
and after 10 min perfusion of 8-cpt-cAMP (100 µM;
n = 6), TPA (50 nM; n = 3),
the inactive phorbol ester 4-PMA (50 nM; n = 3), and DMSO-containing medium (0.05%;
n = 4) (Fig. 8). After perfusion with 8-cpt-cAMP, the response to PLA2 was reduced
by 88 ± 5%, which was significantly different (unpaired
t test; p < 0.01) from the effect of
perfusion with DMSO-containing medium (reduction of 11 ± 15%).
TPA, however, had no effect (Fig. 8). This suggests that PKA inhibits
the agonist-induced potassium current by phosphorylation of a site
downstream of phospholipase activation, e.g., the potassium channel. In
contrast, PKC phosphorylation targets a site upstream of AA, e.g., the
receptors, the G-protein, or the endogenous phospholipase. This last
option cannot be excluded in view of possible differences between the
bee venom PLA2 used in our experiments and the endogenous
phospholipase in LGCs.
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Figure 8.
Potassium current response induced by
PLA2 is inhibited by 8-cpt-cAMP but not by TPA.
A, Effect of 10 min perfusion with 0.1 mM
8-cpt-cAMP (n = 6), 50 nM TPA
(n = 3), and the corresponding controls 0.05% DMSO
(n = 4) and 50 nM 4-PMA
(n = 3) on the potassium current response to
PLA2 (50 U/ml). Responses after perfusion
(I) are normalized to responses before
perfusion (I0). *p < 0.01, significant difference from control; unpaired t test.
B, Current responses to PLA2 before and
after perfusion with 8-cpt-cAMP and before and after perfusion with
TPA, as indicated. Differences in response kinetics are caused by
differences in duration of PLA2 application and perfusion
rate. 8-cpt-cAMP, TPA, and corresponding control solutions were applied
by Y-tube.
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To further specify the site of action of PKC, we induced sustained and
irreversible activation of the G-protein by repeated applications of
FMRFa by means of the nonhydrolyzable GTP-analog GTP--S (200 µM) as described above (Fig. 5B). This
procedure results in the development of a sustained outward current,
which only slowly runs down after washout of the agonist (van Tol-Steye et al., 1997). TPA caused a rapid reduction of this sustained current,
as shown in Figure 9A. Within
4 min of perfusion, TPA (50 nM) reduced the sustained
current by 56 ± 8% (n = 5), which was
significantly different from the reduction of 33 ± 7% under control conditions (perfusion with 4-PMA or DMSO; n = 8; p < 0.05; unpaired t test) (Fig.
9B). These results and those from Figure 8 imply that PKC
must act at a site downstream of the receptor but upstream of AA and
may either target the active G-protein subunit mediating the response
or the endogenous phospholipase.
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Figure 9.
TPA inhibits the potassium current induced by
irreversible activation of the G-protein. A, The
sustained current (measured as Ihold at 40 mV;
see legend of Fig. 5B) as a function of time after
perfusion with TPA (50 nM) or with the inactive phorbol
ester 4-PMA (50 nM). Perfusion of TPA or 4-PMA
(indicated by bars) is started directly after repetitive
FMRFa applications. TPA (left) causes a rapid decrease
of the sustained current compared with the decrease under control
conditions, i.e., with 4-PMA (right).
B, Average reduction of the sustained current after 4 min of perfusion of TPA (56 ± 8%; n = 5) or
control medium with inactive phorbol ester 4-PMA or DMSO (33 ± 7%; n = 8). *p < 0.05;
unpaired t test.
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DISCUSSION |
Three main conclusions can be drawn from the present study. First,
the activation of S-like potassium channels in LGCs by dopamine, APGWa,
and FMRFa involves AA and its metabolism by lipoxygenases. Thus,
convergence of dopamine, FMRFa, and APGWa onto the potassium conductance resides at or upstream of AA. Second, AA release is probably mediated by G-protein subunits, which activate the phospholipase. Third, the responses to the agonists are all affected by
PKA and PKC, of which PKC may target a site upstream of the point of
convergence and PKA downstream of this point. A schematic representation of the most likely signal transduction pathway used by
dopamine, APGWa, and FMRFa to activate S-like K+
channels is given in Figure 10.
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Figure 10.
Minimal hypothetical scheme of the signal
transduction routes involved in regulation of the receptor-driven
K+ channels. Boxes indicate that
different isoforms may be used. Dashed arrows indicate
equally probable options. PL, Phospholipase;
LO, lipoxygenase.
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Dopamine, APGWa, and FMRFa activate a single type of
K+ channel via lipoxygenase metabolites of
arachidonic acid
Several lines of evidence suggest that activation of the common
K+ conductance in LGCs by dopamine, APGWa, and FMRFa
be accomplished by the release of AA and lipoxygenase metabolites.
First, inhibitors of AA formation block the responses to the three
agonists. Second, the responses are mimicked by application of AA and
PLA2, which generates AA from membrane lipids, and
there is occlusion of the responses to PLA2 and FMRFa.
Third, the responses are blocked by inhibitors of lipoxygenases but not
of cyclooxygenases. Apparently, lipoxygenase metabolites cause,
directly or not, opening of the K+ channel. Because
the above results hold for all three agonists, convergence may occur
either at the level of the G-protein, the phospholipase, or AA.
4-BPB not only inhibits AA formation by PLA2 but also by
the combined action of phospholipase C and diacylglycerol lipase (Blackwell and Flower, 1983; Piomelli et al., 1987a). Our results with
4-BPB, therefore, do not specify the type of phospholipase activated by
the three agonists.
The presently described responses are closely related to other
receptor-driven K+ currents that are all activated
by FMRFa via formation of lipoxygenase metabolites, i.e., S and S-like
currents in Aplysia sensory neurons (Piomelli et al., 1987a;
Belardetti et al., 1989; Buttner et al., 1989), in identified neurons
of Helisoma (Bahls et al., 1992), and in the caudodorsal
cells of Lymnaea (Kits et al., 1997). Our data imply that,
apart from FMRFa, dopamine and the neuropeptide APGWa also couple to
the AA pathway. Dopamine-induced activation of PLA2 was
shown to occur after D2 receptor expression in Chinese hamster ovary cells (Vial and Piomelli, 1995) and is suspected to occur
in striatal cells (Schinelli et al., 1994).
Molluscan S-like currents were recently suggested to derive from a
novel class of voltage-independent K+ channels
cloned from mammals and characterized by the occurrence of four
transmembrane segments and two pore-forming P domains per subunit (Fink
et al., 1998; Patel et al., 1998). Overall, these channels (notably
TREK-1) show a strong similarity to S channels. They are
activated by AA and inhibited by PKA-dependent phosphorylation when
expressed in COS cells. However, they do not respond to lipoxygenase
products of AA. Also, the S-like current in Lymnaea differs
from these channels because it does not follow Goldman-Hodgkin-Katz
rectification at potentials negative to 90 mV and shows a much
stronger block by TEA and Ba2+ (Kits et al., 1997;
van Tol-Steye et al., 1997). The limited amount of homology between the
novel channels (~30%) suggests a large potential variability that
may account for these differences.
Effects of -interacting peptide suggest a role for
the G subunit
We tested the effect of peptide G, a -interacting peptide
derived from the -binding sequence of ARK1, on the
dopamine-induced response under standard conditions and on the
prolonged dopamine- and FMRFa-induced responses obtained with GTP--S
in the electrode. In both cases, peptide G inhibited the
K+ current, suggesting that subunits are the
active G-protein subunits mediating the signal from receptor to
effector. The controls with GTP--S showed that the inhibitory effect
of peptide G is not caused by a reduction of the amount of functional
heterotrimeric G-protein, resulting from competition of the peptide
with G for binding .
The simplest hypothesis incorporating both the G-protein subunit
and AA in the signal transduction pathway is to assume that, after
G-protein dissociation, causes activation of a phospholipase. As
discussed above, AA might be released from membrane phospholipids by
PLA2 or by the combined action of PLC and diacylglycerol lipases. PLC can be directly activated by subunits, and
PLA2 also has been suggested to be activated, whether or
not directly, by subunits (Jelsema and Axelrod, 1987; Murayama
et al., 1990; Exton, 1997). Alternatively, AA might activate a
G-protein, after which the K+ channels are directly
activated by G. Such a scheme was proposed to explain AA
modulation of GIRKs in atrial cells (Kurachi et al., 1989). It is,
however, very unlikely that this hypothesis explains our results. The
result that TPA inhibited the K+ current induced by
GTP--S, but not the PLA2-induced current, suggests that
there is no G-protein acting downstream of PLA2. Moreover,
preliminary experiments failed to show an effect of peptide G on the
PLA2-induced current response (K. Kits and J. C. Lodder, unpublished observations).
The concentration of peptide G we used to inject was 5 mM.
We estimate that, at maximum, ~1% of the cell volume was injected, implying that the final concentration of the peptide was always <50
µM. At this concentration, the K+
currents were inhibited by 50% or more. A similar IC50
value (76 µM) was reported for inhibition of
-stimulated ARK1 activity by peptide G in rod outer segment
membranes (Koch et al., 1993), suggesting that the affinity of
for the peptide is similar in both cases, despite possible differences
between molluscan and mammalian . A much lower IC50
value (65 nM), however, was found for inhibition by peptide
G of angiotensin II-mediated stimulation of L-type calcium channels in
rat myocytes (Marcez et al., 1997).
All responses are modulated by phosphorylation at two
different sites in the AA pathway
All three responses are inhibited by (analogs of) cAMP and by
PKC-activating phorbol ester. These effects were relieved in the
presence of PKA and PKC inhibitors, respectively, indicating that
phosphorylation by PKA and PKC mediates the inhibition. Moreover, the
dopamine response was reduced by the phosphatase inhibitor okadaic
acid. Apparently, phosphorylation counteracts the signaling induced by
dopamine, APGWa, and FMRFa.
The observation that 8-cpt-cAMP also inhibited the outward current
induced by exogenous PLA2 suggests that PKA-dependent
phosphorylation occurs at a site downstream of AA release, e.g., the
channel. It cannot be excluded that inhibition of PKA by lipoxygenase
metabolites of AA (or possibly activation of a phosphatase) is the
actual trigger for opening of the K+ channel.
However, if dopamine would inhibit PKA activity, it does not achieve
this via inhibition of adenylyl cyclase, because dopamine does not
inhibit adenylyl-cyclase in LGCs (Werkman et al., 1990). An alternative
and more simple explanation is that PKA-dependent phosphorylation forms
a modulatory pathway. Either way, the fact that not only cAMP but also
okadaic acid affects the responses suggests that a basal level of
phosphorylation controls the activity of the K+
channels. The presumed mammalian S-like channel, TREK-1, has a PKA
consensus site in its cytoplasmic C-terminal region, which is critical
for 8-cpt-cAMP mediated downmodulation of the channel (Patel et
al., 1998).
Interestingly, a similar relationship as found here between
AA-dependent activation of K+ channels and
(de)phosphorylation has been reported in sensory neurons of
Aplysia. The S channels in these cells are activated by
FMRFa via 12-lipoxygenase metabolites of AA, and they are inhibited by
serotonin via PKA-dependent phosphorylation of the channel or an
associated protein (Shuster et al., 1985; Belardetti and Siegelbaum,
1988). In addition to the AA metabolite-dependent effect on the S
channel, which is thought to be independent of (de)phosphorylation
(Belardetti et al., 1989; Buttner et al., 1989), FMRFa also reverses
PKA-dependent phosphorylation in the sensory neurons (Belardetti and
Siegelbaum, 1988; Sweatt et al., 1989; Shi and Belardetti, 1991). There
are indications that dephosphorylation is secondary to AA formation and
not dependent on inhibition of adenylyl-cyclase or stimulation of
phosphodiesterase activity (Volterra and Siegelbaum, 1988; Sweatt et
al., 1989; Shi and Belardetti, 1991). A similar mechanism might be
active in the LGCs.
Although both PKA and PKC suppress the responses to dopamine, APGWa,
and FMRFa, there is a clear differential effect of the two kinases.
Unlike 8-cpt-cAMP, the phorbol ester TPA failed to inhibit
PLA2-induced K+ currents. TPA, however,
inhibited the GTP--S-activated sustained current. Therefore, PKC
most likely targets a site upstream of AA, presumably the G
subunit or the phospholipase. Interestingly, Lymnaea
G-protein subunits contain six consensus sites for PKC phosphorylation (Knol et al., 1994; H. van Heerikhuizen, unpublished observations).
Thus, the responses to all three agonists are affected by PKA and PKC,
of which PKC may act at a site upstream of the putative point of
convergence (AA) and PKA downstream of AA. This suggests a modulatory
role for receptors that stimulate PKA- or PKC-activity in LGCs.
Candidate agonists for such receptors are acetylcholine and conopressin
that induce depolarizations in LGCs, an effect that is also produced by
injections of cAMP (Stoof et al., 1984). The ability of conopressin to
activate PKC in Lymnaea anterior lobe neurons was recently
demonstrated in our lab (P. F. van Soest, unpublished observations).
| |
FOOTNOTES |
Received Oct. 30, 1998; revised March 4, 1999; accepted March 9, 1999.
We thank Dr. Arjen B. Brussaard and Dr. Paul F. van Soest for comments
on this manuscript.
Correspondence should be addressed to Dr. Karel S. Kits, Department of
Neurophysiology, Research Institute Neurosciences, Vrije Universiteit,
De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
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TOP
ABSTRACT
INTRODUCTION
