Sächsische Akademie der Wissenschaften zu Leipzig, D-07743 Jena, Germany
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ABSTRACT |
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Wicher, Dieter. Peptidergic Modulation of an Insect Na+ Current: Role of Protein Kinase A and Protein Kinase C. J. Neurophysiol. 85: 374-383, 2001. The modulation of voltage-gated Na+ currents in isolated somata of dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana was investigated using the patch-clamp technique. The neuropeptide Neurohormone D (NHD), which belongs to the family of adipokinetic hormones, reversibly reduced the Na+ current in concentration-dependent manner (1 pM to 10 nM). At 10 nM, NHD caused an attenuation of the maximum of current-voltage (I-V) relation for peak currents by 23 ± 6%. An analysis of NHD action on current kinetics in terms of the Hodgkin-Huxley formalism revealed that NHD reduces the time constant of inactivation, whereas steady-state activation and inactivation as well as the time constant of activation were not affected. In addition, NHD prolonged the recovery from inactivation. The cAMP analogue 8-bromo-cAMP, forskolin, and the catalytic subunit of protein kinase A mimicked the action of NHD. Furthermore, preincubation of cells with the protein kinase A inhibitor KT 5720 abolished the action of NHD. Thus NHD seems to modify the Na+ current via channel phosphorylation by protein kinase A. Activation of protein kinase C by oleoylacetylglycerol (OAG) also reduced the Na+ current, but it did not occlude the action of NHD. On the other hand, inhibition of protein kinase C by chelerythrine or Gö 6976 did not essentially impair the NHD effects.
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INTRODUCTION |
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In vertebrates the
proteins forming subunits of voltage-gated
Na+ channels are substrates for phosphorylation
by protein kinase A (PKA) and protein kinase C (PKC) (Conley
1999
). Such phosphorylation may be used for signal transduction
if there are exogenous modulators like hormones or transmitter-like
substances that can modify the Na+ current. For
example, in rat hippocampal neurons, activation of muscarinic receptors
or D1-like dopamine receptors decreases the Na+
current (Cantrell et al. 1996
, 1997
,
1999
). The signal transduction pathways involve channel
phosphorylation by PKC or PKA, respectively. In most vertebrate
preparations, phosphorylation by PKA or PKC reduces the
Na+ current (e.g., Gershon et al.
1992
; Godoy and Cukierman 1994a
; Li et
al. 1992
, 1993
; Numann et al.
1991
; Schiffmann et al. 1995
; Smith and
Goldin 1997
; Surmeier et al. 1992
).
In the fruit fly Drosophila melanogaster, the two genes
encoding Na+ channel subunits,
DSC1 and para, are highly homologous to a rat
Na+ channel cDNA (Loughney et al.
1989
; Ramaswami and Tanouye 1989
; Salkoff
et al. 1987
). The para gene product that is
predominantly expressed in the Drosophila nervous system
(Hong and Ganetzky 1994
) contains four potential
phosphorylation sites for PKA (Loughney et al. 1989
). Up
to now there is no published information on the role of these
phosphorylation sites in the fruit fly or in other insects.
In the somata of several groups of neurosecretory insect neurons,
Na+ channels occur (Burrows 1996).
Among them are efferent dorsal unpaired median (DUM) neurons of the
cockroach Periplaneta americana, the somata of which
generate action potentials driven by large Na+
currents (Lapied et al. 1989
, 1990
).
These cells show spontaneous activity resulting from contributions of
many ionic currents (Grolleau and Lapied 2000
).
There is a member of the adipokinetic hormone family (AKHs)
(Gäde et al. 1997) termed neurohormone D (NHD)
(Baumann and Penzlin 1984
) or MI (Witten et al.
1984
) or Pea-CAH I (Raina and Gäde 1988
),
which affects some of these ionic currents. For example, the
potentiation of a voltage-gated Ca2+ current
component by NHD (Wicher and Penzlin 1994
) causes an increase of Ca2+-activated
K+ currents (Wicher et al. 1994
).
The combined effects of this octapeptide lead to accelerated spiking of
these DUM neurons as first observed on intracelllular recording from
their somata (Birkenbeil 1971
).
The present study, performed on acutely isolated DUM neurons, demonstrates for the first time a neuromodulatory modification of a Na+ current in an insect. It was found that, in addition to modulating Ca2+ and K+ currents, NHD also affects Na+ currents in these cells. As will be shown, the signal transduction process seems to involve the cAMP system and channel phosphorylation by PKA.
Some of the results have already been published in abstract form
(Wicher 2000).
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METHODS |
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Cells
Isolation of cells was performed as described previously
(Wicher et al. 1994). Briefly, the fifth abdominal
(i.e., the last unfused) ganglia of adult cockroaches
(Periplaneta americana) were excised, desheathed, and
incubated for 10 min at room temperature in saline (composition, in mM:
190 NaCl, 5 KCl, 5 CaCl2, 2 MgCl2, and 10 HEPES; pH 7.4) containing 0.5 mg/ml
trypsin (type II, Sigma, Deisenhofen, Germany) and 0.5 mg/ml
collagenase (type I, Sigma, Deisenhofen, Germany). After thoroughly
washing off the enzyme, the ganglia were stored in saline for at least
1 h. In the fifth abdominal ganglion, four octopaminergic DUM
neurons form a cluster (Eckert et al. 1992
). From this
DUM cell cluster that can be easily identified, the neurons were
separated using thin metal needles.
Electrophysiology
The ionic currents of the isolated DUM neurons were measured at
room temperature using the patch-clamp method in the whole cell
configuration. Current measurements and data acquisition were performed
using an EPC9 patch-clamp amplifier controlled by the PULSE software
(HEKA Elektronik, Lambrecht, Germany). Data were sampled at 10 kHz and
filtered at 2.9 kHz. Capacitive and leak currents were compensated by a
cancellation routine provided by PULSE. Remaining uncompensated leakage
currents were subtracted using an on-line P/4 protocol (leak holding
potential 110 mV). For off-line data analysis the PULSFIT software
(HEKA) was used. Pipettes having resistances of 0.5-0.8 M
were
pulled with a P-87 micropipette puller (Sutter Instrument, Novato, CA)
from borosilicate capillaries (Hilgenberg, Malsfeld, Germany). The
series resistance remaining after compensation did not exceed 1.3 M
.
The holding potential (Vhold) was
90 mV.
Separation of Na+ currents in these DUM neurons
and evidence that the procedure yields a pure TTX-sensitive
Na+ (and not Ca2+) current
was shown elsewhere (Wicher and Penzlin 1998). The
pipette solution used for this separation contained (in mM) 100 choline methyl sulfate (CMS), 60 CsOH, 30 tetraethylammonium (TEA)-Br, 5 NaCl,
2 Mg-ATP, 1 CaCl2, 10 EGTA, and 10 HEPES. The
bath solution for Na+ current measurements
contained (in mM) 30 Na-isethionate, 120 CMS, 40 TEA-Br, 7 MgCl2, 1 CdCl2, and 10 HEPES. The pH value was adjusted to 7.4 (bath solution) and 7.25 (pipette solution). Liquid junction potentials between pipette and bath
solution were taken into account before establishing the seal. This
combination of bath and pipette solutions allowed measurements of
separated Na+ currents approximately 1 min after
breaking into the cell. Within this time, contaminating
Ca2+ and K+ currents
disappeared completely. The low Na+ concentration
of 30 mM in the bath solution was chosen to reduce the risk of voltage
error due to series resistance. Under these conditions the maximal peak
current did not exceed 5 nA. The Na+ current
recordings were usually stable, i.e., there was no rundown during
experiments. Occasionally, there was a virtual decrease of current due
to increasing series resistance, but data from such experiments were
not taken for further analysis.
Spiking of neurons was measured under current-clamp conditions without
current injection. Neurons were bathed in saline (cf. Cells) and the
patch pipettes (resistance >1.5 M) were filled with a solution
composed of (in mM) 180 K-gluconate, 10 NaCl, 1 CaCl2, 10 EGTA, 2 Mg-ATP, and 10 HEPES (pH 7.25).
Between registrations (duration 1 s) the cells were held under
voltage clamp at a holding potential of
70 mV.
NHD was obtained from Peninsula (Belmont, CA), 8br-cAMP from Sigma (Deisenhofen, Germany), KT 5720 and Gö 6976 from Calbiochem (Bad Soden, Germany), chelerythrine and oleoylacetylglycerol (OAG) from Alexis (Grünberg, Germany), and the catalytic subunit of PKA (porcine heart) from Biomol (Hamburg, Germany). Application or wash out of blocking agents was performed by transferring the cell (attached to the pipette tip) within a glass tube into the various solutions. A complete and fast solution change was achieved by sucking a small amount of solution into the tube. Injection of PKA into the cells was performed with Eppendorf Femtotips while the neuron was sucked to the patch pipette (whole cell mode). The Na+ current was registered before and after penetration of the cell membrane by the Femtotip, and only if the currents were identical the catalytic PKA subunit was injected using the transjector 5246 (Eppendorf, Germany). Approximately 20 pl of pipette solution containing protein kinase A (0.85 units/µl) were injected in each cell tested. Sham injections performed in some cells did not affect the Na+ current.
Data analysis
If not otherwise stated, results are given as means ± SD (n is the number of cells). Statistical significance of differences was estimated using Student's t-test. The evaluation of statistical significance of differences for data sets with variables (voltage, time) was performed with two-way ANOVA. Differences were considered significant if P < 0.05. Mathematical expressions (e.g., Boltzmann equation) were fitted to the means of data sets by a least-square routine using a model with variable Hill coefficent. The parameters obtained from fits are given as means ± SE (n is the number of cells). For data analysis including nonlinear fitting procedures, the software Prism 2 (Graph Pad Software, San Diego, CA) was used.
Current-voltage relationships for Na+ peak currents were fitted taking into account current rectification according to the Goldman-Hodgkin-Katz (GHK) equation.
The Na+ current
INa = G * (V VNa) was described in terms of the
Hodgkin-Huxley formalism using the software PULSEFIT (HEKA Elektronik,
Lambrecht, Germany) (cf. Wicher and Penzlin 1998
). For
the conductance G was assumed
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(5) |
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RESULTS |
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NHD modifies the Na+ current
In cockroach DUM neurons, a voltage jump from 90 to
30 mV
evokes a small, slowly inactivating Na+ current,
and a jump to
10 mV evokes a rapidly decaying current of maximal size
(Fig. 1; cf. Fig. 3). The effect of 10 pM
NHD on these two Na+ currents differing in their
inactivation rate is shown in Fig. 1. Whereas NHD hardly affected the
Na+ current at
30 mV (Fig. 1A1), it
clearly reduced its peak and shortened its duration at
10 mV (Fig.
1A2). At both potentials, NHD did not seem to affect
activation kinetics. An analysis of the NHD effect during a 10-ms
lasting voltage jump revealed that 1 ms after jumping to
30 mV or to
10 mV, there was no change in current (Fig. 1, B1 and
B2). Later, a clear difference in the peptide action became
apparent: for pulses to
30 mV, a slight current reduction by NHD
developed 3 ms after jump and maximal effect was seen at the end of the
jump (Fig. 1B1). However, a strong current reduction
happened already 2 ms after jump to
10 mV, and maximal effect was
reached at 3 ms (Fig. 1A2). The attenuation of
Na+ current by NHD was accompanied by a slight,
but consistent decrease of the time-to-peak. The voltage dependence of
this effect is shown for a representative cell in Fig.
2A. At
10 mV the
time-to-peak under control conditions was 1.69 ± 0.06 (SD) ms and 2 min after application of 10 nM NHD 1.61 ± 0.06 ms (n = 7); the difference was statistically
significant (paired t-test). Compared with data known from
axons where the time-to-peak is usually <0.1 ms, these figures might
appear relatively large and perhaps indicative of bad voltage control.
However, similar figures have been found for other insect
Na+ currents, e.g., those of
Drosophila neurons (O'Dowd and Aldrich 1988
), Drosophila para channels expressed in
Xenopus oocytes (Warmke et al. 1997
), and
cricket neurons (Kloppenburg and Hörner 1998
).
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The voltage dependence of the effect of 10 nM NHD on peak currents is
shown in Fig. 3A. The peak of
currents evoked by voltage jumps ranging from threshold to 25 mV was
not affected. The reducing peptide effect on current size starts at
depolarization to
25 mV and becomes stronger with increasing voltage.
The NHD-induced attenuation of peak currents was concentration
dependent (Fig. 3B). The threshold concentration was
1 pM,
and there was no saturation at the highest tested concentration of 10 nM NHD where the reduction amounted to 23 ± 6%
(n = 9). The concentration dependence of current reduction by NHD was described by an isotherm with an
IC50 of 5 pM and a Hill slope of 0.35.
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The reduction of the peak current after application of 10 nM NHD
developed in a single exponential fashion (time constant = 61 s, n = 8, Fig. 3C). At this
concentration the peptide effect partially reversed by ~80% after
washing off within 5-10 min when NHD was present for ~4 min, but it
disappeared rapidly within half a minute when NHD was applied for
1 min.
In addition to the effect of NHD on Na+ current
size and duration, the peptide led to a slower time course of recovery
from inactivation (Fig. 4). Using a
holding potential of 90 mV and test potential of
10 mV,
Na+ peak currents recovered with a biphasic time
course. Within the first 10 ms, 93% of current recovered under control
conditions and 90% in the presence of NHD. This part of rapid recovery
could be described by single exponentials with time constants
r = 1.6 ± 0.1 ms in the absence and
r = 2.3 ± 0.2 ms in the presence of NHD.
The slow recovery process was complete after 60-80 ms and was not
apparently affected by NHD. After washing off the peptide, the change
of recovery kinetics disappeared with a time course comparable to the
reversal of the peak current.
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Kinetic analysis of the NHD effect
To quantify the NHD-induced changes of kinetics, the Na+ current was described in terms of the Hodgkin-Huxley formalism. The used model includes biphasic, i.e., fast and slow, inactivation kinetics (cf. METHODS). Analysis of the NHD effect on Na+ currents in terms of this model revealed that peptide action was restricted to the fast inactivating current. Therefore no data concerning slow inactivation will be presented, and the parameter describing fast inactivation will be denoted as h.
The steady-state activation m,
taken as the cubic root of maximum conductance, was not affected by 10 nM NHD. The voltage of half-maximal activation
Vm obtained from fitting the Boltzmann equation (Eq. 4, cf. METHODS) to data were
28.8 ± 0.6 mV before versus
30.0 ± 0.7 mV after NHD
application, the slope Km was 6.9 ± 0.5 mV versus 7.0 ± 0.6 mV, respectively. The slight
hyperpolarizing shift seen in the presence of NHD was not statistically
significant (Fig. 5A1; ANOVA).
Similarly, the steady-state inactivation
h
remained unchanged by NHD
(ANOVA). The voltage of half-maximal inactivation
Vh obtained from the Boltzmann
equation (Eq. 5) was
39.0 ± 0.5 mV both in the
presence and in the absence of NHD and the figures for the slope
Kh were 7.0 ± 0.4 mV (Control)
and 6.7 ± 0.4 mV (NHD; Fig. 5B1). There was,
furthermore, no significant change in the activation time constant
m by NHD (Fig. 5A2; ANOVA). The
only one parameter affected by NHD was the inactivation time constant.
Peptide application caused a decrease of
h
within the whole voltage range except for potentials less than
20 mV
(Fig. 5B2). Although relatively small, the differences were
statistically significant (ANOVA, P = 0.0001). The
inset in Fig. 5B2 shows, for depolarizations
evoking maximal currents, that the inactivation time constant decreased
in each of the seven cells analyzed. Again, the differences were
considered statistically significant (paired t-test;
P = 0.003). The changes of
h
reversed within a few minutes after wash out of NHD.
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Thus NHD has two effects on Na+ current kinetics: first, it accelerates the inactivation from open state, and second, it slows down the recovery from inactivation. The first effect leads to a decrease of peak current, to a reduction of the time-to-peak, and to a shortening of the total duration of current. The second effect is not expected to affect the spiking properties since it would lead to adaptive changes only for interspike intervals <0.10 ms (i.e., a spike frequency >100 Hz; cf. Fig. 4).
Signal transduction mechanism
CAMP SYSTEM AND PROTEIN KINASE A.
Most neuropeptide receptors known so far are metabotropic receptors
(Iversen 1995). Possible targets in
Na+ channels for modulatory actions initiated by
activation of metabotropic receptors may be some phosphorylation sites
like those found for PKA in the para channel of the fruit
fly (Loughney et al. 1989
). In DUM neurons, NHD affects
several ionic currents. For example, it potentiates
Ca2+-dependent K+ current
(Wicher et al. 1994
) by enhancing a voltage-activated Ca2+ current (Wicher and Penzlin
1994
). Both effects were also obtained by bath application of
the membane-permeant cAMP analogue 8-bromo-cAMP (8br-cAMP) indicating
that NHD might act via increasing the cAMP level (Achenbach et
al. 1997
).
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PKC.
In vertebrates, Na+ channels may be modulated by
either PKA or PKC (Conley 1996) or in convergent manner
in which phosphorylation by PKC is required for the action of PKA
(Li et al. 1993
). Similarly, the effectiveness of
phosphorylation by PKA can be enhanced by concurrent activation of PKC
(Cantrell et al. 1997
, 1999
). Presently, no phosphorylation sites for PKC are known for insect
Na+ channels. To get an indication for a possible
modification of the Na+ current in DUM neurons by
phosphorylation via PKC, the effect of a PKC activator, the
diacylglycerol analogue OAG, was tested. It was furthermore
investigated whether activation or inhibition of PKC changes the effect
of NHD on the Na+ current.
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Sodium current reduction and action potentials
The significance of the NHD-induced reduction of both peak and duration of Na+ current for shaping action potentials is hardly to reveal experimentally since NHD acts on several currents. Therefore the application of NHD under current-clamp conditions shows always the concerted result of all peptide actions. Under these conditions, a separation of Na+ current effects on spiking by the block of calcium currents (e.g., by Cd2+) is impossible since such manipulation readily stops the discharge of the DUM neurons (unpublished observation). To get a rough estimate for the consequence of a reduced Na+ current for spiking, low amounts of TTX (2.5 and 5 nM) were applied under current-clamp conditions. These experiments revealed that the most prominent effect of TTX was a dose-dependent reduction of action potential overshoot. In addition, there was a mild shift of action potential threshold but almost no effect on undershoot (Table 1). For example, TTX at 2.5 nM blocks 23% of the sodium current, i.e., an effect comparable to the action of 10 nM NHD. The action potential overshoot in this situation is reduced by 7 mV, the threshold is shifted toward depolarization by 3.7 mV, and the undershoot is reduced by <2 mV. At a TTX concentration of 10 nM, which corresponds to 50% block of INa, no long-lasting spiking with large overshoots could be observed.
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DISCUSSION |
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This study presents, for the first time, evidence that an endogenous modulator like the neuropeptide NHD can modify the biophysical properties of voltage-gated Na+ currents in an insect. The investigations of the action of NHD on the Na+ current in cockroach DUM neurons suggest that NHD shortens the time constant of inactivation, which leads to a faster decay and to a reduction of the maximum current. Additionally, NHD prolongs the recovery from inactivation.
Experiments addressing the involved signal transduction process provided some evidence that the action of NHD on the Na+ current is mediated by an increase in the cAMP production and subsequent Na+ channel phosphorylation by PKA. First, the effects of NHD on Na+ current were mimicked by 8-br-cAMP and forskolin. Second, NHD had no effect when it was applied after preincubation of cells with the PKA inhibitor KT5720. Third, injection of the catalytic subunit of PKA changed the Na+ current kinetics in a NHD-like manner.
It has been further shown that also activation of PKC attenuates the Na+ current in DUM neurons without occluding the NHD effect. On the other hand, inhibition of PKC slightly attenuated the response to NHD. Although this point has to be investigated in more detail, this finding may be indicative of a cooperativity of PKC and PKA.
Previous investigations have shown that -conotoxin MVIIC and
-agatoxin IVA, which are known to block P/Q-type
Ca2+ currents in vertebrates, do not only block a
Ca2+ current component in the DUM neurons
(Wicher and Penzlin 1997
), but they also modify the
Na+ current (Wicher and Penzlin
1998
). This modification of the Na+
current was very similar to the action of NHD. Therefore it was tested
whether the application of NHD occludes the effect of
-conotoxin MVIIC and vice versa. This was, however, not the case. In the presence
of 10 nM NHD, 1 µM of the toxin led to an additional reduction of the
Na+ peak current by 22 ± 5%
(n = 3), and 10 nM NHD reduced the peak current in the
presence of 1 µM
-conotoxin MVIIC by 22 ± 7%
(n = 3). Thus, although both agents have similar
effects on Na+ current, they do not appear to
bind at the same receptor.
The mechanism by which adipokinetic hormones AKH I, II, and III, i.e.,
peptides structurally related to NHD, stimulate the release of
carbohydrates due to activation of glycogen phosphorylase from fat body
cells in the African migratory locust, Locusta migratoria, involves an accumulation of cAMP (Vroemen et al.
1998). This accumulation was shown to require extracellular
Ca2+. Furthermore, the stimulating action of the
three AKHs on glycogen phosphorylase could be mimicked by application
of the Ca2+ ionophore A23187. By contrast, the
modulation of the Na+ current in DUM neurons by
NHD is independent of a Ca2+ influx since all
experiments were performed using a bath solution that
contained 1 mM Cd2+ to block all
Ca2+ currents.
In vertebrates, phosphorylation of Na+ channels
by PKA or PKC leads in most cases to a reduction of
Na+ current (Cantrell et al. 1997,
1999
; d'Alcantara et al. 1999
; Gershon et al. 1992
; Godoy and Cukierman
1994a
; Li et al. 1992
, 1993
;
Numann et al. 1991
; Schiffmann et al.
1995
; Smith and Goldin 1992
,
1997
; West et al. 1991
). Only in some
cases phosphorylation induces an increase of Na+
current (Godoy and Cukierman 1994b
; Li et al.
1993
).
The reduction of the DUM cell Na+ current
observed on channel phosphorylation by PKA seems to be similar to most
examples in the vertebrate preparations cited above. On the other hand,
a prolonged recovery from inactivation as seen in DUM cell
Na+ current after activation of PKA was not
reported for Na+ currents in vertebrates (e.g.,
Li et al. 1992). In rat brain Na+
channels, phosphorylation by PKA produced changes in
Na+ current kinetics akin to those observed in
the DUM cell Na+ current (d'Alcantara et
al. 1999
). The rat brain Na+ peak current
was reduced, and the current duration was shortened, but activation
kinetics was not affected. The gating mechanism of this rat brain
Na+ channel could be described by a kinetic model
that involves three closed states, one open state, and two inactivated
states. In terms of this model, activation of PKA accelerated the
transition from open state to an inactivated one (d'Alcantara
et al. 1999
). Such a mechanism might perhaps also account for
the accelerated inactivation kinetics observed for the cockroach
Na+ current.
In hippocampal neurons, the effect of phosphorylation by PKA appeared
to be voltage dependent, i.e., a more depolarized holding potential
gave rise to a stronger reduction of Na+ current.
In addition, the PKA effect was enhanced by concurrent activation of
PKC (Cantrell et al. 1999). Whereas a synergistic action
of PKC and PKA in the regulation of the Na+
current in DUM neurons cannot be excluded, no effect of a change of
holding potential in the range between
50 and
90 mV on the action
of NHD was found (not shown; n = 5).
Although a reduction of Na+ current on activation
of PKC was seen in vertebrate neurons as well as in DUM neurons, some
differences have to be stated. For example, in rat brain neurons the
Na+ peak current reduction was accompanied by
slower inactivation (Numann et al. 1991). The
phosphorylation site for PKC is located in the intracellular loop
between the transmembrane domains III and IV, which is responsible for
inactivation (West et al. 1991
). A part of this loop is
represented by the synthetic peptide SP19, which involves the
phosphorylation site. Interestingly, an antibody against this peptide
recognizes Na+ channels in DUM neurons
(Amat et al. 1998
). Although one might expect to find
this phosphorylation site also in DUM neuron channel, the result of
phosphorylation differs in that the current is reduced but not prolonged.
An accelerated inactivation on activation of PKC was observed in mouse
neuroblastoma cells (Cukierman 1996; Godoy and
Cukierman 1994a
,b
; Renganathan et al. 1995
). But
in these cells (in contrast to DUM neurons) the steady-state parameters
were shifted on the voltage axis toward more negative potentials.
In rat brain Na+ channels, phosphorylation by PKC
is required for a current reduction by phosphorylation via PKA in the
loop between domaine I and II (Li et al. 1993). In DUM
neurons PKC inhibition did not prevent the effect of the NHD-induced
channel phosphorylation by PKA, but the NHD effect appeared somewhat
reduced. More detailed investigations are required to clarify whether
and to what extent there is really a convergent regulation of DUM cell
Na+ channels by PKA and PKC.
What is the functional significance of the NHD-mediated modulation of
the Na+ current for the spike activity of DUM
neurons? The prolonged recovery from inactivation does not seem to
affect neuronal activity since it is significant only in the first 10 ms after repolarization. Although the experiments have been performed
under voltage-clamp conditions using voltage jumps, it is not likely
that the slower time courses during action potentials prolong the time
recovery by an order of magnitude that would be necessary to bring
about adaptation at spike frequencies observed in these neurons (up to
10 Hz) (Lapied et al. 1989). Current-clamp experiments
in DUM neurons have shown that the action potential undershoot is
between
60 and
70 mV. The time constant of recovery from
inactivation in this potential range is between 2 and 3 ms
(Lapied et al. 1990
). Even a prolongation by factor of
two would not be enough to affect spiking.
The TTX-induced reduction of Na+ current produced changes in action potential parameters, which may be considered as rough estimates for the NHD effect (Table 1). Thus it seems likely that the modulation of INa by NHD will reduce the overshoot and perhaps slightly shift the threshold, but it will not affect the hyperpolarization of action potentials. However, it should be kept in mind that TTX does not adequately imitate the effect of NHD.
In the DUM neurons, a Na+ influx activates a
Na+-dependent K+ current
(Grolleau and Lapied 1994). The latter current is
independent of voltage and activates only at an appropriate
concentration of Na+ in the vicinity of the
channel pore (some 10 mM) (cf. Conley 1996
). Therefore
the maximum of the current-voltage relation of the
Na+-activated K+ current is
reached at the potential where the Na+ current is
maximal (i.e., around
15 mV). If in the presence of NHD the
Na+ influx was reduced, the
K+ efflux should become attenuated. This
assumption could be confirmed experimentally (data not shown). Thus NHD
not only reduces a depolarizing contribution to the action potential
(Na+ current), but it also indirectly attenuates
a repolarizing contribution that becomes already activated during the
rising phase of the action potential
(Na+-activated K+ current).
One might speculate whether the superposition of these both NHD effects
in concert with the previously observed potentiation of a
Ca2+ current (Wicher and Penzlin
1994
) as well as a Ca2+-activated
K+ current (Wicher et al. 1994
)
may account for the increased action potential overshoot that is
observed in the presence of NHD.
Thus the modulation of the Na+ current seems to
be part of orchestrated changes of electrical conductances by NHD,
which in turn leads to an increased spike frequency and changes in the shape of action potentials. The efferent DUM cells contain the biogenic
amine octopamine that is released to peripheral structures such as
visceral and skeletal muscles (Stevenson and
Spörhase-Eichmann 1995). The electrical excitability of
the DUM cell somata is thought to be involved in triggering octopamine
release (Burrows 1996
; Finlayson and Osborne
1975
). Modulation of ionic currents contributing to
excitability including the Na+ current would then
provide a mechanism to control neurosecretion according to the
physiological requirements. A possible source of the modulator NHD,
which might regulate the activity of abdominal DUM neurons are some
non-DUM neurons that appear unclustered in the dorsal midline of
abdominal ganglia and show NHD-like immunoreactivity (Eckert et
al. 1997
).
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ACKNOWLEDGMENTS |
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The author thanks Dr. C. Walther for comments on part of the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft (Wi 1422/2-3).
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FOOTNOTES |
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Address for reprint requests: Sächsische Akademie der Wissenschaften zu Leipzig, Erbertstrasse 1, D-07743 Jena, Germany (E-mail: b6widi{at}pan.zoo.uni-jena.de).
Received 26 April 2000; accepted in final form 14 September 2000.
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