Sächsische Akademie der Wissenschaften zu Leipzig, D-07743 Jena, Germany
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ABSTRACT |
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Wicher, Dieter.
Peptidergic Modulation of Insect Voltage-Gated Ca2+
Currents: Role of Resting Ca2+ Current and Protein
Kinases A and C.
J. Neurophysiol. 86: 2353-2362, 2001.
The modulation of voltage-gated Ca2+
currents in isolated dorsal unpaired median (DUM) neurons of cockroach
was investigated using whole cell patch clamp. The neuropeptide
neurohormone D (NHD), a member of the adipokinetic hormone family,
affected Ca2+ currents at pico- to nanomolar
concentrations. It strongly enhanced currents activating at lower
depolarizations, whereas those activating at strong depolarizations
were slightly attenuated. The first effect results from upregulation of
a previously characterized -conotoxin MVIIC- and
-agatoxin
IVA-sensitive "mid/low voltage-activated" (M-LVA)
Ca2+ current. The cAMP-analogue 8-bromo-cAMP,
forskolin, and the catalytic subunit of protein kinase A (PKA) mimicked
the stimulating action of NHD. In addition, preincubation of neurons
with the PKA inhibitor KT 5720 abolished the action of NHD. Thus NHD
seems to upregulate the M-LVA current via channel phosphorylation by
PKA. Activation of protein kinase C by oleoylacetylglycerol (OAG)
mimicked the effect of NHD, and subsequent NHD application only
enhanced the current to a moderate extent. On the other hand,
inhibition of protein kinase C (PKC) by Gö 6976 abolished the NHD
effect. These results indicate that also PKC, too, may play a role in
the peptidergic modulation of the M-LVA Ca2+
current. The reduction of Ca2+ currents in the
high-voltage-range is caused by the NHD-induced upregulation of a
voltage-independent Ca2+ resting current,
ICa,R, which most probably leads to
enhanced Ca2+-dependent inactivation of
voltage-gated Ca2+ currents. To assess the major
consequences of the Ca2+ current changes,
current-clamp investigations were performed. Experiments with
iberiotoxin, a specific blocker of BK-type
Ca2+-dependent K+ currents,
and the M-LVA current-blocking
-toxins suggested that NHD
causes
via increasing Ca2+-dependent
K+ currents
a larger hyperpolarization of action
potentials. The lowering in the action potential threshold produced by
NHD, however, seems to be a direct consequence of the hyperpolarizing
shift of the activation curve of total Ca2+
current resulting from NHD-induced upregulation of the M-LVA current component.
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INTRODUCTION |
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Voltage-gated
Ca2+ channels provide a major
Ca2+ entry pathway in neurons (Parekh and
Penner 1997) and thus play an important role in the regulation
of a wide variety of cellular activities such as electrical activity,
transmitter release, neuronal plasticity or gene expression
(Berridge 1998
). Ca2+ influx
through these channels is tightly regulated by neurotransmitters and
hormones in accord with the varying physiological demands. G proteins
activated by metabotropic receptors may either directly affect channels
(Zamponi and Snutch 1998
) and/or induce signal transduction cascades thereby leading to channel phosphorylation or
dephosphorylation (Catterall 1997
; Rossie
1999
). The effect of a modulator in a neuron depends on the
pattern of expressed receptors and channels. The signaling system
involved in the modulation is determined by the type of G protein
coupled to the receptor. For example, activation of various
muscarinergic receptors in rat striatal neurons modulates L-, N- and
P-type Ca2+ currents via different pathways
(Howe and Surmeier 1995
). On the other hand, a given
type of Ca2+ channel may be modulated by various
signaling pathways. Q-type Ca2+ currents, for
example, are upregulated by channel phosphorylation via protein kinase
A (PKA) (Kaneko et al. 1998
) and protein kinase C (PKC)
(Wheeler et al. 1994
). Separate signaling pathways may act independently or in a synergistic or antagonistic manner
(Selbie and Hill 1998
).
The pore-forming 1 subunits of vertebrate
voltage-gated Ca2+ channels can be grouped into
the three families CaV1 to
CaV3 (Ertel et al. 2000
).
Representatives of each family also occur in invertebrates (Littleton and Ganetzky 2000
). For the properties of
invertebrate Ca2+ channel currents and their
modulation, see recent reviews for mollusks (Kits and Mansvelder
1996
) and insects (Wicher et al. 2001
). Apart
from the exogenous control by modulators, many
Ca2+ channels respond to an increase in
Ca2+ concentration at the inner mouth of their
pore with channel inactivation and thus limit further
Ca2+ influx (e.g., Lee et al.
1999
; Romanin et al. 2000
).
The present study was performed to elucidate the mechanism of a
neuropeptide-mediated modulation of voltage-activated
Ca2+ currents in central insect neurons. The
cells used in these investigations, efferent dorsal unpaired median
(DUM) neurons from the cockroach Periplaneta americana,
belong to one of some groups of neurosecretory insect neurons the
somata of which generate spontaneous action potentials (Burrows
1996).
The electrical activity originating in the soma of these DUM cells is
controlled by the concerted activity of a large variety of ionic
channels, including voltage-gated Ca2+ channels
(Grolleau and Lapied 2000). The octapeptide neurohormone D (NHD) modulates the activity of DUM cells (for review, see
Wicher et al. 2001
). NHD not only affects the spike
frequency but also the shape of action potentials. It lowers the action
potential threshold, increases the overshoot, shortens the duration,
and enhances the hyperpolarization (Wicher et al.
1994
). The analysis of NHD action on the level of ionic
currents revealed a modulation of both depolarizing and repolarizing
currents, including a voltage-independent ("resting")
Ca2+ current (Wicher et al. 1994
),
voltage-gated Ca2+ currents (Wicher and
Penzlin 1994
) and Ca2+-dependent
K+ currents (Wicher et al. 1994
).
Previous studies have shown that the total Ca2+
current in DUM neurons is composed of various components, namely low-voltage-activated (LVA) currents, mid/low voltage-activated (M-LVA)
currents, and high-voltage-activated (HVA) currents (Grolleau and Lapied 1996
; Wicher and Penzlin 1997
).
Therefore before elucidating the signal transduction mechanism(s)
involved in the peptidergic modulation the question has to be answered
which of these components are affected by NHD.
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
ganglia of adult cockroaches (P. 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,
Taufkirchen, Germany) and 0.5 mg/ml collagenase (type I, Sigma). 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, which 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 technique (whole cell configuration). 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). 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 leakage currents were compensated
by a cancellation routine. Remaining uncompensated leakage currents
were subtracted using an on-line P/4 procedure (Bezanilla and
Armstrong 1977
) with a holding potential for leakage measurement of
110 mV. The series resistance remaining after compensation did not exceed 1.3 M
. The holding potential
(Vhold) was
90 mV.
Separation of Ca2+ currents in these DUM neurons
was performed as described elsewhere (Wicher and Penzlin
1997). The pipette solution used for this separation contained
(in mM) 100 choline methyl sulfate (CMS), 60 CsOH, 8 CsCl, 30 tetraethylammonium (TEA)-Br, 2 Mg-ATP, 1 CaCl2, 3 EGTA, and 10 HEPES. The free Ca2+ concentration
was calculated to amount to ~70 nM (Winmaxc v1.80 by Bers et
al. 1994
). The bath solution for Ca2+
current measurements contained (in mM): 190 CMS, 5 CaCl2, and 10 HEPES and 0.5 µM tetrodotoxin
(TTX). When using Ba2+ or
Sr2+ as charge carriers the 5 mM
Ca2+ in the bath solution were substituted for by
3 mM Ba2+ and 3 mM Sr2+,
respectively. For quantitative comparison of Ca2+
currents with such currents, Sr2+ was preferred
to Ba2+ since the current-voltage
(I-V) relationship of Sr2+ currents is
not shifted toward lower voltages like the I-V curve of
Ba2+ currents (Wicher and Penzlin
1997
). 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. The
combinations of bath and pipette solutions allowed measurements of
isolated Ca2+ currents ~2 min after breaking
into the cell. Within this time, contaminating
Na+ and K+ currents
disappeared completely. For data analysis, only such cells were used in
which the run-down of Ca2+ currents was <10%
within the time required for the investigations (~15 min).
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) 190 K-gluconate, 5 NaCl, 1 CaCl2, 3 EGTA, 2 Mg-ATP, and 10 HEPES (pH = 7.25). Between recordings (duration, 1 s) the cells were held
under voltage clamp at a holding potential of
70 mV.
NHD was obtained from Peninsula (Belmont), forskolin and 3-isobutyl-1-methylxanthine (IBMX) from RBI (Natick), oleoylacetylglycerol (OAG) from Alexis (Grünberg, Germany), KT5720 and Gö6976 from Calbiochem (Bad Soden, Germany), the catalytic subunit of PKA (porcine heart) from Biomol (Hamburg, Germany), 8br-cAMP, TTX and TEA from Sigma (Taufkirchen, Germany). Application or washout of blocking agents was performed by transferring the cell (attached to the pipette tip) within a glass tube into the different solutions. A complete and fast solution change was achieved by sucking a small amount of solution into the tube.
Data analysis
Results are given as means ± SD (n = number of cells). The evaluation of statistical significance of
differences was performed with two-way ANOVA. For data analysis
including nonlinear fitting procedures, the software Prism 2 (Graph Pad
Software, San Diego, CA) was used. Current-voltage relationships for
Ca2+ peak currents I(V)
were fitted taking into account current rectification according to a
Goldman-Hodgkin-Katz (GHK)-model of the form
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RESULTS |
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The peptide NHD affected Ca2+ channel currents in cockroach DUM neurons from 5th abdominal ganglion in a complex manner depending on concentration, voltage, and charge carrier. The analysis of the effects of voltage and charge carrier has been performed using a fixed NHD concentration of 10 nM, which was the highest concentration tested.
NHD action and charge carrier
The effects of NHD with Ca2+ as charge
carrier are shown in Fig. 1A.
NHD enhanced Ca2+ currents in the lower voltage
range (from 40 to 0 mV), and it slightly reduced currents obtained by
depolarizations positive to 0 mV (Fig. 1A2). The current
induced by NHD was maximal at
20 mV and it strongly inactivated. The
time of half-maximal decay was <10 ms (Fig. 1A1). Thus the
total rise of current by NHD at this potential was much more pronounced
for the peak current than for the current measured at the end of a
20-ms lasting voltage jump (rise by 3.2 ± 0.6 vs. 0.7 ± 0.3 nA, n = 7). Similar effects of NHD on
Ca2+ currents have been previously found for DUM
neurons from terminal ganglia (Wicher and Penzlin 1994
).
In these cells, the NHD-effect on total Ca2+
current was shown to result from the potentiation of a current component activating positive to
50 mV on the one hand and from the
attenuation of a current component activating positive to
30 mV on
the other hand.
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The total Ca2+ current in DUM neurons is the sum
of various components. There are two LVA currents, a transient
component with an activation threshold of 70 mV and a maintained
component activating positive to
60 mV. The maximum of the
I-V relation of LVA currents is
50 mV (Grolleau and
Lapied 1996
). Furthermore, there is a M-LVA current that
activates positive to
50 mV and reaches maximum at
10 mV. This
current shows Ca2+-induced decay, and it
undergoes strong steady-state inactivation when the holding potential
is depolarized to
40 mV. The M-LVA current is sensitive to the
blockers of mammalian P/Q-type Ca2+ currents,
-conotoxin MVIIC, and
-agatoxin IVA (Wicher and Penzlin 1997
). Finally, there are HVA currents with activation
threshold of
30 mV and maximum of I-V relation at +15 mV.
These currents show weak Ca2+-induced decay, and
they appear hardly reduced at depolarized holding potential of
50 mV.
HVA currents are sensitive to the blockers of mammalian L-type
Ca2+ current, verapamil and diltiazem, and to the
blocker of mammalian N-type Ca2+ current,
-conotoxin GVIA (Wicher and Penzlin 1997
). The
different electrophysiological and pharmacological properties of the
various components of total Ca2+ current should
provide the basis for answering the question whether NHD acts on some
or only one current component and, furthermore, for identifying the
modulated component(s).
Since at voltages between 30 and
10 mV the
Ca2+-dependently decaying M-LVA current is the
dominating component, Ca2+-induced inactivation
of total Ca2+ currents is most pronounced in this
voltage range (Wicher and Penzlin 1997
). The strong and
fast decay of the NHD-induced Ca2+ current
probably reflects such Ca2+-dependent process. It
should be noted that the NHD-induced Ca2+ current
decays considerably faster than the total Ca2+
current. For example, the time of half-maximal decay at
20 mV was
~10 ms for the former (Fig. 1A1), whereas it was ~30 ms
for the latter current (Wicher and Penzlin 1997
).
When Sr2+ was used as charge carrier, the effect
of NHD was somewhat different from that found for
Ca2+. Again, 10 nM NHD enhanced the current in
the lower voltage range with largest effect at 20 mV, but the
NHD-induced Sr2+ current did not inactivate
during the course of the test pulse (Fig. 1B1). Thus in
contrast to Ca2+ currents, the rise of
Sr2+ peak current by NHD at this potential
compared with the rise of the current measured at the end of a 20-ms
lasting voltage jump (rise by 4.3 ± 0.7 vs. 4.2 ± 0.6 nA,
n = 7). Furthermore, NHD did not reduce the
Sr2+ current in the high-voltage-range (Fig.
1B2). Also with Ba2+ there was no
attenuation of currents by NHD (n = 5; not shown). As
stated in the preceding text, in DUM neurons from terminal ganglia NHD
was found to reduce HVA Ca2+ currents
(Wicher and Penzlin 1994
). To test whether NHD also reduces HVA currents carried by Sr2+, a
depolarized holding potential of
40 mV was chosen at which LVA and
M-LVA currents undergo steady-state inactivation. Under these
conditions, NHD hardly affected the remaining
Sr2+ currents (Fig. 4C). For example,
in the presence of 10 nM NHD the peak of Sr2+
currents activated at +20 mV amounted to 94 ± 12% of control (n = 5).
Thus the NHD-induced reduction of HVA currents seems to rely on the
charge carrier Ca2+, and it is possibly caused by
a Ca2+-dependent type of inactivation. There are
two types of Ca2+ channels the upregulation of
which by NHD may induce such inactivation. These are, on the one hand,
voltage-gated Ca2+ channels with a low activation
threshold and on the other hand, voltage-independent
Ca2+ channels that conduct the resting
Ca2+ current
ICa,R (Wicher and Reuter
1993; Wicher et al. 1994
).
ICa,R is sensitive to two organic
compounds which block nonselective cation currents in various
preparations, the chloride channel blocker
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and the
anti-inflammatory drug mefenamic acid (Heine and Wicher
1998
). When NHD (10 nM) was applied in the presence of 10 µM
NPPB, Ca2+ currents appeared generally increased,
i.e., also in the high-voltage-range (Fig.
2). In the voltage range from
40 to
20 mV, the NHD effect on currents obtained in the presence of NPPB
was not significantly different from that obtained in the absence of
NPPB (ANOVA). By contrast, at depolarizations positive to
20 mV NHD
significantly enhanced Ca2+ currents when NPPB
was present (ANOVA, P < 0.05, Fig. 2B).
These results indicate that the increased Ca2+
influx through voltage-gated Ca2+ channels with a
low activation threshold does not cause
Ca2+-dependent reduction of HVA currents. Instead
of this, the enhanced Ca2+ resting conductance
seems to account for this phenomenon. On the other hand, the
NHD-induced increase of currents activating in the lower voltage range
was obviously not affected by the enhanced Ca2+
resting conductance. This finding is remarkable insofar as these currents show pronounced Ca2+-dependent
inactivation (cf. Fig. 1A1). Possibly the channels conducting this type of current are located in some distance from the
Ca2+ resting channels. In this case, the
Ca2+ entering the cells through the resting
conductance might be buffered within the cytosol or sequestered into
intracellular stores before it diffuses to the channels that are
upregulated by NHD (Messutat et al. 2001
). An
application of NHD for several minutes, however, leads to a global
strong rise in the free intracellular Ca2+
concentration [Ca2+]i due
to Ca2+ release from intracellular stores
(Heine and Wicher 1998
). Under such conditions, i.e.,
after application of 10 nM NHD for 4 min, a reduction of
Ca2+ currents in the whole voltage range was
observed (n = 4).
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Effect of NHD on steady-state activation and inactivation
These experiments were performed with Sr2+
as charge carrier to avoid two sources of error. First, the fast
Ca2+-dependent inactivation of
Ca2+ currents may hamper the determination of
steady-state activation, and second, the long 5 s-prepulses necessary
for evaluation of inactivation (Wicher and Penzlin 1997)
may lead to Ca2+-induced run-down. Application of
10 nM NHD caused a shift of both activation and inactivation toward
more negative potentials (Fig. 3). The
activation curves shown in Fig. 3A are described by
Boltzmann equations for the conductance G
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Concentration-dependence of NHD effects
The effects of NHD on Ca2+ channel currents
were concentration dependent. NHD effects appeared ~10 s after
application and reached steady state at 1 min. After washing off the
peptide, the currents returned to the control level within 1 min. At
all concentrations tested, the most prominent changes were obtained in
the voltage range between 30 and 0 mV. At 10 fM, NHD reduced
Ca2+ currents in this range by ~15%
(n = 6). The threshold for potentiation of
Ca2+ currents was ~1 pM. Figure 3C
shows that increasing concentrations of NHD gradually enhanced
Ca2+ currents activated at
20 mV. When
Ca2+ was substituted for by
Ba2+, no attenuation of currents at 10 fM NHD was
observed (106 ± 13% of control at
30 mV, n = 5), and the NHD-induced potentiation of current at higher
concentrations became more pronounced than with
Ca2+ (Fig. 3C). The enhancing effect
of 10 nM NHD on Ba2+ current (increase by
108 ± 38%, n = 6) was very similar to that on
Sr2+ current (increase by 105 ± 29% at
30 mV; Fig. 1B2). Because for both,
Ca2+ and Ba2+ currents, the
10-fold increase in peptide concentration from 1 to 10 nM did not
produce a statistically significant difference in current potentiation,
10 nM is expected to be near a saturating concentration.
NHD potentiates the M-LVA current
Because the current induced by NHD shares some properties with the
M-LVA current such as voltage range of activation and pronounced decay,
it seemed probable that NHD enhances just this current. Indeed, when
the M-LVA channel was blocked by 50 nM -agatoxin IVA
(n = 3) or 1 µM
-conotoxin MVIIC
(n = 4), NHD (10 nM) did not affect the residual
Sr2+ current. A representative example of an
experiment with
-conotoxin MVIIC (
-CmTx) is shown in Fig.
4, A and B. Further
support for the assumption that the M-LVA current
which shows strong
steady-state inactivation at a holding potential of
40 mV
is
selectively affected by NHD was provided by the observation that 10 nM
NHD failed to affect Sr2+ currents obtained by
depolarizations from holding potential of
40 mV (n = 5, Fig. 4C).
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Signal transduction mechanism
CAMP SYSTEM AND PKA.
A previous study has shown that bath application of the
membrane-permeant cAMP analogue 8bromo-cAMP (8br-cAMP) may enhance the M-LVA Ca2+ current (Achenbach et al.
1997). This suggests that one of the parameters regulating the
M-LVA Ca2+ channel might be the cAMP level. Most
neuropeptide receptors presently known are metabotropic (Iversen
1995
) and thus modulate second-messenger systems via G-protein
stimulation. Thus the latter was expected to mimic the effect of NHD
and enhance the M-LVA Ca2+ current. To test this,
the nonhydrolysable GTP analogue guanosine 5-O-3-thiotriphosphate (GTP
S) was added to the pipette
solution (at 5-500 µM). Ca2+ currents obtained
under this condition appeared enlarged with most pronounced differences
in the voltage range between
40 and 0 mV. For example, the peak
current at
20 mV amounted to 8.2 ± 3 nA (n = 7)
in the presence of 500 µM GTP
S versus 4.8 ± 1.5 nA in the
control. Direct evidence for the current-enhancing effect of GTP
S
was obtained by injection into neurons after a control registration of
currents. Again, Ca2+ currents were enhanced with
the largest effect at
20 mV whereby the increase amounted to 25%
(n = 3). Characteristically, GTP
S enhanced
Ca2+ currents at all voltages positive to
40
mV, which is in contrast to the current reduction in the HVA range
obtained with 10 nM NHD (cf. preceding text).
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PKC.
Q-type channels, i.e., those vertebrate Ca2+
channels being sensitive to -conotoxin MVIIC were upregulated by PKA
(Kaneko et al. 1998
) as well as by PKC (Wheeler
et al. 1994
). Insect Ca2+ channels may
also contain potential phosphorylation sites for both, PKA and PKC, as
was shown in the house fly Musca domestica (Grabner
et al. 1994
). To evaluate a possible role of PKC in the control
of Ca2+ channels in DUM neurons, the effect of an
activator of PKC, the diacylglycerol analogue, OAG, was tested. When
applied at a concentration of 1 µM for 30 s to 2 min, OAG
enhanced Sr2+ currents similar to NHD (Fig.
6; n = 6). Subsequent
application of NHD only slightly but not statistically significantly
enhanced the currents (n = 5). On the reverse, when NHD
was applied at first, a second application of OAG did not further
increase currents (n = 4, not shown). Preincubation of
cells for
15 min with the inhibitor of PKC, Gö6976 (1 µM), prevented the current increase by NHD and 8br-cAMP (Fig.
6B). This raises the question of why inhibition of PKC
disrupts the NHD-signal transduction cascade which in the preceding
text was shown to critically depend on activation of PKA. One reason
might be that the availability of M-LVA currents requires basal
activity of PKC. When, however,
-conotoxin MVIIC was applied after
preincubation of cells with Gö6976, the toxin caused a
reversible reduction of currents by 75% at
20 mV (n = 5, not shown). Thus the M-LVA current was not suppressed by
inhibition of PKC. Alternatively, PKC may either cross-react with PKA
phosphorylation sites at the M-LVA channel or PKC may phosphorylate the
PKA thereby activating the enzyme. If the latter was true, inhibition
of PKA e.g., by KT5720, should depress the response to OAG. Indeed,
when OAG (1 µM) was applied 2 min after administration of 10 µM
KT5720 (which attenuated currents, cf. the preceding text), OAG no
longer increased the currents. For example, 90 s after OAG
application, the size of Sr2+ currents at
20 mV
amounted to 75 ± 8% of control (Fig. 6; n = 5).
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Potentiation of the M-LVA current and action potentials
To assess the major consequences of the NHD-induced Ca2+ current changes on spiking, current-clamp investigations have been performed. NHD not only increases the spike frequency of DUM neurons but also causes changes in the shape of action potentials (APs, Fig. 7A). NHD (10 nM) induced, for example, a hyperpolarizing shift of AP threshold by 3.9 ± 0.9 mV and an increase of hyperpolarization by 5.5 ± 1.0 mV (n = 7). As shown in Fig. 1A2, the current-voltage relation for Ca2+ peak currents in the presence of 10 nM NHD is shifted by ~5 mV toward hyperpolarization (cf. Fig. 3A). Thus with NHD, the Ca2+ currents can provide for an additional pacemaker shift and/or contribute to the early phase of the AP upstroke thereby lowering the AP threshold.
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The deeper AP hyperpolarization obtained with NHD, however, cannot be
readily explained by the change of the Ca2+
activation curve. Previous investigations on DUM neurons, involving 10 mM TEA or application of Ca2+ channel blockers
indicated that Ca2+-dependent
K+ (KCa)
currents play an important role in the control of AP duration and
hyperpolarization (Lapied et al. 1989; Wicher et
al. 1994
). These KCa currents
are sensitive to charybdotoxin (Grolleau and Lapied
1995
; Wicher et al. 1994
) and iberiotoxin
(Wicher and Walther 1999
), the latter of which is a
specific blocker of BK-type KCa currents (Kaczorowski and Garcia 1999
). To confirm and
to extend the preceding findings on the role of the repolarizing
KCa currents by more specific means,
they were blocked by 100 nM iberiotoxin (IbTx, Fig. 7B).
This lead to larger overshoots (increase by 5 ± 2 mV) and
significant prolongation of APs, and the level of hyperpolarization
attained after the spike became more positive (by 9 ± 2 mV;
n = 4). Block of the M-LVA Ca2+
current by
-agatoxin IVA (50 nM) changed the shape of the AP much
like IbTx (Fig. 7C; overshoot increased by 7 ± 3 mV;
hyperpolarization reduced by 9 ± 3 mV; n = 4).
Interestingly, diltiazem (10 µM) and
-conotoxin GVIA (1 µM),
which block Ca2+ current components different
from the M-LVA current (Wicher and Penzlin 1997
), had
little effect on APs (not shown). With diltiazem, the overshoot was not
increased but somewhat reduced (by 5 ± 2 mV), and the
hyperpolarization was reduced clearly to a lesser extent than by
-agatoxin IVA (by 4 ± 1 mV, n = 4).
-conotoxin GVIA did not significantly affect neither overshoot nor
hyperpolarization (n = 6). These findings indicate that
the M-LVA current plays a crucial role in the activation of the
KCa currents and thus in the control
of AP duration and hyperpolarization. Taking into account that NHD
potentiates just the M-LVA current the observed increase in AP
hyperpolarization by NHD might be accounted for by this fact.
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DISCUSSION |
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This study shows that the effects of the neuropeptide NHD on
voltage-activated Ca2+ currents in cockroach DUM
neurons arise from two independent mechanisms. The large,
concentration-dependent potentiation of currents activated by moderate
depolarizations results from upregulation of the M-LVA
Ca2+ current. The identification of M-LVA current
as the target of modulation was indicated by voltage dependence and
kinetics of the NHD-generated current and could be convincingly
demonstrated by the use of the M-LVA current blockers -conotoxin
MVIIC and
-agatoxin IVA, which abolished the NHD-effect.
The reduction of Ca2+ currents within the whole voltage range at low NHD concentrations (<1 pM) and within the high-voltage range at higher concentrations (10 nM) could be attributed to the NHD-induced potentiation of the resting Ca2+ current ICa,R. There are three lines of evidence for this suggestion. First, NHD did not reduce HVA currents carried by Sr2+ or Ba2+. Second, NHD did not attenuate Ca2+ currents in the HVA range when ICa,R was blocked by NPPB. Third, agents mimicking the enhancing effect of NHD at lower voltages such as 8br-cAMP, forskolin, or PKA did not depress HVA Ca2+ currents.
The experiments addressing the signal transduction mechanism that
mediates the NHD-induced upregulation of the M-LVA
Ca2+ current demonstrated a central role of the
cAMP system and PKA. The transduction process most probably starts with
the activation of a metabotropic peptide receptor on NHD binding
thereby activating a G protein. There was no indication for a direct
effect of the activated G protein on Ca2+ current
like it was found for vertebrate N-type and P/Q-type channels
(Catterall 1997). This G protein presumably belongs to the Gs class and rises
via stimulation of
adenylyl cyclase
the cAMP level. cAMP subsequently activates the
cAMP-dependent protein kinase (PKA) that eventually phosphorylates the
channel passing the M-LVA Ca2+ current.
Regulation of Ca2+ currents by channel
phosphorylation via PKA is a mechanism found both in invertebrates
(Kits and Mansvelder 1996
; Wicher et al. 2001
) and vertebrates (Brammar 1999
). Although
there is no strong similarity in the electrophysiological and
pharmacological properties between the cockroach M-LVA
Ca2+ current and any type of vertebrate currents,
mammalian P/Q-type Ca2+ currents share some
properties with this insect current such as the sensitivity to the
specific blockers
-agatoxin IVA and
-conotoxin MVIIC
(Wicher and Penzlin 1997
). Interestingly, both P- and
Q-type currents were upregulated by phosphorylation via PKA
(Gubitz et al. 1996
; Kaneko et al. 1998
).
On the other hand, Q-type currents may also be increased by PKC as was
shown in the presynapse of hippocampal neurons (Wheeler et al.
1994
).
The findings that in DUM neurons, on one hand, activation of PKC mimics
the NHD effect and mainly occludes further current potentiation by NHD
and that, on the other hand, PKC activation was ineffective when
performed after NHD application indicate a role of PKC in the
transduction process of the NHD signal. This was further supported by
the observation that NHD and 8br-cAMP failed to potentiate
Ca2+ channel currents when PKC was inhibited. On
the reverse, inhibition of PKA did not only depress the action of NHD
but also that of OAG, i.e., neither the activation of the cAMP cascade
without PKA nor activation of PKC alone can enhance the currents. A
consistent explanation of these data would be the proposal that
activation of PKA might require preceding phosphorylation by PKC. A
similar convergence of signaling systems occurs in hippocampal neurons where concurrent activation of PKC enhanced the effect of PKA on the
Na+ current (Cantrell et al.
1999). Also in the serotonergic modulation of rat motoneurons,
inhibition of PKA blocked the effect of PKC and vice versa
(Inoue et al. 1999
). One might speculate whether the PKA
in DUM neurons exhibits several consensus sites for PKC and whether
phosphorylation might regulate the affinity to cAMP. The latter
suggestion would explain why NHD fails to further enhance Ca2+ channel currents after activation of PKC. In
that case, the basal cAMP level would already be sufficient to induce
activation of PKA. However, to verify these suggestions, further
experiments are required that are beyond the scope of the present study.
Generation and maintenance of spiking in DUM neurons depends both on
Na+ and Ca2+ currents
(Grolleau and Lapied 2000). Interestingly, in addition to the modulation of Ca2+ currents, NHD also
affects the Na+ current in these cells. NHD
attenuates the Na+ current via reducing the time
constant of inactivation, and this effect is most probably mediated by
channel phosphorylation by PKA (Wicher 2001
). The
investigation of the modulatory actions of NHD thus have shown that
Na+ and Ca2+ currents were
differently affected. The reduction of the Na+
current contrasts to the potentiation of the M-LVA
Ca2+ current. In both cases, the signal
transduction pathway includes activation of PKA. There seems to be,
however, a difference in the putative role of PKC. Whereas it plays, if
at all, only a minor role in the modification of the
Na+ current (Wicher 2001
), at
least a basal activity of this enzyme seems to be required for the
potentiation of the M-LVA Ca2+ current.
In DUM neurons, an influx of Na+ or
Ca2+ activates Na+- or
Ca2+-dependent K+ currents,
respectively (Grolleau and Lapied 2000). Both types of
K+ currents, KNa
and KCa, activate rapidly thereby
providing for a fast repolarization of the action potential
(Wicher et al. 2001
). However, there is a main
difference between them, namely in the dependence on voltage.
KNa is independent of voltage
and therefore peaks when the Na+ influx
is maximal, i.e., at around
15 mV (Grolleau and Lapied 1994
). By contrast, KCa is
voltage-gated and attains the maximum at much more depolarized
potentials (Grolleau and Lapied 1995
; Wicher et
al. 1994
). The NHD-induced reduction of
Na+ influx implies a reduction of
KNa (Wicher 2001
),
whereas the potentiation of the M-LVA
Ca2+ current enhances
KCa (Wicher et al.
1994
) (to check whether there is also a direct effect of NHD on
KCa was not yet performed). When the
BK-type blocker iberiotoxin and the M-LVA current blocker
-agatoxin
IVA were tested to assess the roles of
KCa and the M-LVA
Ca2+ current in spiking, it became clear that
KCa participates in adjusting AP
hyperpolarization and AP duration and that the M-LVA Ca2+ current plays a central role in the
activation of KCa. Because block of
both, the KCa and the M-LVA
Ca2+ current, prolonged APs and considerably
reduced hyperpolarization, it seems reasonable to conclude that
potentiation of these currents would have contrary effects on the APs,
i.e., effects that were indeed observed on application of NHD. Thus the
potentiation of the M-LVA Ca2+ current by the
peptide has two main effects on shaping APs, namely it decreases the AP
threshold and it deepens the undershoot.
The modulation of the Ca2+ current is part of
orchestrated changes of electrical conductances by NHD that lead to
accelerated spiking and changed shape of action potentials. Efferent
DUM neurons release the biogenic amine octopamine, which modulates
peripheral structures such as visceral and skeletal muscles
(Stevenson and Spörhase-Eichmann 1995). The
electrical excitability of the DUM cell somata is thought to play a
role in triggering octopamine release (Burrows 1996
;
Finlayson and Osborne 1975
). Modulation of ionic
currents that control excitability including Ca2+
currents would then provide a mechanism to adjust neurosecretion according to the physiological demands.
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ACKNOWLEDGMENTS |
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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 20 March 2001; accepted in final form 18 July 2001.
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REFERENCES |
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