Peptidergic Modulation of an Insect Na+ Current: Role of Protein Kinase A and Protein Kinase C

Dieter Wicher

Sächsische Akademie der Wissenschaften zu Leipzig, D-07743 Jena, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In vertebrates the proteins forming alpha  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 alpha  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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega 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 MOmega . 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 MOmega ) 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
<IT>G</IT><IT>=</IT><IT>G</IT><SUB>max</SUB> ∗ <IT>m</IT><SUP><IT>3</IT></SUP><IT> ∗ </IT>[<IT>a </IT><IT>∗ </IT><IT>h</IT><SUB><IT>1</IT></SUB><IT>+</IT>(<IT>1−</IT><IT>a</IT>)<IT> ∗ </IT><IT>h</IT><SUB><IT>2</IT></SUB>] (1)
where Gmax is the maximum conductance, m is the activation parameter, and h1, h2 are inactivation parameters. The two inactivation parameters are necessary to model the biphasic time course of Na+ current inactivation where in addition to the usual, fast decay (time constant tau h1 = 0.5-5 ms) there is also a slow decay (tau h2 ~ 20 ms) (Lapied et al. 1990). The parameter a is the fraction of the fast inactivation (for potentials positive to -20 mV, a is ~0.9). As stated in RESULTS, the slowly decaying component remained unaffected in the presence of NHD. Therefore data analysis was performed using two inactivation time constants, but only data for the fast inactivation h1 are presented (referred to as simply h). The time dependencies of m and h are described in the usual fashion, i.e.,
<IT>m</IT>(<IT>t</IT>)<IT>=</IT><IT>m</IT><SUB><IT>∞</IT></SUB><IT>−</IT>(<IT>m</IT><SUB><IT>∞</IT></SUB><IT>−</IT><IT>m</IT><SUB><IT>0</IT></SUB>)<IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>m</SUB></IT>) (2)

<IT>h</IT>(<IT>t</IT>)<IT>=</IT><IT>h</IT><SUB><IT>∞</IT></SUB><IT>−</IT>(<IT>h</IT><SUB><IT>∞</IT></SUB><IT>−</IT><IT>h</IT><SUB><IT>0</IT></SUB>)<IT> exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>h</SUB></IT>) (3)
At very negative potentials (less than -90 mV) m0 and h0, the steady-state values before a voltage jump, are 0 and 1, respectively; i.e., no channels are activated and none are inactivated. The voltage dependence of minfinity and hinfinity is described by the Boltzmann equations
<IT>m</IT><SUB><IT>∞</IT></SUB><IT>=1/</IT>{<IT>1+exp</IT>[(<IT>V</IT><SUB><IT>m</IT></SUB><IT>−</IT><IT>V</IT>)<IT>/</IT><IT>K</IT><SUB><IT>m</IT></SUB>]} (4)

<IT>h</IT><SUB><IT>∞</IT></SUB><IT>=1/</IT>{<IT>1+exp</IT>[(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>h</IT></SUB>)<IT>/</IT><IT>K</IT><SUB><IT>h</IT></SUB>]} (5)
Vm and Vh are the potentials of half-maximal activation and inactivation, respectively; and Km and Kh are slope parameters.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Neurohormone D (NHD) modifies the Na+ current in cockroach dorsal unpaired median (DUM) neurons. A: Na+ currents activated by depolarizing pulses from -90 to -30 mV (A1) and -10 mV (A2) were recorded before (Control) and 2 min after application of 10 pM NHD. Note that the slowly inactivating current obtained at -30 mV is hardly affected by NHD, whereas the fast inactivating current obtained at -10 mV is reduced and appears somewhat shortened in the presence of NHD. The time course of current activation remains unchanged. B: effect of 10 pM NHD on the time course of currents activated by 10-ms lasting depolarizations to -30 mV (B1) and -10 mV (B2). Bars represent normalized currents (NHD/Control) measured at the indicated time intervals t from start of jumps (means ± SD from n = 7 cells).



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Fig. 2. Effect of 10 nM NHD on time-to-peak of Na+ current. A: voltage dependence of time-to-peak in absence (Control) and presence of NHD for a representative neuron. B: time-to-peak for currents obtained by jumps from -90 to -10 mV in absence (Control) and presence of NHD. Means ± SD from n = 7 cells. The asterisk marks significant difference (t-test).

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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Fig. 3. A: current-voltage (I-V) relationships for Na+ peak currents before (Control) and 2 min after application of 10 nM NHD. The currents were normalized to the maxima of I-V relations obtained under control conditions. Symbols, means of 8 cells; bars, SD. B: concentration-response relationship for the reduction of maxima of I-V relations (-10 mV) by NHD. Circles, means of n = 5-8 cells; bars, SD. The isotherm fitted to the data are described by IC50 = 5 pM and Slope = 0.35. C: time course of the Na+ peak current reduction by 10 nM NHD. Currents were obtained by depolarizing voltage steps from -90 to -10 mV. NHD application is indicated by the arrow. Circles, means of 8 cells; bars, SD. The time course of the toxin effect was described reasonably well with a single-exponential function (time constant tau  = 61 s).

The reduction of the peak current after application of 10 nM NHD developed in a single exponential fashion (time constant tau  = 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 tau r = 1.6 ± 0.1 ms in the absence and tau 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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Fig. 4. Recovery from inactivation of Na+ peak currents before (Control) and 2 min after application of 10 nM NHD. Time courses were obtained using a double pulse protocol as shown in the inset. Currents obtained at the test pulse were normalized by the conditioning prepulse. Data points, means of n = 7 cells; bars, SD. The data sets are significantly different (ANOVA, P < 0.001). Single-exponential functions with delayed onset (Delta t) were fitted to data points. The curves are described by time constant of recovery tau r = 1.6 ms and Delta t = 0.78 ms (Control), and tau r = 2.3 ms and Delta t = 0.78 ms (NHD).

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 minfinity , 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 hinfinity 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 tau m by NHD (Fig. 5A2; ANOVA). The only one parameter affected by NHD was the inactivation time constant. Peptide application caused a decrease of tau 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 tau h reversed within a few minutes after wash out of NHD.



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Fig. 5. Effect of NHD on activation (A) and inactivation (B) properties of Na+ currents under control conditions (black-square------black-square) and 2 min after application of 10 nM NHD (- - -). A1: steady-state activation minfinity . Data were obtained as cubic root of maximal conductance. Squares, means of n = 7 cells; bars, SD. Curves were fitted to the data according to the Boltzmann equation minfinity  = 1/{1 + exp[(Vm - V)/Km]}. They are described by voltage of half-maximal minfinity , Vm = -29 mV (Control) and -30 mV (NHD), and slope Km = 6.9 mV (Control) and 7.0 mV (NHD). Data obtained in the presence of NHD do not significantly differ from those obtained under control conditions (ANOVA). A2: activation time constant tau m. Data were derived from analysis of Na+ currents in terms of a Hodgkin-Huxley model (see METHODS). Squares, means of n = 7 cells; bars, SD. Differences between the data obtained under control conditions and in presence of NHD were statistically not significant (ANOVA). B1: steady-state inactivation hinfinity . Data were obtained using a double-pulse protocol as shown in the inset. Squares, means of n = 7 cells; bars, SD. Curves were fitted to the data according to the Boltzmann equation hinfinity  = 1/{1 + exp[(V - Vh)/Kh]}, and they are described by voltage of half-maximal hinfinity , Vh = -39 mV (Control) and -39 mV (NHD), and slope Kh = 7.0 mV (Control) and 6.7 mV (NHD). NHD did not affect the steady-state inactivation (ANOVA). B2: inactivation time constant tau h. Squares, means of n = 7 cells; bars, SD. The asterisks mark significant differences according to a paired t-test with P < 0.05. Inset: scatter plot of tau h for jumps to -10 mV, the maximum of the I-V curve, before (Control) and after application of NHD. The data sets are significantly different (paired t-test, P = 0.003).

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).

To get a hint whether the cAMP system may be involved in the NHD-induced Na+ current modification too, the effect of 8br-cAMP on this current was tested. Indeed, 1 µM 8br-cAMP mimicked the effect of NHD; i.e., it reduced the Na+ current duration and attenuated the peak at -10 mV by 24 ± 7% (n = 5; Fig. 6A1). Like with NHD, the current-voltage relation for peak currents was unchanged from threshold up to -25 mV, and it was reduced for stronger depolarizations (not shown). Furthermore, the recovery from inactivation became significantly slower (ANOVA, P = 0.0002). The time constant of recovery tau r was raised from 1.8 ± 0.2 ms (Control) to 2.8 ± 0.4 ms (8br-cAMP; Fig. 6B1). Similar results were obtained with the activator of adenylate cyclase, forskolin. In the presence of 10 µM forskolin, the Na+ current activation kinetics was not changed, whereas the inactivation became faster and the peak current at -10 mV was reduced by 20 ± 7% (n = 5; Fig. 6A1). In addition, forskolin increased tau r from 1.7 ± 0.1 ms before to 3.0 ± 0.2 ms after its application (Fig. 6B1).



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Fig. 6. Effects of Na+ channel phosphorylation by protein kinase A (PKA) on the Na+ current. A1: Na+ peak currents (depolarization from -90 to -10 mV; Control = 100%) were reduced by bath application of 10 nM NHD, 1 µM 8-br-cAMP, 10 µM forskolin, and injection of catalytic subunit of PKA. NHD did not affect the Na+ current in the presence of the PKA inhibitor KT 5720. Means of 5-9 cells; bars, SD. Asterisks mark significant differences to currents obtained under Control (paired t-test; P < 0.05). A2: I-V relations for Na+ peak current before (Control) and 2 min after injection of catalytic subunit of PKA. Means of n = 5 cells; bars, SD. Inset: current traces recorded on jumps from -90 to -10 mV before (Control) and 2 min after injection of PKA. B1: time constant of recovery from inactivation tau r (obtained as described in Fig. 4) were increased by bath application of 10 nM NHD, 1 µM 8-br-cAMP, 10 µM forskolin, and injection of catalytic subunit of PKA. NHD did not affect tau r in the presence of the PKA inhibitor KT 5720. Means of 5-9 cells; bars, SE. Asterisks mark significant differences to data obtained under Control (ANOVA). B2: recovery from inactivation of Na+ peak currents before (Control) and 2 min after injection of catalytic subunit of PKA. Data and curves were obtained as described in Fig. 4. Means of n = 5 cells; bars, SD. The data sets are significantly different (ANOVA, P < 0.001). The curves (cf. Fig. 4) are described by time constant of recovery tau r = 1.6 ms and Delta t = 0.78 ms (Control), and tau r = 3.0 ms and Delta t = 0.70 ms (PKA).

The Na+ channel modulation might result from a direct cAMP effect or from activation of a cAMP-dependent protein kinase (PKA). To decide which alternative was realized in the DUM cell Na+ channels, catalytic subunit of PKA was injected into cells. The PKA injection produced the same modulatory effects on the Na+ current as observed in the presence of NHD, 8br-cAMP, and forskolin (Fig. 6). The current traces in the inset in Fig. 6A2 show that PKA does not affect activation but accelerates inactivation (compare with Fig. 1A2). PKA reduced the peak current at -10 mV by 23 ± 4% (n = 5; Fig. 6A1) and caused a rise of tau r from 1.6 ± 0.1 ms to 3.0 ± 0.2 ms (Fig. 6, B1 and B2).

Furthermore, preincubation of cells with KT5720, a specific inhibitor of PKA, suppressed the effect of 10 nM NHD on Na+ current (Fig. 6, A1 and B1). Taken together with these results, the NHD effect on the Na+ current seems to be produced by PKA-mediated channel phosphorylation.

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.

Activation of PKC by 25 µM OAG reduced the Na+ current and shortened its duration at depolarizations positive to -30 mV (Fig. 7A2, and inset). The amount of reduction was 26 ± 9% at -10 mV (n = 5), i.e., a figure similar to that obtained with 10 nM NHD (Fig. 7A1). The activation of Na+ current, estimated from measured peak conductance, was not affected by OAG (ANOVA). The conductance was half-maximal at -19 mV both under control conditions and in the presence of OAG; the slope was 4.7 mV (Control) and 5.3 mV (OAG; n = 5; not shown). Also the steady-state inactivation was not changed by OAG (Vh was shifted from -37 to -39 mV and Kh from 7.5 to 7.7; statistically not significant according to ANOVA; not shown). The recovery from inactivation became slowed down (Fig. 7, B1 and B2). The time constant of recovery tau r increased from 1.8 ± 0.1 ms before (n = 4) to 2.2 ± 0.1 ms after OAG application (n = 4). Although this change was considered statistically significant (ANOVA, P < 0.05), it was clearly weaker than the change caused by 10 nM NHD (increase of tau r from 1.6 to 2.3 ms).



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Fig. 7. Effects of Na+ channel phosphorylation by protein kinase C (PKC) on the Na+ current. A1: Na+ peak currents (depolarization from -90 to -10 mV; Control = 100%) were reduced by bath application of 10 nM NHD and 1 µM oleoylacetylglycerol (OAG). The presence of OAG did not occlude the effect of NHD (since NHD caused further reduction of Na+ peak current by 19%). Currents were also reduced by NHD in the presence of 1 µM chelerythrine and 1 µM Gö 6976. Means of 5-7 cells; bars, SD. Asterisks mark significant differences to peak currents obtained under Control (paired t-test; P < 0.05). A2: I-V relations for Na+ peak current before (Control) and 2 min after application of 1 µM OAG. Means of n = 5 cells; bars, SD. Inset: current traces recorded on jumps from -90 to -10 mV before (Control) and 2 min after application of OAG. B1: time constant of recovery from inactivation tau r (obtained as described in Fig. 4) were increased by 10 nM NHD, 1 µM OAG, and 10 nM NHD in the presence of 1 µM OAG, 1 µM chelerythrine, and 1 µM Gö 6976. Means of 5-7 cells; bars, SE. Asterisks mark significant differences to data obtained under Control (ANOVA). B1: recovery from inactivation of Na+ currents before (Control) and 2 min after application of 1 µM OAG. Data and curves were obtained as described in Fig. 4. Means of n = 5 cells; bars, SD. The data sets are significantly different (ANOVA, P < 0.001). The curves are described by time constant of recovery tau r = 1.8 ms and Delta t = 1.0 ms (Control), and tau r = 2.2 ms and Delta t = 1.0 ms (PKA).

Nevertheless, application of 10 nM NHD after preincubation with 25 µM OAG caused an additional attenuation of the current (Fig. 8). The further reduction by 19 ± 7% (n = 5) produced by NHD in this situation was somewhat but not significantly smaller than the reduction of 23 ± 9% obtained in the absence of OAG (Fig. 7A1). Also the kinetics of recovery from inactivation was (in the presence of OAG) further slowed down by NHD (statistically significant, ANOVA, P = 0.0001). The time constant tau r was raised from 2.2 ± 0.2 ms before to 2.8 ± 0.2 ms after NHD application (Fig. 7B1).



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Fig. 8. Effect of preincubation of 1 µM OAG (A) and 1 µM Gö 6976 (B) on the effect of 10 nM NHD on the Na+ current. Currents were activated by jumps from -90 to -10 mV. NHD was added 3 min after application of OAG (A) or 15 min after incubation with Gö 6976.

These results show that PKC is capable of modulating the Na+ current in DUM neurons in a manner comparable to the action of NHD. This modulation, however, does not seem to impair the effect of NHD on the Na+ current. To test whether inhibition of PKC might affect the NHD action, the peptide was applied after 15 min preincubation of cells with two inhibitors of PKC, chelerythrine, and Gö 6976. In this situation, the Na+ peak current was reduced after application of 10 nM NHD by 17 ± 7% (n = 4) and 16 ± 6% (n = 5), respectively (Fig. 7A1). These reductions are significantly weaker than those observed in absence of PKC inhibitors (t-test, P < 0.02). For both agents, NHD prolonged the time constant of recovery from inactivation, although statistically significant (ANOVA), but again the effect was somewhat weaker than with NHD alone (Fig. 7B1). These first data obtained with the PKC inhibitors show that the NHD effect was not essentially impaired by PKC inhibition. Nevertheless, a synergistic effect of phosphorylation by PKA and PKC cannot be readily excluded.

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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Table 1. Effect of Na+ current reduction by TTX on action potential parameters


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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 omega -conotoxin MVIIC and omega -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 omega -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 omega -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).


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

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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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society