Peptidergic Modulation of Insect Voltage-Gated Ca2+ Currents: Role of Resting Ca2+ Current and Protein Kinases A and C

Dieter Wicher

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 omega -conotoxin MVIIC- and omega -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 omega -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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega 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 MOmega . 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 MOmega ) 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
<IT>I</IT>(<IT>V</IT>)<IT>=</IT><IT>GV</IT>{[<IT>1−exp−</IT>(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>rev</IT></SUB>)<IT>/25 mV</IT>]<IT>/</IT>[<IT>1−exp−</IT><IT>V</IT><IT>/25 mV</IT>]}

<IT>×</IT>{<IT>1/</IT>[<IT>1+exp−</IT>(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>0.5</IT></SUB>)<IT>/</IT><IT>S</IT>]}<IT>,</IT>
where G is the total Ca2+ conductance, Vrev is the Ca2+ reversal potential, V0.5 is the potential for half-maximal activation of total Ca2+ conductance, and S is the slope factor of activation (cf. Hille 1992).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Effect of 10 nM neurohormone D (NHD) on Ca2+ (A) and Sr2+ currents (B). A1: recordings of Ca2+ currents obtained by depolarizations from -90 to -20 mV (top left) and to +20 mV (top right). The NHD-induced current activated at -20 mV (NHD - control, bottom left) shows strong inactivation during the depolarizing pulse. A2: I-V relationships for Ca2+ peak currents before (control) and 2 min after application of NHD.  and , means of 7 cells; bars, SD. Curves were fitted to the data according to a GHK model (cf. METHODS). B1: recordings of Sr2+ currents obtained by depolarizations from -90 to -20 mV (top left) and to +20 mV (top right). Note that the NHD-induced attenuation of Ca2+ current in the high-voltage range is not observed with Sr2+ as charge carrier. The NHD-induced current activated at -20 mV (NHD - control, bottom left) does not show any remarkable inactivation during the depolarizing pulse. B2: I-V relationships for Sr2+ peak currents before (control) and 2 min after application of NHD.  and , means of 7 cells; bars, SD.

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



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. Effect of 10 nM NHD in the presence of 10 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). A: recordings of Ca2+ currents activated by jumps from -90 to -20 mV (left) and to +20 mV (right). B: I-V relationships for Ca2+ peak currents before (NPPB) and 2 min after application of NHD (NPPB + NHD).  and , means of 7 cells; bars, SD. Curves were fitted to the data according to a Goldman-Hodgkin-Katz (GHK) model (cf. METHODS).

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
<IT>G</IT><IT>/</IT><IT>G</IT><SUB><IT>max</IT></SUB><IT>=1/</IT>{<IT>1+exp</IT>[<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>0.5</IT></SUB>]<IT>/</IT><IT>S</IT>} (1)
where the potentials of half-maximal activation V0.5 are -22 mV (control) and -27 mV (NHD), and the slope factors S are 5.6 mV (control) and 4.2 (NHD). Thus NHD not only shifted the activation curve by 5 mV, but it also increased its steepness. The inactivation curves in Fig. 3B are described by Boltzmann equations for the current I
<IT>I</IT><IT>/</IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=1/</IT>{<IT>1+exp</IT>[<IT>V</IT><SUB><IT>0.5</IT></SUB><IT>−</IT><IT>V</IT>]<IT>/</IT><IT>S</IT>} (2)
where the figures for V0.5 are -41 (control) and -48 mV (NHD) and for S are 8.4 (control) and 9.1 mV (NHD).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. A and B: effect of NHD on activation and inactivation of Sr2+ currents. A: activation was determined from peak currents activated by depolarizing pulses from -90 mV to the indicated potentials and is given as normalized conductance G/Gmax before () and 2 min after application of 10 nM NHD ().  and , means of 7 cells; bars, SD. Differences between the data obtained under control conditions and in presence of NHD were statistically significant (ANOVA, P < 0.001). Curves were fitted to the data according to the Boltzmann Eq. 1 (see text). They are described by voltage of half-maximal activation, V0.5 = -22.0 (control) and -27.1 mV (NHD), and slope factor S = 5.6 (control) and 4.2 mV (NHD). B: steady-state inactivation was determined using a double-pulse protocol as shown in the inset. After conditioning prepulses to the potentials indicated, normalized Sr2+ peak currents I/Imax were obtained on jumps to -10 mV before () and 2 min after application of 10 nM NHD ().  and , means of n = 8 cells; bars, SD. Differences between the data obtained under control conditions and in presence of NHD were statistically significant (ANOVA, P < 0.001). Curves were fitted to the data according to the Boltzmann Eq. 2 (see text). They are described by voltage of half-maximal activation, V0.5 = -40.8 (control) and -48.1 mV (NHD), and slope factor S = 8.3 (control) and 9.1 mV (NHD). C: concentration dependence of the NHD effect on Ca2+ () and Ba2+ current (). Data represent means of peak currents obtained by depolarizations from -90 to -20 mV (Ca2+) or to -30 mV (Ba2+); n = 4 to 6 cells; bars, SD. The difference of NHD effect on Ca2+ vs. Ba2+ currents is statistically significant (P < 0.05, 2-way ANOVA). The isotherms fitted to the data points are described by EC50 = 24 (Ca2+) and 8 pM (Ba2+), and Hille slope S = 0.67 (Ca2+) and 0.53 (Ba2+).

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 omega -agatoxin IVA (n = 3) or 1 µM omega -conotoxin MVIIC (n = 4), NHD (10 nM) did not affect the residual Sr2+ current. A representative example of an experiment with omega -conotoxin MVIIC (omega -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).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4. NHD acts on the mid/low-voltage-activated (M-LVA) current component. A: I-V-relations of Sr2+ currents in one DUM neuron obtained by voltage ramps from -55 to +50 mV (in 500 ms). Block of the M-LVA current by application of 1 µM omega -conotoxin MVIIC reduces the total current. In the presence of the toxin, NHD (10 µM) fails to increase the current. B: Sr2+ currents activated by voltage steps as indicated. omega -conotoxin MVIIC (1 µM) strongly reduces the current, and, again, NHD (10 nM) does not enhance the remaining current. C: NHD (10 nM) fails to affect the Sr2+ current activated by a voltage step from depolarized holding potential of -40 to +20 mV, i.e., the peptide has no direct effect on HVA currents.

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 (GTPgamma 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 GTPgamma S versus 4.8 ± 1.5 nA in the control. Direct evidence for the current-enhancing effect of GTPgamma 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, GTPgamma 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).

To get more evidence for an involvement of the cAMP system in the signal tranduction process activated by NHD, various modulators acting on components of this system were tested. Figure 5B shows the effects of these modulators on peak currents obtained at -20 mV in comparison with the effect of 10 nM NHD. Forskolin (10 µM), an activator of adenylyl cyclase, enhanced Ca2+ currents similar to GTPgamma S or 8br-cAMP, i.e., at all voltages positive to -40 mV thereby attaining the largest effect in the lower voltage range. At -20 mV, forskolin enhanced the Ca2+ peak current by 63 ± 33% (n = 4), a figure that compares to the response obtained on application of 8br-cAMP (rise by 73 ± 37%, n = 5). The inhibitor of phosphodiesterase, IBMX (10 µM), increased Ca2+ currents in a manner similar to forskolin (n = 3, data not shown). When, on the other hand, NHD (10 nM) was applied in the presence of 8br-cAMP no significant further increase in current size was observed (Fig. 5B).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 5. Effects of Ca2+ channel phosphorylation by protein kinase A (PKA) on Ca2+ currents. A: currents activated by jumps from -90 to -20 mV. Left: current before (control) and 2 min after injection of the catalytic subunit of PKA. Right: inhibition of PKA by KT5720 (10 µM) attenuates the current, and in its presence, NHD (10 nM) fails to enhance the current. B: Ca2+ peak currents (obtained on depolarization to -20 mV; control = 100%) were enhanced by bath application of 10 nM NHD, 1 µM 8-br-cAMP, 10 µM forskolin and by injection of catalytic subunit of PKA. When NHD (10 nM) was applied in the presence of 1 µM 8-br-cAMP, it did not further increase the current. Preincubation of cells with the PKA inhibitor KT5720 (10 µM) for 3-4 min not only prevents NHD (10 nM) from increasing the current but also decreases the current. Means of 5 to 8 cells; bars, SD. *, significant differences to the control (P < 0.05). C: I-V relations for Ca2+ peak currents before (control, black-lozenge ) and 2 min after injection of catalytic subunit of PKA (diamond ). black-lozenge  and diamond , means of 6 cells; bars, SD. Curves were fitted to the data according to a GHK model (cf. METHODS). D: I-V relations for Ca2+ peak currents before (control, ) and 2 min after application of KT5720 (), and 1 min after application of NHD (10 nM) in the presence of KT5720 for 3-4 min (). Note that the effect of PKA inhibition is less pronounced in the lower voltage range. open circle , , and , means of 8 cells; bars, SD. Curves were fitted to the data according to a GHK model.

The effect of cAMP on Ca2+ channel currents might be produced either directly or by activation of cAMP-dependent protein kinase (PKA). To decide which of these alternatives was realized in DUM neurons, the catalytic subunit of PKA was injected into the somata. This PKA injection caused similar current changes as obtained with cAMP and forskolin (Fig. 5, A-C). At -20 mV, PKA enhanced the Ca2+ peak current by 62 ± 21% (n = 5). On the other hand, in the presence of KT5720, a specific inhibitor of PKA, 10 nM NHD failed to enhance the Ca2+ currents. On the contrary, the currents appeared reduced and the attenuation to 62 ± 17% of control at -20 mV (n = 5) was statistically significant (paired t-test, P < 0.05; Fig. 5B). Similar results for the action of KT5720 and NHD were obtained with Sr2+ as charge carrier. For example, Sr2+ currents evoked by depolarization to -20 mV were attenuated to 63 ± 11% of control (n = 4). The effects on Ca2+ channel currents of PKA on the one hand and of KT5720 plus NHD on the other hand suggest that the mechanism of NHD action involves Ca2+ channel phosphorylation via PKA.

Interestingly, inhibition of PKA by KT5720 caused an attenuation of Ca2+ peak currents. As shown in Fig. 5D, 2 min after application of KT5720 this reduction was more pronounced in the higher voltage range (). In the lower voltage range e.g., at -20 mV, the reduction to 83 ± 12% of control was not statistically significant (paired t-test, Fig. 5B). This might indicate that some HVA Ca2+ channels require constitutive phosphorylation by PKA for maintained function. Prolonged presence of KT5720 for 4 min produced a further reduction of currents that compared with that observed after application of KT5720 plus NHD (Fig. 5B). Thus the above-mentioned reduction of currents observed with these two agents seems to result from the slow development of the KT5720 effect.

PKC. Q-type channels, i.e., those vertebrate Ca2+ channels being sensitive to omega -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, omega -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).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6. Effects of Ca2+ channel phosphorylation by protein kinase C (PKC) on Sr2+ currents. A: currents activated by jumps from -90 to -20 mV. Left: current before (control) and 2 min after application of 1 µM oleoylacetylglycerol (OAG). Application of 10 nM NHD in the presence of OAG leads to an only weak current increase. Right: inhibition of PKC by Gö9676 (1 µM) prevents NHD (10 nM) from enhancing the current. B: Ca2+ peak currents (obtained on depolarization to -20 mV; control = 100%) were enhanced by bath application of 10 nM NHD and 1 µM OAG. Application of NHD (10 nM) in the presence of OAG has no significant further effect. After preincubation of cells with the PKC inhibitor Gö9676 (1 µM), application of NHD (10 nM; Gö + NHD) or 8br-cAMP (1 µM; Gö + cAMP) had no significant effect on currents. OAG (1 µM) fails to enhance the current in the presence of the PKA inhibitor KT5720 (10 µM; KT + OAG). Means of 5 to 8 cells; bars, SD. *, significant differences to the control (P < 0.05). C: I-V relations for Sr2+ peak currents before (control, ) and 2 min after application of 1 µM OAG (). open circle , the effect of 10 nM NHD on peak currents in the presence of OAG. , , and open circle , means of 6 cells; bars, SD. Curves were fitted to the data according to a GHK model (cf. METHODS).

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.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7. Effects of NHD, iberiotoxin, and omega -agatoxin IVA on spiking. A: 10 nM NHD accelerates spiking (A1) and changes the shape of action potentials (APs; A2). It lowers the AP threshold, increases the overshoot, and enhances the hyperpolarization. B1: the specific BK-type KCa current blocker iberiotoxin (IbTx, 100 nM) prolongs the AP and reduces hyperpolarization. B2: omega -agatoxin IVA (AgaTx, 50 nM), which blocks the M-LVA Ca2+ current, also prolongs the AP and reduces hyperpolarization. The control was recorded after washing off the toxin because the toxin also affects the Na+ current in these cells but in irreversible manner (Wicher and Penzlin 1998). Thus with this sequence of recording, the effect of the toxin on the M-LVA current (which is reversible) could be separated. Current-clamp recordings; for solutions see METHODS; between registrations the cells were held under voltage-clamp conditions at -70 mV.

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 omega -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 omega -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 omega -agatoxin IVA (by 4 ± 1 mV, n = 4). omega -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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 omega -conotoxin MVIIC and omega -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 omega -agatoxin IVA and omega -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 omega -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.


    ACKNOWLEDGMENTS

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 20 March 2001; accepted in final form 18 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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