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Article

Ca2+/Calmodulin-Dependent
Protein Kinase Regulates GABA-Activated Cl− Current in Cockroach
Dorsal Unpaired Median Neurons

Philippe
Alix,

Francoise
Grolleau, and

Bernard
Hue

1 Laboratoire de Neurophysiologie Unité Propre de
Recherche de l'Enseignement Supérieur Equipe d'Accueil 2647, Université
d'Angers, Unité de Formation et de Recherche Sciences, F-49045 Angers Cedex,
France

Submitted 31 July 2001.

accepted in final form 4 February 2002.

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Abstract

We studied γ-aminobutyric acid (GABA)-mediated currents in short-term
cultured dorsal unpaired median (DUM) neurons of cockroach Periplaneta
americana using the whole cell patch-clamp technique in symmetrical
chloride solutions. All DUM neurons voltage-clamped at −50 mV displayed inward
currents (I GABA) when 10−4 M of GABA was applied
by pneumatic pressure-ejection pulses. The semi-logarithmic curve ofI
GABA amplitude versus the ejection time yielded a Hill coefficient of
4.0.I GABA was chloride (Cl−) because the
reversal potential given by the current-voltage (I-V) curve varied
according to the value predicted by the Nernst equation for
Cl−dependence. In addition, I GABA was almost
completely blocked by bath application of the chloride channel blockers
picrotoxin (PTX) or
3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile (BIDN). The
I-V curve forI GABA displayed a unexpected biphasic
aspect and was best fitted by two linear regressions giving two slope
conductances of 35.6 ± 2.1 and 80.9 ± 4.1 nS for potentials ranging from 0 to
−30 and −30 to −70 mV, respectively. At −50 mV, the current amplitude was
decreased by cadmium chloride (CdCl2, 10−3 M) and
calcium-free solution. The semi-logarithmic curve for
CdCl2-resistantI GABA gave a Hill coefficient of
2.4. Hyperpolarizing voltage step from −50 to −80 mV was known to increase
calcium influx through calcium-resting channels. According to this protocol, a
significant increase ofI GABA amplitude was observed.
However, this effect was never obtained when the same protocol was applied on
cell body pretreated with CdCl2. When the calmodulin
blockerN-(6-aminohexyl)-5-chloro-1-naphtalene-sulfonamide or the
calcium-calmodulin-dependent protein kinase blocker
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) was added in the pipette
solution,I GABA amplitude was decreased. Pressure ejection
application of the cis-4-aminocrotonic acid (CACA) on DUM neuron cell
body held at −50 mV, evoked a Cl− inward current which was
insensitive to CdCl2. The Hill plot yielded a Hill coefficient of
2.3, and the I-V curve was always linear in the negative potential
range with a slope conductance of 32.4 ± 1.1 nS. These results, similar to those
obtained with GABA in the presence of CdCl2 and KN-62, indicated that
CACA activated one subtype of GABA receptor. Our study demonstrated that at
least two distinct subtypes of Cl−-dependent GABA receptors were
expressed in DUM neurons, one of which is regulated by an intracellular
Ca2+-dependent mechanism via a calcium-dependent protein kinase. The
consequences of the modulatory action of Ca2+ in GABA receptors
function and their sensitivity to insecticide are discussed.

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INTRODUCTION

γ-aminobutyric acid (GABA)-gated chloride channel receptors are
largely widespread in the CNS of insects where their physiological role is to
mediate fast inhibitory neurotransmission (Anthony et al.
1993; Sattelle
1990). Although insect ionotropic receptors have been shown to share some
functional analogies with their vertebrate counterparts, many studies have
demonstrated that insect GABA-operated Cl− channels are
pharmacologically and structurally distinct from vertebrate
GABAAreceptors (Sattelle et al.
1991). In addition, the ionotropic GABA receptors described in insect CNS
are of considerable interest because they form an important molecular targets
for distinct chemicals classes of insecticidally active compounds such as
picrotoxin (PTX), dieldrin, fipronil, and BIDN (Bloomquist
1996;Eldefrawi and
Eldefrawi 1987; Sattelle
1990). In these regards, differences between vertebrate and insect GABA
receptors have always been proved to be exploited for designing novel more
selective insecticide molecules. During the last decade, molecular biology
studies of insect ionotropic GABA receptors have contributed not only to further
understand their structural and functional organization but also to support
their heterogeneity. To date, three GABA receptors subunit genes have been
cloned fromDrosophila melanogaster including the resistance to
dieldrinRdl gene (Ffrench-Constant
et al. 1993), the ligand-gated chloride channel homologue 3, LCCH3 β-like
gene (Henderson et al.
1993), and the glycine-like receptor GRD gene (Harvey et al.
1994). In addition, 12 GABAA/glycine-like receptor subunit genes
have been recently identified in the D. melanogaster genome (Rubin et al.
2000) confirming the existence of a multiplicity of GABA receptor subunits
in insects.

Neuronal cell lines and oocyte expression system have been mainly used
for establishing the pharmacological properties of insect recombinant RDL
receptors (Buckingham et al.
1994a; Grolleau and
Sattelle 2000; Hosie and Sattelle
1996;Millar et al.
1994; Zhang et al.
1994). Insect neuronal preparations have also been developed that allowed
studies on native GABA receptors (Aydar and Beadle
1999; Buckingham et al.
1994b;Dubreil et al.
1994; Hue 1991;Lees et al.
1987; Sattelle
1990;Shimahara et al.
1987; Watson and Salgado
2001; Zhang et al.
1994). For instance, in cockroach CNS, bicuculline-insensitive GABA
receptors coupled to Cl− channels have been detected in a ventral
giant interneuron (GI) (Hue 1991), in
an identified motor neuron Df (Buckingham et al.
1994b;David and Pitman
1996) and in dorsal unpaired median (DUM) neuron (Dubreil et al.
1994; Goodman and
Spitzer 1980; Le Corronc and Hue
1999). In GI preparation, two GABA-gated Cl− channel receptor
subtypes have been further distinguished on the basis of their differences in
Cl− conductance and GABA and picrotoxin sensitivity (Hue 1998).
Although the presence of two GABA receptor-operated Cl− channel
subtypes have been hypothesized in cockroach DUM neurons (Le
Corronc and Hue 1999), the detailed electrophysiological and pharmacological
properties of these receptor subtypes remain to be investigated.

In addition, there is now substantial evidence that intracellular
messengers and regulatory proteins can modulate vertebrate GABA-activated
Cl− currents (Moran and Dascal
1989). In this context, phosphorylation/dephosphorylation process is
currently regarded as an important mechanism for modulating the function of GABA
receptors (Moss et al.
1992; Raymond et al.
1993;Swope et al.
1999) and consequently for controlling synaptic plasticity (Smart 1997).
Many GABA receptor subtypes contain consensus sites for phosphorylation (Shofield et al.
1987), and most intracellular regulatory mechanisms, described for
vertebrate GABAA or GABAC receptors, involved
Ca2+-sensitive proteins such as protein kinase C,
Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM K II), and/or
calcineurin (Browning et al.
1990;Chen et al.
1990; Feigenspan and
Bormann 1994; Filippova et al.
1999; Huang and Dillon
1998; Krishek et al.
1994; Swope et al.
1999). Functional studies have demonstrated that phosphorylation might lead
to depression or potentiation of the GABA response according to the subunit
composition of receptors, their locations (synaptic or extrasynaptic), and the
type of cells which expressed the receptors. In contrast to vertebrate,
excepting a recent study performed on nonidentified cockroach neurons (Watson and Salgado
2001), the characterization of the intracellular mechanisms involved in the
regulation of the functional properties of the insect ionotropic GABA receptors
is still limited.

Consequently, we have studied, in this paper, the electrophysiological
and pharmacological properties of the GABA responses on cockroach DUM neurons, a
well-known insect neuronal preparation (Grolleau and
Lapied 2000). This study has been also focused on the influence of
Ca2+ ions on the modulation of GABA receptor-gated Cl−
currents. We report that Ca2+ positively regulates a component of the
GABA response via a CaM protein kinase. Preliminary report of some of these
findings has been published elsewhere in abstract form (Alix et al.
2000).

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METHODS

Preparation

All experiments were performed on DUM neuron cell bodies isolated from
the dorsal midline of the terminal abdominal ganglion (TAG) of the nerve cord of
adult male cockroach Periplaneta americana. Insects are taken from our
laboratory stock colonies maintained at 29°C with a photoperiod of 12 h light:12
h dark. Cockroaches were immobilized dorsal side up on a dissection dish. The
dorsal cuticle, gut, and some dorsolongitudinal muscles were removed to allow
access to the ventral nerve cord. The TAGs were carefully dissected and placed
in normal cockroach saline containing (in mM) 200 NaCl, 3.1 KCl, 5
CaCl2, 4 MgCl2, 50 sucrose, and 10 HEPES; pH was adjusted
to 7.4 with NaOH.

Cell isolation

Isolation of adult DUM neuron cell bodies was performed under sterile
conditions using enzymatic digestion and mechanical dissociation of the median
parts of the TAG as previously described (Grolleau and
Lapied 1996; Lapied et al.
1989). Briefly, the dorsal median parts were incubated for 40 min at 29°C in
cockroach saline containing collagenase (type IA, 1.5 mg/ml, Worthington
Biochemical). The ganglia were then rinsed twice in normal saline and
mechanically dissociated by repetitive gentle suctions through fire-polished
Pasteur pipettes. The DUM neurons, suspended in normal saline supplemented with
fetal calf serum (5% by volume, Life Technologies, Cergy Pontoise, France),
penicillin (50 IU/ml), and streptomycin (50 μM/ml), were allowed to settle on
poly-d-lysin hydrobromide (MW, 70,000–150,000, Sigma
Chemicals) coating the bottom of 35-mm tissue-culture petri dishes.

Electrophysiological recordings

Whole cell patch-clamp recording (Hamill et al.
1981) was carried out in DUM neuron cell bodies 24 h after dissociation.
Only those having diameter of ∼60 μm and a bright appearance under phase
contrast microscope were selected. The GABA-induced currents were recorded using
an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and filtered at 5
kHz (−3 dB, 4-pole low-pass Bessel filter). Patch pipettes were pulled from
borosilicate glass capillary tubes (Clark Electromedical Instruments, Reading,
UK) with a Narishige puller (PP-83, Tokyo, Japan) and had resistance ranging
from 0.9 to 1.2 MΩ when filled with the internal solution (see composition in
the following section). The liquid junction potential between bath and internal
pipette solution was always corrected before the formation of a gigaohm seal
(>3 GΩ). Signals were stored on-line on the hard disk of a NEC celeron 333
computer connected to a 125-kHz labmaster DMA acquisition system (TL-1–125
interface, Axon Instruments). The pClamp package (version 6.04, Axon
Instruments) was used for data acquisition and analysis (sampling frequency, 2
kHz). When necessary, a SMP-300 programmable stimulator (Biologic, Echirolles,
France) was used to apply hyperpolarizing pulses.

Solutions and chemicals

The cells were continuously superfused with a Cl−-isotonic
solution containing (in mM): 167 NaCl, 33 d-gluconic acid,
3.1 KCl, 4 MgCl2, 5 CaCl2, and 10 HEPES; pH was adjusted
to 7.4 with NaOH. The saline was supplied by a gravity perfusion system at a
constant rate (0.1 ml/min) through a plastic tubing positioned near the cell
body (≈100 μm). The pipette solution consisted of (in mM) 170 KCl, 15 NaCl, 1
MgCl2, 0.5 CaCl2, 3 ATP-Mg, 10 EGTA, 20 HEPES, and 10
phosphocreatine diTris; pH was adjusted to 7.4 with KOH. Antagonists
[picrotoxin, 3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile
(BIDN), cadmium chloride] were diluted in the bathing solution and modulators of
the secondary effectors
[N-(6-aminohexyl)-5-chloro-1-naphtalene-sulfonamide (W7) and
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62)] were added in the
internal solution. Stock solution of BIDN, W7, and KN-62 were previously
dissolved in dimethylsulfoxide (DMSO). The final concentration of DMSO never
exceeded 0.1%. For extracellular Ca2+-free solution,
MgCl2was substituted for CaCl2 in equivalent amount. BIDN
was obtained from DuPont Agrochemical. All other pharmacological agents and
chemicals were obtained from Sigma Chemicals (L'Isle d'Abeau Chesnes, France).
Experiments were carried out at room temperature (20°C).

Pneumatic pressure ejection application of agonist and data analysis

GABA (10−4M) and CACA (10−3M) were dissolved in
the extracellular saline solution and were delivered by pressure ejection (15
lb./in.2 gauge) with a pneumatic pressure ejection system (Miniframe
PPS-2, Medical System, Greenvale, NY). Agonists were ejected through a glass
micropipette (resistance, 2 MΩ when filled with agonist) placed at ∼40 μm of the
cell body. When droplets were ejected under oil and the diameter was measured
with an ocular micrometer, a linear relationship was established between the
volume delivered and the pulse duration parameters (McCaman et al.
1977). Consequently, we constructed an agonist dose-response relationship by
increasing the ejection time at constant pressure. In these conditions, we
assumed that the amount of agonist delivery by pressure was linearly related to
ejection pressure (Di Angelotonio and
Nistri 2001; Lapied et al.
1990; McCaman et al.
1977; Raymond et al.
2000). Pressure application from fine-tipped micropipette was preferentially
used to apply agonist to minimize the risk of desensitization and to avoid large
exposition of all other cells in the chamber. In no experiment did the pressure
ejection of normal saline affect the current baseline. The steady-state
recordings were made 2 min after setting of the whole cell recording
configuration and repeated applications of GABA were made with an interval of 2
min between the end of one application and the beginning of the next. Under
these conditions, amplitude of I GABA, normalized to the
value of the response to 120-ms pulse duration, was then plotted against
increasing pulse duration. The dose-response curves were analyzed using GraphPad
Prism and were fitted to a sigmoid function with four-parameter logistic
equation (sigmoid concentration response) with a variable slope. The equation
used to fit the concentration-response relationship was
where I was the normalized GABA-activated current,I
max was the maximal normalized GABA current, T the duration
of the pulse,T50 the pulse duration inducing 50% of the
I max, and n the Hill slope factor. Dose-response
curves were obtained from the mean of the normalized values calculated for each
neuron. Data are expressed as means ± SE. Using a nonparametric Student's
t-test assessed statistical significance of data.

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RESULTS

Transient inward current evoked by pressure ejection application of
GABA

Whole cell recordings were performed in symmetrical chloride ion
solutions ([Cl−]out = [Cl−]in = 188
mM). In all DUM neuron cell bodies tested, GABA induced a transient inward
current at a holding membrane potential of −50 mV. The amplitude of the inward
current increased when the GABA dose was elevated by progressively raising the
length of the pressure ejection pulse. Figure1 A
illustrates typical examples of GABA-activated currents (I
GABA) evoked by pulse of various duration (from 30 to 300 ms).
Normalized amplitudes ofI GABA were plotted against the
logarithm of increasing pulse duration (Fig. 1 B). A
T50 value of 86.2 ms was revealed [—, which corresponded to the best
fit through the mean data points (r = 0.9995) according to the Hill
equation (seemethods)]. The Hill slope factor determined
by a linear regression analysis of the Hill plot (Fig. 1
B,inset) was 4.0 ± 0.2 (n = 9,r = 0.9995).
The amplitude of the inward current was maximum for pressure ejection duration
>200 ms (Fig. 1 B). In
the following experiments, the pulse duration was adjusted to give a
half-maximal response (e.g., 80 ms). In these conditions, the amplitude of
I GABA evoked by repeated puffs separated by 2 min remained
stable over 30 min.

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Fig. 1.

GABA-induced current (I GABA)
in isolated dorsal unpaired median (DUM) neurons in Cl− isotonic
conditions. A: typical examples of current traces recorded in response
of increasing GABA doses at a holding membrane potential of −50 mV. GABA
(10−4 M) was delivered from the pipette at various pressure-pulse
durations indicated next to each trace. B: dose-response curve. Points
correspond to the amplitude of I GABA measured on the same
cell body for the different pressure-pulse durations and were normalized to the
value obtained in response to the 120-ms pulse of GABA. —, the best fit of the
mean data (n = 9) using a 4-parameter logistic equation (sigmoid curve
in log scale). Inset: the Hill plot of data gathered in the same way. The Hill
coefficient, determined from the slope of the line, is clearly >2.

Voltage dependence and ionic selectivity of IGABA

Figure 2 Aa
shows inward currents activated by a 80-ms pulse of GABA recorded at different
steady-state membrane potentials ranging from −70 to +30 mV in 10-mV increments.
The relationship between amplitude ofI GABA and membrane
potential (I-V curve) was constructed by averaging data from a total of
12 cells (Fig. 2Ab). The I-V curve exhibited inward rectification at
positive membrane potentials and gave a reversal potential of +1.8 mV. The curve
shows that the currents were inwardly directed for potentials more negative than
+1.8 mV and became outward above +1.8 mV. When [Cl−] in the internal
solution was reduced to 13 mM by replacing KCl with 170 mM potassium aspartate,
the reversal potential was shifted to −66 mV, according to the value (−67.3 mV)
predicted by the Nernst equation under our experimental conditions (Fig. 2 Ab, □).
With symmetrical Cl− concentration inside the patch and in the bath,
the I-V relationship displayed a biphasic aspect in the negative
membrane potential range with an inflection at about −30 mV (Fig. 2 Ab, ●).
To ensure that the total inward current evoked by GABA was completely due to
Cl− ions, we tested Cl− channel blockers acting on insect
and vertebrate ionotropic GABA receptors such as PTX and BIDN. As seen in Fig.
2 Ba,
10−6 M BIDN depressedI GABA elicited at −50 mV by
90.8 ± 0.1% (n = 3). PTX (10−4 M) also markedly reduced the
response by 98.9 ± 1.1% (n = 3, Fig. 2Ba, b). Taken together, these
results confirmed that the inward current elicited by GABA was carried by
Cl− ions.

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Fig. 2.

Chloride dependence of GABA-induced responses in
DUM neurons.Aa: whole cell currents evoked by locally applied GABA at
various membrane holding potentials increasing in 10-mV increments from −70 to
+30 mV. GABA (10−4 M) was ejected on the cell for a duration of 80
ms. Ab: plot of the current-voltage relationship of GABA-activated
current. The I-V curve displayed a biphasic aspect resulting from an
increase of the current amplitude at potentials more negative than −30 mV and
exhibited inward rectification at potential more positive than 0 mV. The
reversal potential (+ 1.8 mV) was very close to the calculatedE
Cl (−0.1 mV) with symmetrical [Cl−] inside and outside the
cell (●) but shifted to −66 mV when [Cl−] in the patch pipette was
reduced to 13 mM (○). Ba: representative current evoked by 80-ms pulse
of GABA (10−4 M) at −50 mV before, during, and after application of
either 10−6 M
3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile (BIDN;
top) or 10−4 M picrotoxin (bottom). The current did
not fully recovered following the washout of each blocker. Bb:
comparative histogram of the percentage of residual GABA-induced current
amplitude after application of BIDN and PTX. Each column represents the mean
value of 3 experiments from different cells and vertical bars represent 1 SE
(the SE for BIDN of 0.025 is indistinguishable).

Effect of Ca2+ ions on IGABA

As mentioned in introduction, we were
interested by examining the role of Ca2+ in the regulation
ofI GABA. Consequently, we next tested the effect of bath
saline containing CdCl2 or Ca2+-free solution on DUM
neuron cell body held at −50 mV. The amplitude of I GABA was
decreased by 42.9 ± 4.1% (n = 13;P < 0.01) under
CdCl2(10−3 M). This effect occurred within 6 min and was
reversible. Treatment of the cell with Ca2+-free solution also
reduced the current by 26.0 ± 7.1% (n = 3; P < 0.05; Fig.
3 A, a
and b). Figure 3 B shows
the dose/response relationship constructed under 10−3 M
CdCl2; the — corresponds to the best fit through the mean data points
(r = 0.9993). The mean value of the Hill coefficient was estimated to
be 2.4 ± 0.1 (n = 6), a value that was much smaller than one determined
under control condition (see Fig. 1 B). In
the literature, it is well documented that two molecules of GABA are necessary
for activation of the native receptor channel (MacDonald and
Olsen 1994). In our case, it was unclear if the high Hill slope factor value
(4.0) calculated under control conditions resulted of the simultaneous binding
of more than two molecules of GABA or if two different GABA receptor subtypes
could be involved in the GABA-induced current. Based on results obtained under
CdCl2 treatment, it was suggested that two receptor subtypes were
expressed in DUM neurons. However, only one of them could be still activated in
the presence of CdCl2. To confirm this hypothesis, we compared the
I-V curve between −70 and 0 mV in control and under CdCl2
(10−3 M). In the last case, it was clear that the voltage dependence
ofI GABA did not display the biphasic aspect (Fig. 3 C) as
described in the preceding text. Although two linear regressions were necessary
to fit mean data points in control (Table 1), only one
linear regression was used under CdCl2 treatment between 0 and −70 mV
(Fig. 3 C and
Table 1). Furthermore,
the parameters of the linear regression used under CdCl2 were very
similar to that of the control between 0 and −30 mV. In conclusion, comparisons
of both Hill coefficient values together with the I/V curves obtained in control
and under CdCl2 argued for the activation of two different receptor
subtypes.

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Fig. 3.

Ca2+ dependence of GABA-induced
responses recorded from isolated DUM neuron cell body. Aa:I
GABA was reversibly decreased during bath application of cadmium
chloride (CdCl2, 10−3M). · · · , the amplitude of the
control response.Ab: I GABA was similarly reduced
when Ca2+ was withdrawn from the normal bathing solution. B:
dose-response curve under 10−3M CdCl2. Each point
corresponds to theI GABA amplitude measured on the same cell
body for different pressure-pulse duration and is normalized to the value
obtained in response to the 120-ms pulse of GABA. Solid line, the best fit of
the mean data (n = 6) using a 4-parameter logistic equation.
C: superimposed voltage dependence of I GABA
amplitude curves in control conditions (closed squares, n = 13) and in
the presence of 10−3 M CdCl2 (open squares,n =
3). Two linear regressions were used to fit the data in control conditions
(black dashed line from 0 to −30 mV and solid line from −30 to −70 mV). The I-V
curve under CdCl2was best fitted with only one linear regression
between 0 and −70 mV (gray dashed line). Note that the parameters of the last
regression were very close to those of that one used for the control curve
between 0 and −30 mV (see Table 1 for more
details). D: effect of activating resting Ca2+ channels by
applying a hyperpolarizing voltage step before GABA ejection either in control
conditions or in the presence of CdCl2 (10−3M). The 1-min
hyperpolarizating voltage pulses from −50 to −80 mV are symbolized on the
drawing up to the current traces, and the solid bar indicated the time of
perfusion with CdCl2. Note that in control condition, I
GABA was always increased in amplitude when GABA ejection was
preceded by a hyperpolarization, but it was never the case under
CdCl2.E: comparative histogram of the variation ofI
GABA amplitude with CdCl2, free calcium bathing saline and
after hyperpolarizing step either in control conditions or during
CdCl2 treatment.

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Table 1.

Slope conductance

The restricted negative potential range in which CdCl2 was
active suggested a possible implication of CdCl2-sensitive calcium
resting channels, known in DUM neurons to be mainly activated in the
hyperpolarizing potential range between −50 and −110 mV (Heine and Wicher
1998; Wicher et al.
1994). These authors demonstrated that increases of intracellular calcium
concentration following opening of Ca2+ resting channels were
observed in DUM neurons, when the holding potential was hyperpolarized from −50
to −70 or −90 mV (Heine and Wicher
1998). Accordingly, it was decided to stimulate Ca2+ entry
through these Ca2+ resting channels by stepping the membrane from −50
to −80 mV for 1 min before ejecting GABA.I GABA were
recorded within 3 s after the termination of the hyperpolarizing steps. In all
cell tested,I GABA were always significantly potentiated by
20.8 ± 4.9% (n = 6;P < 0.01; Fig. 3
D) with a recovery period of ∼4 min. By contrast, when the same
hyperpolarizing voltage step was applied in the presence of
CdCl2(10−3 M), amplitude ofI GABA
recorded at −50 mV did not increase (2.2 ± 1.4% of reduction, n =
3,P > 0.1, Fig. 3 D). The
histogram illustrated in Fig. 3 E
summarizes these results. It clearly shows that one of the components
ofI GABA at −50 mV was dependent on Ca2+ entry
through Ca2+resting channels.

Implication of Ca-dependent proteins for maintaining IGABA

The Ca2+ sensitivity of the GABA response might be
explained by either a direct interaction of Ca2+ with the GABA
receptors or by an activation of Ca-dependent enzymatic processes that
co-activate the GABA receptors. Because direct effect of Ca2+ ions on
GABA receptors have been reported in vertebrate to be mainly involved in the
suppression of GABA-activated current (e.g., Inoue et al.
1986; Martina at al.
1994) rather than a potentiation of the response (our study), we performed
additional experiments using an internal pipette solution containing
Ca2+-dependent messenger inhibitors. Calmodulin is a ubiquitous
intracellular protein that regulates the activity of various enzymes in a
Ca2+-dependent manner. To explore the putative participation of
calmodulin in DUM neuron,I GABA was recorded at a holding
potential of −50 mV using a pipette solution containing 10−3 M of the
calmodulin inhibitor W7. In this condition, the recordings were performed over a
time period of 30 min after establishing the whole cell configuration to allow
compounds sufficient time to diffuse into the cell.I GABA
was then normalized to the value measured 4 min after the initial GABA
application. In five cells tested, the main effect obtained with W7 after 20 min
consisted of a reduction of I GABA (17.7 ± 2.7%, P
< 0.05). The plot of the mean values ofI GABA versus time
was illustrated in Fig. 4 A.
However, in three cells, I GABA was increased by 56.0 ± 7.5%
and in other three cells, there was no variation (3.0 ± 1.0%). The current
amplitude over time in the absence of the drug (○, Fig. 4
A) did not change and had a value of 97.3 ± 5.1% of its initial value
after 20 min (n= 5). Because heterogeneous effects were obtained with
W7, we tested the effect of the specific inhibitor of the
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), the
KN-62. At a holding potential of −50 mV, KN-62 (5.10−6 M)
reducedI GABA by 38.3 ± 8.4% (n = 9, P
< 0.05) within 20 min in all cells tested (Fig. 4
B). The voltage dependence ofI GABA was studied in
the presence of 5.10−6 M KN-62. As shown in Fig. 4
C, the discontinuity of the I-V curve disappeared in the
presence of KN-62, and data were fitted by a single linear regression between 0
and −70 mV, yielding a slope factor close to those previously obtained under
CdCl2 and in control condition (Table 1). In addition,
when CdCl2 was tested on the remaining current under KN-62 (Fig. 4 D), no
additional reduction of I GABA was observed (99.5 ± 5.4%,
n = 4, P> 0,1). These results indicated that a CaM kinase
II-like protein was implicated in the regulation of the
CdCl2-sensitive component ofI GABA.

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Fig. 4.

Effect of different inhibitors of intracellular
secondary messengers on GABA-induced currents. All compounds are included in the
pipette solution. A and B: plot ofI GABA
amplitude vs. time and representative current traces (inset) at −50 mV
in the absence (○) and in the presence of 10−3 M W7 (A, ■,
n = 5) and 5.10−6 M
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62, B, ■, n
= 9). Points represents means ± SD. C: current-voltage relationship of
GABA responses in the presence of 5.10−6 M of KN-62 (■, n =
3). The curve was linearized between 0 and −70 mV (- - -). Note that the linear
regression for the curve under KN-62 is close to the linearization of the
control curve between 0 and −30 mV (see Table 1).D:
absence of blocking effect onI GABA of CdCl2
applied after 30 min of intracellularly dialysis with 5.10−6 M KN-62.

Activation of only one component by CACA

To substantiate the hypothesis for the co-existence of two distinct
subtypes of GABA Cl−-gated receptor in DUM neurons, the
cis-4-aminocrotonic acid (CACA), the well known agonist of vertebrate
GABAC receptor (Johnston 1996)
and insect GABA receptor such as RDL receptor (Millar et al.
1994) or native giant interneuron GABA receptor (Hue 1998), was
tested. Pressure application of 10−3 M CACA, for various ejection
duration pulses, onto the isolated cell body (holding potential −50 mV),
elicited a transient inward current (Fig.5 A). The
amplitudes of the peak current (I CACA) were normalized to
the value of the response to 300-ms ejection pulse for each cell. The mean data
points (n = 4) were then plotted against the logarithm of increasing
pressure ejection duration (Fig.5 B). The
solid line corresponded to the best fit (correlation coefficient r =
0.9996) according to the Hill equation. The mean value of the Hill coefficient,
determined by a linear regression analysis of the Hill plot (Fig. 5 B,
inset) was 2.3 ± 0.1 and corresponded to a value close to that already
calculated for I GABAunder CdCl2 treatment. In
addition, as illustrated in Fig. 5 C,
10−3 M CdCl2 did not significantly reduce the response
evoked by 200-ms pulse of CACA (5.1 ± 1.9%, n = 3, P >
0.05). The I-V plot of the current evoked by CACA, constructed from
five cells, confirmed that the CACA-induced current was chloride (Fig. 5 D). The
estimated reversal potential was −3.5 mV, a value very close toE
Cl in our experimental conditions. However, the I-V curve
did not display the biphasic aspect between −10 and −70 mV (see Table 1) but showed a
voltage dependence that was similar to those observed with CdCl2 and
KN-62. These results support the idea that two different receptor subtypes
contributed to the global inward current evoked by GABA at −50 mV. Either GABA
or CACA might activate one receptor subtype, shown to be not regulated by a CaM
kinase II-like protein.

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Fig. 5.

cis-4-Aminocrotonic acid (CACA)-induced
current (I CACA) in isolated DUM neurons in Cl−
isotonic conditions. A: typical examples of current traces recorded in
response of increasing CACA doses at a holding membrane potential of −50 mV.
CACA (10−3 M) was delivered from the pipette at various
pressure-pulse durations indicated next to each trace.B: dose-response
curve. Points correspond to the amplitude of I CACA measured
on the same cell body for the different pressure-pulse durations and were
normalized to the value obtained in response to the 300-ms pulse of CACA. —, the
best fit of the mean data (n = 4) using a 4-parameter logistic
equation. Inset: the corresponding Hill plot of data. —, the best fit
to a linear regression with a slope of 2.3. C: CACA-induced currents
recorded in the absence or presence of CdCl2 (10−3 M). DUM
neuron was clamped at −50 mV and CACA (10−3 M) was applied for a
duration of 200 ms D: voltage dependence of the currents induced by
200-ms pulses of CACA. Linear regression through the mean data points gave a
reversal potential of −2 mV and yielded a slope which is close to that obtained
for GABA in control between 0 and −30 mV (see Table 1).
Inset: superimposed whole cell currents evoked by locally applied CACA
at various membrane holding potentials increasing in 20-mV increments from −70
to +10 mV.

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DISCUSSION

The present study provides evidences for the existence in DUM neuron
cell body of more than one ionotropic GABA receptor subtype including a
Cl−-dependent GABA receptor that is regulated by an intracellular
Ca2+-dependent mechanism.

Evidence for the co-existence of two distinct Cl−-dependent GABA
receptor subtypes

Although the voltage dependence of the whole cell response evoked by
GABA usually results in a uniphasic I-V relationship in vertebrates
(e.g., Filippova et al.
1999; Kapur et al.
1999; Tietz et al.
1999) as well as in insect (e.g., Wafford and
Sattelle 1986;Zhang et al.
1994), our results show a more complexI-V curve because a
discontinuity was observed for negative potentials. The reversal potential is
very closed to the equilibrium potential for Cl− ions, indicating
that the current is chloride. This conclusion, reinforced by the complete
blocking effect of both PTX and BIDN, indicated thatI GABA
is mediated by an ionotropic Cl−-dependent GABA receptor and not a
GABAB receptor. Although an unequal chloride activity across the
membrane would be responsible for the rectification property, it was suggested
that more than one conductance probably underlie the discontinuity of the
I-V relationship. Three sets of experiment have been performed to
determine the number of receptor subtypes involved. First, the dose-response
curve that have been established at −50 mV by varying the pressure ejection time
(seemethods) yielded a high Hill coefficient (i.e., 4).
Second, analysis of the pharmacological profile indicated that the inward
current recorded at a membrane potential more negative than −30 mV could be
further dissociated into two components including a Ca2+-sensitive
Cl− current and a Ca2+-insensitive Cl−current.
Finally, we found that only the Cd2+-insensitive component of
IGABA is activated by the GABAC agonist, CACA. However,
CACA was ≥10-fold weaker as a agonist to elicit the Ca2+-insensitive
Cl−current in DUM neurons and consequently, it was difficult to
assess a relative contribution of both receptors. Although it was never
demonstrated to our knowledge that Hill coefficients should be additive, we
conclude that at least two GABA receptor types are expressed in DUM neuron cell
body. In this context, a bimodal dose-response curve for the combined current
would be expected. Instead, the Hill analysis suggested that the two receptors
have identical dissociation constant and showed a degree of positive
cooperativity comparable to that reported for instance for DUM neuron
cholinergic receptors (i.e., n Hill = 4.28) (Lapied et al.
1990). Among others possibilities, we can speculate that the high value of
Hill coefficient may reflect the interaction of receptor molecules. In fact,
there is growing evidence that a positive cooperativity exists between a few
nearby receptors (Bornhorst and Falke
2000). Such cooperativity between receptors was proposed as an alternative
of signal transduction and particularly in amplifying signal transduction events
(Bornhorst and Falke
2000; Chen et al.
2000; Liu et al.
2000; Yonekura et al.
1991).

Based on the literature, the characterization of two different
ionotropic GABA receptor subtypes within individual cell body seems to be unique
in an insect neuronal preparation. To date, two kinds of picrotoxin-sensitive
GABA receptor were differentiated according to their location on DUM neurons (Dubreil et al.
1994). It was shown that an extrasynaptic GABA receptor was located onto the
soma of DUM neuron, and another synaptic receptor was also revealed on the
neuritic arborization of these cells. In vertebrates, there are only few
examples reporting that two Cl− currents mediated by different GABA
receptors could be recorded in the same cell (e.g., Han and Yang
1999; Tietz et al.
1999). For instance, Tietz et al.
(1999)suggested that the biphasic response to GABA from rat CA1 pyramidal
cells reflected the presence of molecular heterogeneous GABA receptors. In our
case and because the CdCl2-resistant Cl− current activated
by GABA was also activated by CACA, it is tempting to assume that one of the two
receptors identified might correspond to a GABAC-like receptor. The
presence of such receptor subtype has previously been suggested in cockroach
ventral giant interneuron (Hue 1999).
Another possibility, which cannot be ruled out, is the putative existence of an
RDL-like receptor on DUM neurons. Subunit composition of GABA receptors in
cockroach CNS remains unknown. However, several studies have suggested that
RDL-like receptor might exist in cockroach. First, expression of Rdl
subunit inXenopus oocytes or S2 cells give functional GABA-gated
Cl− channels exhibiting a pharmacological profile that shares many of
the properties of native GABA receptors on cockroach giant interneuron (Buckingham et al.
1994b) or motor neuron Df (Sattelle
1990). In addition, immunocytochemical staining with polyclonal antibody
raised against the predicted C-terminal sequence of the cloned RDL receptor has
been found largely distributed in the cockroach head ganglia (Sattelle et al.
2000). It should be noted that the homo-oligomericDrosophila GABA
RDL receptor expressed in Xenopusoocyte or in S2 cells has been shown
to be also activated by the GABAC agonists TACA and CACA (Millar et al.
1994; Grolleau, personal observation).

Ca2+/calmodulin-dependent protein kinase regulated one component
of IGABA

Overall, our data demonstrate that Ca2+ is necessary to
maintain one component ofI GABA. We have found that CdCl2 or
Ca2+-free solution inhibits the GABA response. Conversely, I
GABA is increased when the intracellular Ca2+
concentration is raised artificially. This has been confirmed by using
hyperpolarizing voltage step known to increase Ca2+ influx through
Ca2+ resting channels (Heine and Wicher
1998). Amplitude of I GABA was dependent either of
increase or decrease of intracellular Ca2+ concentration. These
observations together with the reversibility of the blocking action of
Cd2+ on I GABAsuggest that Ca2+ did
not likely involved through proteolysis of GABA receptor by
Ca2+-dependent proteases. In vertebrate, many studies have described
an effect of variation in intracellular Ca2+ concentration on the
GABAA receptor-gated Cl− current (Akaike 1990;Inoue et al.
1986; Llano et al.
1991;Martina et al.
1994; Mouginot et al.
1991). In these cases, it was suggested that Ca2+ changed the
apparent affinity of the receptor or acted through a diffusible
Ca2+-dependent messenger. In our study, because the effect induced by
Cd2+ was mimicked by W7 or KN-62, which are specific inhibitors of
calmodulin and CaM kinase II, respectively, we concluded that it occurred
through a CaM kinase II-like protein.

Phosphorylation mechanisms were well documented in vertebrates as a
major intracellular pathway that regulates neurotransmitter receptor function
(Raymond et al.
1993; Swope et al.
1999). Conserved serine and threonine residues on the GABAA
receptor β and γ subunits respectively have been identified as a site of protein
phosphorylation by PKA, PKC, and CaM kinase II showing the
importance of these subunits in GABA receptor activity (Krisheck et al.
1994; MacDonald and
Olsen 1994; McDonald and Moss
1997; Raymond et al.
1993; Swope et al.
1999). The effects of PKC or PKA on GABAA
receptors have been shown to involve a positive as well as a negative regulation
depending on the specific preparation and probably on the existence of receptors
composed of different subunits. By contrast, only few reports demonstrated that
CaM kinase II protein is important for receptor activation. In rat acutely
isolated spinal neurons, it has been shown that injection of CaM kinase II
enhanced GABA-induced Cl− current (Wang et al.
1995). This effect was associated with a reduction of the desensitization of
the GABA response. In forebrain synaptosomal membranes, Ca2+ and CaM
kinase II were implicated in the enhancement of the binding of agonist (Churn and
DeLorenzo 1998). In mouse cortical neurons, regulation of GABAA
receptor function also involved a CaM kinase II (Aguayo et al.
1998). These authors demonstrated that both calmodulin and CaM kinase II
inhibitors blocked the effect of increasing intracellular Ca2+ on
responses to GABA.

Functional significance and interest in pest control strategy

We have demonstrated in this study that the GABA response was
sensitive to changes in intracellular Ca2+concentration. From these
results emerges an interesting question concerning the relationship between the
rise in intracellular Ca2+ concentration and the function of the
receptor in the control of the membrane potential. Of particular interest is the
consequence on the firing pattern because DUM neurons are well characterized by
an endogenous pacemaker activity, which is closely related to their
neurosecretory function (Grolleau and
Lapied 2000). Among the different ionic currents underlying this pacemaker
activity, several classes of voltage-dependent ionic currents have been
identified that are regulated by change in intracellular Ca2+
concentration (Grolleau and
Lapied 2000). Here it was demonstrated that an increase of intracellular
Ca2+ concentration enhanced a GABA-gated Cl− current
through a CaM kinase II protein. Because the action of GABA is known to be
inhibitory on the soma of DUM neuron (Dubreil et al.
1994; Goodman et Spitzer
1980; Washio 1994),
such current would probably influence the resting membrane potential after
various stimuli that elevate intracellular Ca2+concentration, in a
more hyperpolarizing direction compared with a Ca2+-independent GABA
receptor current. In this condition, the participation of the CaM kinase
II-like-regulated GABA receptor will help in potentiating the inhibitory action
of neurotransmitter on DUM neuron.

Because GABA receptors are one of major targets in insect for
chloride channel-blocking insecticidally active molecules such as cyclohexane,
cyclodienes, or fipronil (Bloomquist
1996; Hainzl et al.
1998), the evidence of phosphorylation mechanism for maintaining insect GABA
receptor activation is of considerable importance for anticipating the action of
insecticides but also for designing new molecules with insecticidal properties.
The structure-activity relationship of GABA receptor binding insecticide
molecules may be strongly dependent of the occurrence of phosphorylation system,
and for this reason, significant difference in toxicity may be expected. For
instance, it has been suggested from electrophysiological studies on drosophila
S2 cell expressing RDL receptor (Grolleau and
Sattelle 2000) or on rat DRG neuron (Ikeda et al.
2001) that receptor activation facilitates fipronil binding to the receptor.
In this condition, involvement of CaM kinase II-like protein in keeping
ionotropic GABA receptor fully operative should undergo change of insecticide
effect in efficacy.

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Acknowledgments

We are grateful to Pr. Bruno LAPIED of our laboratory for helpful
discussions and critical reading of the manuscript.

P. Alix is supported by a doctoral fellowship from Régions Pays de la
Loire.

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Footnotes

Address for reprint requests: F. Grolleau, Laboratoire de
Neurophysiologie Unité Propre de Recherche de l'Enseignement Supérieur Equipe
d'Accueil 2647, Université d'Angers, Unité de Formation et de Recherche
Sciences, 2 boulevard Lavoisier, F-49045 Angers Cedex, France (E-mail:francoise.grolleau{at}univ-angers.fr).

Copyright © 2002 The American
Physiological Society

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