J Neurophysiol
87: 2972–2982, 2002; 10.1152/jn.00631.2001.
Ca2⫹/Calmodulin-Dependent Protein Kinase Regulates
GABA-Activated Cl⫺ Current in Cockroach Dorsal
Unpaired Median Neurons
PHILIPPE ALIX, FRANCOISE GROLLEAU, AND BERNARD HUE
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
Received 31 July 2001; accepted in final form 4 February 2002
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: [email protected]).
2972
calcium-dependent protein kinase. The consequences of the modulatory action of Ca2⫹ in GABA receptors function and their sensitivity
to insecticide are discussed.
INTRODUCTION
␥-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
GABAA receptors (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 from Drosophila
melanogaster including the resistance to dieldrin Rdl gene
(Ffrench-Constant et al. 1993), the ligand-gated chloride channel homologue 3, LCCH3 ␤-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
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Alix, Philippe, Francoise Grolleau, and Bernard Hue. Ca2⫹/Calmodulin-dependent protein kinase regulates GABA-activated chloride
current in cockroach dorsal unpaired median (DUM) neurons. J Neurophysiol 87: 2972–2982, 2002; 10.1152/jn.00631.2001. We studied
␥-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 (IGABA) when 10⫺4 M of GABA
was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of IGABA amplitude versus the ejection time yielded a
Hill coefficient of 4.0. IGABA 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, IGABA 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 for IGABA 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 calciumfree solution. The semi-logarithmic curve for CdCl2-resistant IGABA
gave a Hill coefficient of 2.4. Hyperpolarizing voltage step from ⫺50
to ⫺80 mV was known to increase calcium influx through calciumresting channels. According to this protocol, a significant increase of
IGABA amplitude was observed. However, this effect was never obtained when the same protocol was applied on cell body pretreated
with CdCl2. When the calmodulin blocker N-(6-aminohexyl)-5chloro-1-naphtalene-sulfonamide or the calcium-calmodulin-dependent protein kinase blocker 1-[N,O-bis(5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was added in the
pipette solution, IGABA 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
CA2⫹ REGULATION OF INSECT GABA-ACTIVATED CHLORIDE CURRENT
METHODS
Preparation
All experiments were performed on DUM neuron cell bodies isolated from the dorsal midline of the terminal abdominal ganglion
J Neurophysiol • VOL
(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
␮M/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 ␮m 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 M⍀ 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 G⍀). 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 ␮m). 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-2,2diacarbonitrile (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
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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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P. ALIX, F. GROLLEAU, AND B. HUE
Ca2⫹-free solution, MgCl2 was 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
I ⫽ Imax/关1 ⫹ 共T50/T兲n兴
where I was the normalized GABA-activated current, Imax was the
maximal normalized GABA current, T the duration of the pulse, T50
the pulse duration inducing 50% of the Imax, 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.
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. Figure 1A illustrates typical examples
of GABA-activated currents (IGABA) evoked by pulse of various duration (from 30 to 300 ms). Normalized amplitudes of
IGABA were plotted against the logarithm of increasing pulse
duration (Fig. 1B). A T50 value of 86.2 ms was revealed [—,
which corresponded to the best fit through the mean data points
J Neurophysiol • VOL
FIG. 1. GABA-induced current (IGABA) 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 IGABA 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.
(r ⫽ 0.9995) according to the Hill equation (see METHODS)].
The Hill slope factor determined by a linear regression analysis
of the Hill plot (Fig. 1B, 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. 1B). 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 IGABA evoked by repeated puffs separated by 2
min remained stable over 30 min.
Voltage dependence and ionic selectivity of IGABA
Figure 2Aa shows inward currents activated by a 80-ms
pulse of GABA recorded at different steady-state membrane
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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 M⍀ when filled with
agonist) placed at ⬃40 ␮m 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 IGABA, 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
CA2⫹ REGULATION OF INSECT GABA-ACTIVATED CHLORIDE CURRENT
2975
potentials ranging from ⫺70 to ⫹30 mV in 10-mV increments.
The relationship between amplitude of IGABA 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.
2Ab, ▫). 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. 2Ab, F). 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. 2Ba, 10⫺6 M BIDN depressed IGABA 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.
J Neurophysiol • VOL
Effect of Ca2⫹ ions on IGABA
As mentioned in INTRODUCTION, we were interested by examining the role of Ca2⫹ in the regulation of IGABA. 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 IGABA 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. 3A, a and b). Figure 3B 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.
1B). 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
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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
calculated ECl (⫺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 (E). 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).
2976
P. ALIX, F. GROLLEAU, AND B. HUE
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 of IGABA did not display the biphasic
J Neurophysiol • VOL
aspect (Fig. 3C) 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. 3C and Table 1).
Furthermore, the parameters of the linear regression used under
CdCl2 were very similar to that of the control between 0 and
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2⫹
FIG. 3. Ca
dependence of GABA-induced responses recorded from isolated DUM neuron cell body. Aa: IGABA was reversibly
decreased during bath application of cadmium chloride (CdCl2, 10⫺3 M). 䡠 䡠 䡠 , the amplitude of the control response. Ab: IGABA
was similarly reduced when Ca2⫹ was withdrawn from the normal bathing solution. B: dose-response curve under 10⫺3 M CdCl2.
Each point corresponds to the IGABA 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 IGABA 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 CdCl2 was
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⫺3 M). 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, IGABA
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 of IGABA amplitude with CdCl2, free calcium bathing saline and after
hyperpolarizing step either in control conditions or during CdCl2 treatment.
CA2⫹ REGULATION OF INSECT GABA-ACTIVATED CHLORIDE CURRENT
TABLE
2977
1. Slope conductance
Control
No. of linear regressions
Potential range, mV
No. of cells
Slope, nS
r
0 to ⫺30
13
35.6 ⫾ 2.1
0.9965
2
⫺30 to ⫺70
13
80.9 ⫾ 4.1
0.9962
10⫺3 M CdCl2
5.10⫺6 M KN-62
10⫺3 M CACA
1
0 to ⫺70
3
32.4 ⫾ 1.1
0.9962
1
0 to ⫺70
3
34.8 ⫾ 0.8
0.9985
1
0 to ⫺70
5
32.4 ⫾ 1.1
0.9962
Slope conductance was calculated from the linear regressions of the I-V relationships obtained for GABA [in control condition and in the presence of CdCl2
or 1-[N,O-bis(5-isoquinolinesulfonyl-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62)] and for cis-4-aminocrotonic acid (CACA). Values are means ⫾ SE.
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 GABAactivated 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, IGABA 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. IGABA was then normalized to the value
measured 4 min after the initial GABA application. In five cells
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tested, the main effect obtained with W7 after 20 min consisted
of a reduction of IGABA (17.7 ⫾ 2.7%, P ⬍ 0.05). The plot of
the mean values of IGABA versus time was illustrated in Fig. 4A.
However, in three cells, IGABA 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 (E,
Fig. 4A) 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) reduced IGABA by 38.3 ⫾ 8.4%
(n ⫽ 9, P ⬍ 0.05) within 20 min in all cells tested (Fig. 4B).
The voltage dependence of IGABA was studied in the presence
of 5.10⫺6 M KN-62. As shown in Fig. 4C, 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. 4D), no additional reduction of IGABA 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 of IGABA.
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.
5A). The amplitudes of the peak current (ICACA) 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. 5B). 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. 5B, inset) was 2.3 ⫾
0.1 and corresponded to a value close to that already calculated
for IGABA under CdCl2 treatment. In addition, as illustrated in
Fig. 5C, 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
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⫺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.
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. IGABA were recorded within 3 s after the
termination of the hyperpolarizing steps. In all cell tested,
IGABA were always significantly potentiated by 20.8 ⫾ 4.9%
(n ⫽ 6; P ⬍ 0.01; Fig. 3D) 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 of IGABA
recorded at ⫺50 mV did not increase (2.2 ⫾ 1.4% of reduction, n ⫽ 3, P ⬎ 0.1, Fig. 3D). The histogram illustrated in
Fig. 3E summarizes these results. It clearly shows that one of
the components of IGABA at ⫺50 mV was dependent on Ca2⫹
entry through Ca2⫹ resting channels.
2978
P. ALIX, F. GROLLEAU, AND B. HUE
current was chloride (Fig. 5D). The estimated reversal potential
was ⫺3.5 mV, a value very close to ECl 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.
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
complex I-V curve because a discontinuity was observed for
negative potentials. The reversal potential is very closed to the
J Neurophysiol • VOL
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 that IGABA 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 (see METHODS) 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 ⱖ10-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
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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 of IGABA amplitude vs. time and representative current traces (inset) at ⫺50 mV in
the absence (E) 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-Ltyrosyl]-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 on IGABA of CdCl2 applied after 30 min of intracellularly dialysis with 5.10⫺6 M KN-62.
CA2⫹ REGULATION OF INSECT GABA-ACTIVATED CHLORIDE CURRENT
2979
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., nHill ⫽
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
J Neurophysiol • VOL
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 RDLlike receptor might exist in cockroach. First, expression of Rdl
subunit in Xenopus oocytes or S2 cells give functional GABAgated 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-oligo-
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⫺
FIG. 5. cis-4-Aminocrotonic acid (CACA)-induced current (ICACA) 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 ICACA 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.
2980
P. ALIX, F. GROLLEAU, AND B. HUE
meric Drosophila GABA RDL receptor expressed in Xenopus
oocyte 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
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.
J Neurophysiol • VOL
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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Here it was demonstrated that an increase of intracellular Ca2⫹
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properties. The structure-activity relationship of GABA receptor binding insecticide molecules may be strongly dependent of
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