Camp. Biochem. Physiol. Vol. 103C, No. 3, pp. 429438, Printed in Great Britain
1992 0
0306-4492/92 $5.00 + 0.00 1992 Pergamon Press Ltd
MINI-REVIEW RECEPTORS FOR L-GLUTAMATE AND GABA THE NERVOUS SYSTEM OF AN INSECT (PERIPLANETA AMERICANA) D. B.
IN
SATTELLE
AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EH, U.K. Tel. (0223) 336678; Fax (0223) 461954 (Received 14 April 1992; accepted for publication 22 May 1992) Abstract-The
nervous system of the cockroach Peripluneta americana is well suited to studies of invertebrate amino acid receptors. Using a combination of radioligand binding and electrophysiological techniques, several distinct receptors have now been identified. These include an L-glutamate-gated
chloride channel which has no known counterpart in the vertebrate nervous system, and a putative kainate/quisqualate receptor with pharmacological properties different from those of the existing categories of vertebrate excitatory amino acid receptors. GABA receptors have also been characterized in the cockroach nervous system. Bicuculline, benzodiazepines and steroids have revealed important differences between certain insect GABA-gated chloride channels and vertebrate GABA receptors. Identifiable neurones may facilitate the allocation of specific functions to amino acid receptor subtypes. In view of the existence of subtypes of amino acid receptors in insects, it is of interest to examine how this is reflected at the molecular level in terms of receptor subunit composition and amino acid sequence. Preliminary molecular cloning studies on insect GABA receptors are described.
1-amino-cyclopentylamino-4-phosphonobutyrate; 1,3-dicarboxylate (ACPD). Apart from the ACPD receptor all are ligand-gated channel molecules, with an integral ion channel (type I). The ACPD receptor (type II) is linked to an inositol trisphosphate second messenger pathway. Molecular cloning has revealed a complex gene family and several subunit members of the family of L-glutamate-gated ion channels have been cloned and sequenced (Hollman et al., 1990). Among the receptors for GABA described in vertebrates two major classes can be recognized, and these have been defined using specific antagonists and agonists. GABA, receptors of vertebrates, which are of the ligand-gated type, are in most cases blocked by bicuculline, and possess modulatory sites for benzodiazepines, barbiturates and steroids (Stephenson, 1988). Such receptors are present on most neurones in the brain of vertebrates. Molecular cloning techniques have identified several subunits cc,,; /I-r; y,_r; 6,; p, (Seeburg et al., 1990; Cutting et al., 1991). GABA, receptors which mediate the effects of GABA on potassium and calcium conductances via G proteins have also been described (Bowery, 1989). Much less is known of the biochemistry, physiology and molecular biology of L-glutamate and GABA receptors of insects. Here a summary is presented of recent data obtained on the nervous system of the cockroach Periplaneta americana, where biochemical and electrophysiological studies can be performed on the same tissue. Work on other insect species is also referred to where appropriate. Some recent findings for the fruit fly Drosophila melanogaster, are also presented. This organism is
INTRODUCTION
The excitatory amino acid L-glutamate is considered to be the neurotransmitter at most excitatory synapses in the well-studied vertebrate nervous systems (Monaghan et al., 1989). In contrast to the findings, for vertebrates, the major insect excitatory neuromuscular junction transmitter appears to be L-glutamate (Usherwood, 1980) and insect neurones exhibit several distinct responses to this amino acid (Gerschenfeld, 1973; Walker, 1980). Unlike the case for vertebrates, insect neurones can exhibit both excitatory and inhibitory responses to L-glutamate. 4-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the nervous system of vertebrates, with up to one third of all synapses estimated to be GABAergic (Bloom and Iversen, 1971). In insects less detailed information is available, but there is growing evidence that GABA is an inhibitory neurotransmitter at many synapses in the nervous system and in muscle (Walker, 1980). Thus, considerable interest has developed in the receptor molecules that detect and mediate the responses to such amino acid neurotransmitters. To date five distinct L-glutamate receptors can be recognized in vertebrate central nervous tissues. The basis for assigning subclasses is their differential sensitivity to a variety of antagonists: kainate (KA); cl-amino-3-hydroxy-5-methyl-isooxazol-4-propionate (AMPA); N-methyl-o-aspartate (NMDA); L-2Review paper presented at The 3rd International Congress of Comparative Physiology and Biochemistry, Tokyo, August, 1991. 429
430
D. B. SATTELLE 25w ,
8
L-homocysteate suppress by 5545% the binding of L-glutamate, suggesting the possible existence of Lglutamate receptor subtypes in insect nervous tissue (Table 1). To date, no evidence has been obtained for the presence of an insect neuronal NMDA (Nmethyl-D-aspartate)-sensitive receptor. Using radiation inactivation techniques, a molecular weight estimate of 77,800 has been obtained for cockroach nervous system L-glutamate binding sites (Sepulveda and Sattelle, 1989b). Electrophysiological studies on identifiable neurones in the metathoracic ganglion of Periplaneta have provided evidence for the existence of more than one type of L-glutamate receptor (Fig. 2). On motor neurone & for example, an L-glutamate gated Clchannel has been detected (Wafford and Sattelle, 1986, 1989; Wafford et al., 1989). The actions of L-glutamate are mimicked by ibotenate and blocked by picrotoxin. Endrin, which blocks GABA-gated Cl- channels on the same cell, does not block these hype~olarizing responses to L-glutamate. On the same cell biphasic responses to L-glutamate are sometimes detected, the initial, rapid hyperpolarization being followed by a slow depolarization. The depolarization is mimicked by kainate and quisqualate. Preliminary experiments on dorsal midline neurones (DM) cells of the same ganglion have revealed a hy~~lari~ng response to ibotenate and a rapid depolarizing response to L-glutamate. Thus, several L-glutamate receptor subtypes are present in the cockroach nervous system (Wafford et al., 1991), and work is in progress to characterize their pharmacology in detail. Several distinct signal transduction systems appear to be involved in L-glutamate signalling in the insect nervous system. Messenger RNA prepared from locust (Sehistocerca gregaria) embryonic tissues has resulted in expression of functional L-glutamate receptors following injection into oocytes of Xenopus laeuis (Fig. 3). A rapid response to L-glutamate which
P
/-------
Time (mint
Fig. I. Time-course of the specific [ 3H]L-glutamate binding to cockroach CNS membranes. Membranes were incubated at &4”C in the presence of 50 nM [3H]-L-glutamate and the reaction stopped by centrifugation at various times. The experiment shown is representative of three, each run in duplicate. The line is the least squares fit of the points to a single increasing exponentiaf. and has an amplitude of 1969 cpm and a time constant (X0,) of 0.205 min-I. From Sepulveda and Sattelle (1989a).
well suited for the application and genetics. L
of molecular biology
Gfutamate receptors
A putative ~3H]-L-gIutamate receptor binding site has been characterized in membrane extracts prepared from the nervous system of Periplaneta americana (Sepulveda and Sattelle, 1989a). A dissociation constant of 0.83 + 0.35 PM and a binding capacity (I$,,,,) of 20.5 _+ 10.8 pmol rng--’ protein have been obtained in saturation experiments (Fig. 1). Affinities determined for a range of L-glutamate receptor ligands reveal a pharmacological profile that is not readily assigned to any of the existing vertebrate classes of central nervous system L-glutamate receptors. L-glutamate and ibotenate are the most effective ligands tested; quisqualate, aspartate, L-cysteate and
Table 1. Binding and physioiogicaal actions of neuroactive amino acids in (he insect central ner”O”S system
Amino acid L-Glutamate L-Aspartate Ibotenate L-Cyst&e L-Homocysteate Quisqualate
Maximum bound [‘H]L-Glutamate displaced (%) (1) 100 63 97 49 61 62
(2) 100 O-10 60-80
1.8
n.t. n.t. 3.5
>lOOO > 1000 >looO >looO >I000
55 34 32 27
50 O-10 2s 25 n.t.
AMPA o-APV
17 3
O-IO 35
26
1~x0(FM) (3) 0.8 8.3 27.0 455.0 530.0
D-Glutamate o-Asp&ate NMDA Kainate L-APB
Changes in membrane potential
-
(4) 0.8 15.0 13.0 14.0 56.0 30.0
200.0 n.t. 200.0 n.t. wt.
(5) 0.25
(6) +
n.t. at. n.t.
+ _ _
17) +
+ at.
_ t
n.t. n.t. n.t.
4.40 2.75 4.05 n.t. n.t.
n.t. n.t. _ + n.t.
n.t. n.t. n.t. + n.t.
n.t. n.t.
n.t. n.t.
n.t. n.t.
ns. 33.0
Specific [IH] L-glutamatebinding displaced by 1.0 mM of several amino acids (expressed as percentage of total); lcw values (pM) for the same ligands and their physiological effect in insect CNS. not tested: n.t.; effect +; no effect Species and tissues from which data were obtained: (I) and (3) Pe?i~l~e~ff um~ri~anQ, nerve cords, Sepulveda and Sattelle, 1989a; (2) Locusta gregaria nervous system, Usoh et al., 1988; (4) Musco domestica heads, Sherby er al., 1987; (5) Drosophila mefanogaster heads, Fiedler et al., 1986; (6) Peripfanera americana D, motor neurone, Wafford and Sattelle, 1989; (7) Schisrocerca gregaria isolated neurone somata, Giles and Usherwood, 1985.
Insect amino acid receptors Motor
neurone
431
(Df)
DUM Neurone
L-Glutamate
L-Glutamate
v
v
L
-65 mV
lbotenate
lbotenate v
v
-65 mV
Guisqualate
ouisqualate
V
V
-65 mV
SmV
5mV
1 min
5 min _1
_I
Fig. 2. Bath application of excitatory amino acids to the cell body of cockroach motor neurone D, and to a dorsal unpaired median (DUM) neurone show different responses. Three types of responses are shown: L-glutamate (1.0 x 10e4 M) generates a hyperpolarizing response on motor neurone D, but depolarizes DUM neurones; ibolenate (1.0 x 10e4 M) elicits a hyperpolarizing response on both cells; quisqualate (1.0 x lo-‘) M induces a slow depolarization when applied to both cell types. From Wafford et al. (1991).
to reverse at I?,,_ has been detected. In some oocytes a slower, sometimes oscillatory, response has been detected in expression studies. Similar responses have been detected in RNA isolated from locust muscle (Harris et al., 1990). Further experiments are needed to characterize these receptors. For insects such as locust and cockroach, further expression studies using tissue-specific RNA will be of considerable interest.
Table
Patch clamp investigations of cockroach GABA-gated
Data are obtained in cell-attached patch recordings from dissociated adult neurons (20-25 PM in diameter) of the cockroach Periplanera americano. Pipette solution (in mM): tetraethylammonium235: I; HEPES, with 10.0: pH 7.3 adjusted Cl, tetramethylammonium-OH. External solution (in mM): sucrose 50.0; pH 7.4 adjusted with NaOH. EC,- is -88.8 mV at 20°C. assuming [Cl-], = 7.0 mM. GABA (2.0 x IO-‘M) is applied via the patch pipette. Based on Malecot and Sattelle (1990).
appears
Cl - channels Unitary conductance properties of insect GABAgated Cl- channels have been reported recently by several laboratories. Noise analysis has been used to estimate a conductance ( 19 pS) and a mean open time
2. Open
Open/closed of channel Gpe” Closed
and closed
state
lifetimes for insect GABA-gated channels
Cl-
Mean open and closed time for Periphnefa neuronal GABA-gated Cl channels (ms) I, 0.28 + 0.03 I, I .43 + 0.20 I, 0.25 + 0.07 fZ 2.22 + 0.21 I, 43.73 + 5.44
432
D.B.
SATTELLE
(a)
+ mRNA
(1.0~ IdeM)
L-Glutamate
5mV
Control
I
d
(1.0% ld”M)
(b)
L-Glutamate
Bicuculline
GABA
GABA
u y
lOnA&
Fig. 3. (a) Upper
trace shows the results of an experiment in which messenger RNA isolated from locust tissue was injected into the cytoplasm of an oocyte of Xenopus laezris. Functional L-glutamate receptors were expressed. As shown in the lower trace control oocytes injected with the equivalent volume of distilled water did not respond to L-glutamate. From Sattelle et al. (1991). (b) Messenger RNA isolated from the nervous system of the cockroach Periplanetaamericana results in
Schistocercu gregariu embryonic
expression of bicuculline-insensitive GABA receptors when injected into the cytoplasm of oocytes of Xenopus Inevis. From Lummis et al. (1991).
(11.8 ms) for the Cl channels activated by GABA (5.0 x 10e5 M) on cultured embryonic cockroach neurons (Shimahara et al., 1987). Recently, Malecot and Sattelle (1990) have obtained unitary conductances for Cl- channels operated by GABA in dissociated adult cockroach neurones. Conductances of 11 pS and 17 pS are detected in the cell-attached recording configuration. Two exponentials are required to fit the open-time distributions and three exponentials are needed to fit the closed-time distributions (Table 2). Channel currents reverse their direction close to the estimated Ec,_ . Openings are brief and occur in bursts. Channel activity is blocked by picrotoxin. The behaviour of GABA-gated Cl- channels in dissociated cockroach neurones resembles that of GABA-gated Cl- channels in chick cerebral neurons (Weiss, 1988; Weiss et al., 1988).
Work on single channel recordings is currently being extended to Drosophila melanogaster. Studies on dissociated neurones and cell lines are being pursued with the aim of characterizing in situ channels of Drosophila. Recently, Clchannels of 20-25 pS conductance which display multi-level conductances and remain in the open state for many seconds have been detected in dissociated Drosophila neurones (Yamamoto and Suzuki, 1988) and a Drosophila cell line 1182-4 (Miller et al., 1991). Further single channel work on GABA-gated Clchannels is needed to assist with classification of insect GABA receptor subtypes. Agonist actions on cockroach channels Studies depressor
on an identified motor neurone,
GABA-gazed
Cl-
neurone (the fast coxal Dr) in the cockroach.
Insect amino acid receptors
(6)
Control
433
+Picrotoxin
+Bkuculllne
1 /Fnfl 7 F
Tf-
GABA-induced 36CI- uptake (% con1roI)
(b)
0
40
80
120
160
ZOO
Control 7 +GABA (1.0 x lOaM) +GABA (1.0 x 10-6M) +Picrotoxin (1.0 x 10” M)
loglo
Fig. 4. Bicuculline fails to block GABA-gated Cl- channels in the cockroach Periplaneta americana. (a) Ionophoretic application of GABA (70 nA for 2 s) onto the cell body of the fast coxal depressor (D,) motor neurone. Input resistance changes are shown by injecting 2.0 nA current pulses of 250 ms duration delivered every 1.5 s. Responses to GABA are recorded in normal saline, following 30 min bath application of 10 JoM bicuculline, and after 30 min bath application of 1OpM picrotoxin. (b) 36C1- uptake in cockroach nerve cord membrane preparation as percentage increase of control (150-200cpm) after 4 s incubation with “Cl- and l.OpM GABA. From Sattelle er al. (1991). et al., 1988) have shown that GABA activates a Cl _ channel. The use of identified neurones such as D, has permitted a systematic analysis of the response to the natural transmitter and a range of ligands. Accumulating data in this way for a defined population of GABA-gated Cl - channels, on a cell of known function, may eventually assist in ascribing particular roles to GABA receptor subtypes. Hill coefficients greater than one suggest that at least two GABA molecules are required for activation of the insect receptors (Roberts et al., 1981; Sattelle et al., 1988) as has been previously determined for vertebrate GABA, receptors. Agonist profiles of several insect GABA receptors share some similarities with those described for vertebrate GABA, receptors. The potency of muscimol is equal to or greater than that of GABA (Roberts et al., 1981; Sattelle et al., 1988) and isoguvacine is also a potent agonist, but 3-aminopropane sulphonic acid (3-APS) is much less effective. Although there are reports describing bicuculline antagonism of insect GABAergic neuronal activity (Roberts et al., 1981; Waldrop et aI., 1987), many insect GABA responses are insensitive to bicuculline (Lees et al., 1987; Sattelle et al., 1988). Bicuculline-insensitive GABA-gated Cl -channels are abundant in the cockroach nervous system (Fig. 5). Also, of the potent GABA, receptor antagonists, including pitrazepin and the steroid derivative RU5 135, only picrotoxin will block GABA responses (Sattelle et al., 1988). Baclofen, the GABA, receptor agonist, has so far proved to be ineffective in insects. It remains to be seen if insects possess a second messenger-linked GABA receptor system. (Pinnock
Actions of receptors
benzodiazepines
2
I
+GABA (1.0 x lo+) +Bicuculline (1.0 x lo4 M)
on
cockroach
GABA
Actions of benzodiazepines and barbiturates have been reported in several insect preparations. Fluni-
GABA
3
dose (nc)
Fig. 5. Benzodiazepines enhance the responses to GABA recorded from the cell body of the cockroach fast coxal depressor motor neuron (Dr). Following a 60 min exposure to flunitrazepam (1.0 x 1O-6 M), the dose-response curve for ionophoretically-applied GABA is shifted to the left. Mean values f standard error are plotted, and the number of cells used for each data point is indicated. (nC = nanocoulombs). From Sattelle et al. (1988).
trazepam enhances GABA-evoked responses in both cockroach (Fig. 5) and locust neuronal preparations (Lees et al., 1987; Sattelle et al., 1988), and noise analysis reveals that its action appears to be mediated by an increased frequency of opening of GABAoperated channels (Shimahara et al., 1987), as in vertebrates. Although there have been few studies on the effects of barbiturates in insects, there is some evidence for enhancement by pentobarbital of GABA responses in locust somata (Lees et al., 1987). However, since some studies have failed to detect interactions of benzodiazepines and barbiturates with insect GABA receptors, it is not yet clear if these modulators interact with insect GABA receptors in precisely the same way in which they act to generate the observed potent enhancement of vertebrate GABAA receptor responses. The pharmacology of insect [ ‘HI-flunitrazepam binding sites differs from that of vertebrate GABA, receptor-linked benzodiazepine binding sites (Table 3). Indeed, in some respects insect receptors resemble more closely vertebrate peripheral benzodiazepine binding sites. Clonazepam and flumazenil (Ro 15-l 788), potent inhibitors of vertebrate GABA, receptor linked sites, have very little effect on specific insect [‘HI-flunitrazepam binding (Robinson et al., 1986), whereas Ro 5-4864, a ligand specific for vertebrate peripheral (non-GABA receptor-linked) benzodiazepine binding sites, is a potent inhibitor Table
3. Displacement by benzodiazepines of (‘HI-flunitrazepam binding to insect and vertebrate tissues
Liaand Ro 5-4864 Diazepam Flunitrazepam Clonazepam Flumazenil Reference n.t., not tested.
Cockroach nervous svstem 560 1000 1600 25,000
100,000 Lummis and Sattelle (1986)
lcso (nM) Human brain
Rat kidnev
> 100,000 30 3
I n.t. Speth er al. (1978)
2 19 16 2500 n.t. Regan et al. (1981)
D. B. SATIELLE
434
(Ozoe et al., 1984). However, the electrophysiological evidence discussed above indicates that at least some insect flunitrazepam binding sites are linked to GABA receptors.
One interpretation of these findings is that insects possess GABA receptor-linked benzodiazepine sites with a pharmacological profile distinctly different from that on the vertebrate receptor-linked sites.
Table 4. ICY values for steroid displacement of [“SJ-TBPS binding Rat membranes 1% (NW -GABA +GABA
5a-Pregnan-3a-ol-20-one
5P-Pregnan-3a&20-one
5a-Pregnan-3a,21
5
>lOO
Fly membranes tc, luW -GABA +GABA
0.04
>I00
0.08
>I00
36
>iO
-&of-2O-one
8
0.14
22
10
5a-Pregnan-Ba-ol-11.20-dione
13
0.52
29
47
inactive
77
5a-Pregnan-3P-ol-20-one
>I00
Inactive
Insect amino acid receptors
435
!5a-pregnan-3a-ob204me (SO)r M)
5a-pre1gnan-3a-ol-l1,2O-dione (100pM) 5a-pregnan3asl-20-one
(100~ M)
5mV
L2 min
Fig. 6. Pregnane steroids 5a-pregnan-3a-ol-20-one and 5a-pregnan-3a-01-11, 20-dione exhibit little or no effect on responses to GABA of the cell body membrane of cockroach motor neuron Dr. GABA application is indicated by the solid bars; steroid application is indicated by the cross-hatched bars. From Sattelle et al. (1991). Further support for this interpretation derives from the observation that insect benzodiazepine binding sites can be modulated by GABA, in a similar way to vertebrate receptor-linked sites, and in contrast
to vertebrate peripheral benzodiazepine sites (Le Fur, 1988). In the insect species examined, binding at this site is enhanced by GABA (Ozoe et al., 1989; Robinson et al., 1986). The enhancement appears to be biphasic, however, since it is reduced at high GABA concentrations (Abalis et al., 1983). An alternative explanation is that in insects, as there are receptor-linked and in vertebrates,
non-receptor-linked benzodiazepine sites which have different pharmacological profiles. Additional studies are needed to discriminate between these possibilities. Recently, the capacity of flunitrazepam to photoaffinity label its receptor has been exploited (Robinson et al., 1986). This work on the locust Schistocerca gregaria has revealed two binding proteins of M, 45,000 and M, 59,000. These results may indicate the existence of two protein subunits, each containing a benzodiazepine site, although later experiments using sarmazenil
HUM@J(l) PI BOVlNE(2) 11 82 RAT (2)
:: !:
SILITILSWVSF!m' ILQTYNPSTIITILSWVSFW ILQTYNPSIIITI ILQTYMPSIMITI moo""
DROSOPHlL4
HUMAN(l)
EMU
IDIYIJCCFVWFLALL ILX4YLMGCFVFVFtULL
TSARVALCITTVLlMlTIS -.."
BOVINE
SMRvALG1mm1 SAARvALGIm~I
rn"..
(3)a, a2 a3
01 n
Fig. 7. Amino acid alignment for (90) residues encompassing membrane spanning regions (MI, M2, M3) of GABA receptor subunits of mammalian brain and a putative GABA receptor of Drosophila melanogaster. The following mammalian GABA receptor subunit sequences are used in the alignment: human a,, /3,, y2; bovine a,.), B,.,; rat /3_,. Filled circles indicate identity of residues in the Drosophila sequence with human a,, /I, and y2. Some of the sequences used for alignment are as follows (1) Pritchett et al. (1989); (2) Ymer et al. (1989); (3) Levitan et al. (1988). From Sattelle et al. (1991).
D. B. SATTELLE
436
(Ro 15-45 13) suggest that the smaller band may be a breakdown product. Actions of steroids on insect and vertebrate GABA receptors Steroids can modulate vertebrate GABA, receptor function by acting at a distinct binding site (see, for example, Turner et al., 1989). Several naturally occurring steroids have been shown to enhance GABAactivated and barbiturate-activated %Zl~ uptake, and to inhibit [35S]-TBPS binding in vertebrate CNS preparations. A series of pregnane steroids are potent inhibitors of [%I-TBPS binding to rat brain membranes. and all are more effective in the presence of GABA (Table 4). However, when the same steroids are tested against insect [“S]-TBPS binding sites in the presence or absence of GABA they are two to three orders of magnitude less potent (Rauh et al., 1991; 1992). Thus, if a unique steroid-sensitive site is present on insect GABA receptor molecules, it possesses a quite different pharmacological profile from the corresponding vertebrate site. The insect-specific steroids ecdysone and 20hydroxyecdysone are also virtually without effect on [35S]-TBPS binding to housefly and rat brain membranes. Like the pregnane steroids, the insect steroids tested to date are also largely ineffective on electrophysiological responses to GABA recorded from the cockroach Dr motor neurone (Fig. 6). It is possible that other insect steroids or endogenous steroid metabolites may be found which are able to modulate insect receptors. Cloning and sequencing of putative melanogaster GABA receptor subunits
modulator neurones. L-glutamate receptors can be expressed in oocytes of Xenopus laevis. GABA-gated Cl- channels that are bicuculhneinsensitive are abundant in the cockroach nervous system. Bicuculline-sensitive GABA receptors have also been described, and these too probably gate Clchannels. GABA-gated Cl- channels are present on cell body and neuropile membranes in the insect nervous system. The sites of action of benzodiazepines, steroids and convulsants on a cockroach GABA-gated Cl- channel differ strikingly in their pharmacology from corresponding sites on vertebrate GABA, receptors. Block by picrotoxin, TBPS and many polychlorocycloalkane insecticides has been observed in the case of cockroach GABA-gated Cl - channels. Patch-clamp studies reveal single channel conductances of 11 pS and 17 pS for cockroach GABA-gated Cl- channels. The recent cloning of Drosophila melanogaster putative GABA receptor subunits offers the prospect of an enhanced understanding of structure-function relations of insect ion channels operated by amino acids. The combination of biochemistry, electrophysiology and molecular biology is enhancing our understanding of the properties and functions of GABA and L-glutamate receptors of the insect nervous system. Acknowledgements-The author is indebted to the following, all of whom contributed to aspects of the work reviewed here: K. A. Wafford. M.-I. Sepulveda, D. Bai. S. C. R. Lummis, N. M. Anthony, K. W. P. Miller, J. H. Wong and J. J. Rauh. The support of the Agricultural and Food Research Council of the U.K. and a grant from E. I. du Pont de Nemours is gratefully acknowledged.
Drosophila
Two putative GABA receptor subunits from Drosophila melanogaster have been reported. In our laboratory oligonucleotides have been prepared, based on the well-characterized mammalian GABA receptor sequences (Barnard et al., 1987; 1989). These synthetic oligonucleotides, designed using the highly conserved transmembrane regions. have been employed to probe a Drosophila melanogaster cDNA library from i sep (Stratagene). From the Drosophila nucleotide sequence an amino acid sequence has been deduced (Sattelle et al., 1991) and this is shown (Fig. 7) for the membrane spanning regions (MlM3). By extending this study, we aim to provide the complete sequence of a putative insect, p-like, GABA receptor subunit. Recently another laboratory has reported a Drosophila GABA receptor subunit. This subunit was discovered by identifying and sequencing a gene responsible for cyclodiene-resistance in the fruitfly (ffrench-Constant et al., 1991). CONCLUSIONS
L-glutamate binding sites have been characterized using cockroach nervous system membranes, and by means of radiation inactivation a MW of 77,800 has been established. L-glutamate gated Cl channels have been demonstrated in cockroach neurones, as well as several other distinct L-glutamate receptor responses. Cell-specific L-glutamate-gated channels are associated with functionally-distinct motor and
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M. E. and Eldefrawi A. T. (1983) Biochemical identification of putative GABA/benzodiazepine receptors in house fly thorax muscles. Pestic.
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Barnard E. A., Darlison M. G. and Seeburg P. H. (1987) Molecular biology of the GABA, receptor: the receptor/ channel superfamily. TINS 10, 502-509. Barnard E. A., Darlison M. G., Marshall J. and Sattelle D. B. (1989) Structural characteristics of cation and anion channels directly operated by agonists In ton Transport (edited by Keeling D. and Benham C.), pp. 1599182. Academic Press, London. Bowery N. G. (1983) Classification of GABA receptors In The GABA Receptors (edited by Enna S. J.), pp. 1733213. Humana Press, Clifton. New Jersey. Cutting G. R., Lvo L., O’Hara B. F., Kasch L. M.. Monstrose-Rafizadell C., Donovan D. M.. Shimada S., Antonarakis S. E., Guggino W. B.. Uhl G. R. and Kazaziam H. H. (1991) Cloning of one y-aminobutyric acid (GABA)p cDNA: a GABA receptor subunit highly expressed in the retina. Proc. Natl Acad. Sci. (U.S.A.) 88, 2673-2677. Fiedler J. L., Inestrosa N. C. and Bustos G. (1986) Putative glutamate receptors in membranes obtained from heads of Drosophila melanogaster. J. Neurosci. 16, 50555 15. ffrench-Constant R. H., Mortlock D. P.. Shaffer C. D., MacIntyre R. J. and Roush R. T. (1991) Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate r-aminobutvric acid subtype A receptor locus. Proc Nati Acad Sci (U.S.A.) 88, 7269-7213. Gerschenfeld H. M. (1973) Chemical transmission in invertebrate central nervous system and neuromuscular junctions. Physiol. Rev. 53, Ill 19.
Insect amino acid receptors Giles D. and Usherwood P. N. R. (1985) The effects of putative amino acid neurotransmitters on somata isolated from neurons of the locust central nervous system. Comp. Biochem. Physiol. 8OC, 231-236.
Hollman M., Rogers S. W., O’Shea-Greenfield A., Deneris E. S., Hughes T. E., Gasic G. P. and Heinemann S. (1990). Glutamate receptor GluR-KI: structure, function and expression in the brain. Cold Spring Harbour Symposia on Quaruitative Biology LV, 41-55. Lees G., Beadle D. J., Neumann R. and Benson J. A. (1987) Responses to GABA by isolated insect neuronal somata: pharmacology and modulation by a benzodiazepine and a barbiturate. Brain Res. 401, 267-278. Le Fur G. (1988) Periuheral benzodiazenine bindina sites In GABA and Benzodiazepine Receptors (Edited by Squires R. F.), pp. 15-34. CRC Press Boca Raton, Florida. Levitan E. S., Schofield P. R., Burt D., Rhee L. M., Wisden W., Kohler M., Fujita N., Rodriguez H. F., Stephenson A., Darlison M. G., Barnard E. A. and Seeburg, P. H. (1988) Structural and functional basis for GABA, reccptar heterogeneity. Nature (1986) 335, 76-79. .. _ Lummis S. C. R. and Sattelle D. B. (1986) Binding sites for [)H]-GABA, [)H]-flunitrazepam and [“S]-TBPS in insect CNS. Neurochem. Int. 9, 287-293. Lummis S. C. R.. Riina H. A. and Sattelle D. B. (1991) Functional expression in Xenopus oocytes of a bicucullinel insensitive insect GABA receptor. Pesric Sci. 32, 505P. Malecot C. and Sattelle D. B. (1990) Single channel recordings from insect neuronal GABA-activated chloride channeis. J. exp. Biol. 151, 495-501. Miller K. W. P.. Leech C. A. and Sattelle D. B. (1991) Sinele channel chloride currents in Drosophila melanogasfer primary cultured neurones and cell lines. Pestic. Sci. 32, 497-499. Monaghan D. J., Bridges R. J. and Cotman C. W. (1989) The excitatory amino acid Receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system. Ann. Rev. Pharmacol. Toxicol. 29, 365402. Ozoe Y., Fukuda K., Mochida K. and Nakamura T. (1989) Actions of benzodiazepines on the housefly. 3. In vitro
binding of [‘H]Ro 54864 responding to GABA receptor ligands. Comp. Biochem. Physiol. 93C, 193-199. Pinnock R. D., David J. A. and Sattelle D. B. (1988) Ionic events following GABA receptor activation in an identified insect motor neuron. Proc. R. Sot. Lond. B 232, 457-470.
Pritchett D. B., Sontheimer H., Shivers B. D., Ymer S., Kettenman H., Schofield P. R. and Seeburg P. H. (1989) Importance of a novel GABA, receptor subunit for benzodiazepine pharmacology. Namre 338, 582-585. Rauh J. J.. Lummis S. C. R. and Sattelle D. B. (1990) Pharmacological and biochemical properties of insect GABA receptors. Trena!s Pharmacol. Sci. 11, 325-329. Regan J. W., Yamamura H. I., Yamada S. and Roeske W. R. (1981) High-affinity renal [‘HI-flunitrazepam binding; characterization, localization and alteration in hypertension. Life Sci. 28, 991-998. Roberts C. J., Krogsgaard-Larsen P. and Walker R. J. (198 I) Studies of the action of GABA, muscimol, and related compounds on Periplaneta and Limulus neurons. Comp. Biochem. Physiol. 69C, 7-l 1. Robinson T., MacAllan D., Lunt G. G. and Battersby M. (1986) y-Aminobutyric acid receptor complex of insect CNS: characterization of a benzodiazcpine binding site. J. Neurochem. 47, 195551962.
Sattelle D. B. (1990) GABA receptors of insects. Adv. Insect. Physiol. 22, l-113. Sattelle D. B., Pinnock R. D., Wafford K. A. and David J. A. (1988) GABA receptors on the cell-body membrane of an identified insect motor neuron. Proc. R. Sot. Lond. B 232, 443456.
437
Sattelle, D. B., Marshall J., Lummis S. C. R., Leech C. A., Miller K. W. P., Anthony N. M. A., Bai D., Wafford K. A., Harrison J. B., Chapaitis L. A., Watson M. K., Benner E. A., Vassallo G., Wong J. F. H. and Rauh J. J., (1991) y-Aminobutyric acid and L-glutamate receptors of insect nervous tissue. In Transmitter Amino Acid Receptors: Structures, Transduction and Modelsfor Drug Development (edited by Barnard E. A. and Costa E.),
pp. 273-291. Thieme Medical Publishers, New York. Seeburg P. H., Wisden W., Verdoom T. A., Pritchett D. B., Werner P., Herb A., Luddens H., Sprengel R. and Sakmann B. (1990) The GABA, receptor family: molecular and functional diversity. Cold Spring Harbour Symposia on Quantitative Biology LV, 29-40.
Sepulveda M.-I. and Sattelle D. B. (1989a) Pharmacology of an insect central nervous system [ 3H]-L-glutamate binding site. Neurochem. Int. 14, 471-475. Sepulveda M.-I. and Sattelle D. B. (1989b) Molecular target size of an L-glutamate receptor from the cockroach central nervous system. Neurosci. Z&r. 100, 21&214. Sherby M. S., Eldefrawi M. E., Wafford K. A., Sattelle D. B. and Eldefrawi A. T. (1987) Pharmacology of putative glutmate receptors from insect skeletal muscle, insect central nervous system and rat brain. Comp. Biochem. Physiol. 8X,
99-106.
Shimahara T., Pichon Y., Lees G., Beadle C. A. and Beadle D. J. (1987) y-Aminobutyric acid receptors on cultured cockroach brain neurones. J. exp. Biol. 131, 231-244. Speth R. C., Wastek G. J., Johnson P. C. and Yamamura H. I. (1978) Benzodiazepine binding in human brain: characterization using [3H]-flunitrazepam. Life Sci. 22, 859-866. Stephenson A. (1988) Understanding the GABA, receptor: a chemically gated ion channel. Biochem. J. 249, 21-32. Turner D. M.. Ransom R. W.. Yana S.-J. and Olsen R. W. (1989) Steroid anaesthetics and naturally occurring analogs modulate the y-aminobutyric acid receptor complex at a site distinct from barbiturates. J. Pharmac. exp. Ther. 248, 960-966.
Usherwood P. N. R. (1980) Neuromuscular transmitter receptors of insect muscle. In Receptorsfor Neurotransmit ters. Hormones and Pheromones in Insects (edited by Sattelle D. B., Hall L. M. and Hildebrand J. G.), DD. 141-153. Elsevier/North Holland. Amsterdam. U.&h G., Duce I. R. and’usherwood P. N. R. (1989) Binding of glutamate to a membrane fraction from locust (Schislocerca gregaria) central nervous system. Pestic. Sci. 24, 71-73.
Watford K. A. and Sattelle D. B. (1986) Effects of amino acid neurotransmitter candidates on an identified insect motorneurone. Neurosci. Letr. 63, 135-140. Wafford K. A., Sattelle D. B., Abalis I., Eldefrawi A. T. and Eldefrawi M. E. (1987) y-Aminobutyric assay for CNS GABA receptors of insects. J. Neurochem. 49, 177-180. Wafford K. A., Sattelle D. B., Gant D. B., Eldefrawi A. T. and Eldefrawi M. E. (1989) Noncompetitive inhibition of GABA receptors in insect and vertebrate CNS by endrin and lindane. Pestic. Biochem. Physiol. 33, 213-219. Wafford K. A. and Sattelle D. B. (1989) L-glutamate receptors on the cell membrane of an identified insect motor neurone. J. exp. Biol. 144, 449462. Wafford K. A., Bai D., Sepulveda M. I. and Sattelle D. B. (1991) L-glutamate receptors in the insect central nervous system. In Excitafory Ammo Acids (Edited by Meldrum B. S., Moroni F., Simon R. P. and Words J. J.), pp. 275-279. Raven Press, New York. Waldrop B., Christensen T. A. and Hildebrand J. G. (1987) GABA-mediated synaptic inhibition of projection neurons in the antenna1 lobes of the sphinx moth Manduca sexta. J. camp. Physiol. 161, 23-32.
Walker R. J. (1980) Chemical neurotransmission in invertebrates. In Receptors for Neurotransmitters, Hormones and Pheromones (edited by Sattelle D. B., Hall L. M. and
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Hildebrand J. G.), pp. 3149. Elsevier/North Holland, Amsterdam. Weiss D. S. (1988) Membrane potential modulates the activation of GABA-gated channels. J. Neurophysiol. 59, 514-521.
Weiss D. S., Barnes E. M. and Hablitz J. J. (1988) Wholecell and single-channel recordings of GABA-gated currents in cultured chick cerebral neurons. J. Neurophysiol. 59, 495-513.
SATTELLE
Yamamoto D. and Suzuki N. (1987) Single channel recordings of chloride currents in primary cultured Drosophila neurons. Arch. Insect Biochem. Physiol. 6, 151-158. Ymer S., Schofield P. R., Draguhn A., Wenner P., Kohler M. and Secburg P. H. GABA, receptor /I subunit heterogeneity: functional expression of cloned cDNAs. EMBO J. 8, 1665.
1992 0
0306-4492/92 $5.00 + 0.00 1992 Pergamon Press Ltd
MINI-REVIEW RECEPTORS FOR L-GLUTAMATE AND GABA THE NERVOUS SYSTEM OF AN INSECT (PERIPLANETA AMERICANA) D. B.
IN
SATTELLE
AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EH, U.K. Tel. (0223) 336678; Fax (0223) 461954 (Received 14 April 1992; accepted for publication 22 May 1992) Abstract-The
nervous system of the cockroach Peripluneta americana is well suited to studies of invertebrate amino acid receptors. Using a combination of radioligand binding and electrophysiological techniques, several distinct receptors have now been identified. These include an L-glutamate-gated
chloride channel which has no known counterpart in the vertebrate nervous system, and a putative kainate/quisqualate receptor with pharmacological properties different from those of the existing categories of vertebrate excitatory amino acid receptors. GABA receptors have also been characterized in the cockroach nervous system. Bicuculline, benzodiazepines and steroids have revealed important differences between certain insect GABA-gated chloride channels and vertebrate GABA receptors. Identifiable neurones may facilitate the allocation of specific functions to amino acid receptor subtypes. In view of the existence of subtypes of amino acid receptors in insects, it is of interest to examine how this is reflected at the molecular level in terms of receptor subunit composition and amino acid sequence. Preliminary molecular cloning studies on insect GABA receptors are described.
1-amino-cyclopentylamino-4-phosphonobutyrate; 1,3-dicarboxylate (ACPD). Apart from the ACPD receptor all are ligand-gated channel molecules, with an integral ion channel (type I). The ACPD receptor (type II) is linked to an inositol trisphosphate second messenger pathway. Molecular cloning has revealed a complex gene family and several subunit members of the family of L-glutamate-gated ion channels have been cloned and sequenced (Hollman et al., 1990). Among the receptors for GABA described in vertebrates two major classes can be recognized, and these have been defined using specific antagonists and agonists. GABA, receptors of vertebrates, which are of the ligand-gated type, are in most cases blocked by bicuculline, and possess modulatory sites for benzodiazepines, barbiturates and steroids (Stephenson, 1988). Such receptors are present on most neurones in the brain of vertebrates. Molecular cloning techniques have identified several subunits cc,,; /I-r; y,_r; 6,; p, (Seeburg et al., 1990; Cutting et al., 1991). GABA, receptors which mediate the effects of GABA on potassium and calcium conductances via G proteins have also been described (Bowery, 1989). Much less is known of the biochemistry, physiology and molecular biology of L-glutamate and GABA receptors of insects. Here a summary is presented of recent data obtained on the nervous system of the cockroach Periplaneta americana, where biochemical and electrophysiological studies can be performed on the same tissue. Work on other insect species is also referred to where appropriate. Some recent findings for the fruit fly Drosophila melanogaster, are also presented. This organism is
INTRODUCTION
The excitatory amino acid L-glutamate is considered to be the neurotransmitter at most excitatory synapses in the well-studied vertebrate nervous systems (Monaghan et al., 1989). In contrast to the findings, for vertebrates, the major insect excitatory neuromuscular junction transmitter appears to be L-glutamate (Usherwood, 1980) and insect neurones exhibit several distinct responses to this amino acid (Gerschenfeld, 1973; Walker, 1980). Unlike the case for vertebrates, insect neurones can exhibit both excitatory and inhibitory responses to L-glutamate. 4-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the nervous system of vertebrates, with up to one third of all synapses estimated to be GABAergic (Bloom and Iversen, 1971). In insects less detailed information is available, but there is growing evidence that GABA is an inhibitory neurotransmitter at many synapses in the nervous system and in muscle (Walker, 1980). Thus, considerable interest has developed in the receptor molecules that detect and mediate the responses to such amino acid neurotransmitters. To date five distinct L-glutamate receptors can be recognized in vertebrate central nervous tissues. The basis for assigning subclasses is their differential sensitivity to a variety of antagonists: kainate (KA); cl-amino-3-hydroxy-5-methyl-isooxazol-4-propionate (AMPA); N-methyl-o-aspartate (NMDA); L-2Review paper presented at The 3rd International Congress of Comparative Physiology and Biochemistry, Tokyo, August, 1991. 429
430
D. B. SATTELLE 25w ,
8
L-homocysteate suppress by 5545% the binding of L-glutamate, suggesting the possible existence of Lglutamate receptor subtypes in insect nervous tissue (Table 1). To date, no evidence has been obtained for the presence of an insect neuronal NMDA (Nmethyl-D-aspartate)-sensitive receptor. Using radiation inactivation techniques, a molecular weight estimate of 77,800 has been obtained for cockroach nervous system L-glutamate binding sites (Sepulveda and Sattelle, 1989b). Electrophysiological studies on identifiable neurones in the metathoracic ganglion of Periplaneta have provided evidence for the existence of more than one type of L-glutamate receptor (Fig. 2). On motor neurone & for example, an L-glutamate gated Clchannel has been detected (Wafford and Sattelle, 1986, 1989; Wafford et al., 1989). The actions of L-glutamate are mimicked by ibotenate and blocked by picrotoxin. Endrin, which blocks GABA-gated Cl- channels on the same cell, does not block these hype~olarizing responses to L-glutamate. On the same cell biphasic responses to L-glutamate are sometimes detected, the initial, rapid hyperpolarization being followed by a slow depolarization. The depolarization is mimicked by kainate and quisqualate. Preliminary experiments on dorsal midline neurones (DM) cells of the same ganglion have revealed a hy~~lari~ng response to ibotenate and a rapid depolarizing response to L-glutamate. Thus, several L-glutamate receptor subtypes are present in the cockroach nervous system (Wafford et al., 1991), and work is in progress to characterize their pharmacology in detail. Several distinct signal transduction systems appear to be involved in L-glutamate signalling in the insect nervous system. Messenger RNA prepared from locust (Sehistocerca gregaria) embryonic tissues has resulted in expression of functional L-glutamate receptors following injection into oocytes of Xenopus laeuis (Fig. 3). A rapid response to L-glutamate which
P
/-------
Time (mint
Fig. I. Time-course of the specific [ 3H]L-glutamate binding to cockroach CNS membranes. Membranes were incubated at &4”C in the presence of 50 nM [3H]-L-glutamate and the reaction stopped by centrifugation at various times. The experiment shown is representative of three, each run in duplicate. The line is the least squares fit of the points to a single increasing exponentiaf. and has an amplitude of 1969 cpm and a time constant (X0,) of 0.205 min-I. From Sepulveda and Sattelle (1989a).
well suited for the application and genetics. L
of molecular biology
Gfutamate receptors
A putative ~3H]-L-gIutamate receptor binding site has been characterized in membrane extracts prepared from the nervous system of Periplaneta americana (Sepulveda and Sattelle, 1989a). A dissociation constant of 0.83 + 0.35 PM and a binding capacity (I$,,,,) of 20.5 _+ 10.8 pmol rng--’ protein have been obtained in saturation experiments (Fig. 1). Affinities determined for a range of L-glutamate receptor ligands reveal a pharmacological profile that is not readily assigned to any of the existing vertebrate classes of central nervous system L-glutamate receptors. L-glutamate and ibotenate are the most effective ligands tested; quisqualate, aspartate, L-cysteate and
Table 1. Binding and physioiogicaal actions of neuroactive amino acids in (he insect central ner”O”S system
Amino acid L-Glutamate L-Aspartate Ibotenate L-Cyst&e L-Homocysteate Quisqualate
Maximum bound [‘H]L-Glutamate displaced (%) (1) 100 63 97 49 61 62
(2) 100 O-10 60-80
1.8
n.t. n.t. 3.5
>lOOO > 1000 >looO >looO >I000
55 34 32 27
50 O-10 2s 25 n.t.
AMPA o-APV
17 3
O-IO 35
26
1~x0(FM) (3) 0.8 8.3 27.0 455.0 530.0
D-Glutamate o-Asp&ate NMDA Kainate L-APB
Changes in membrane potential
-
(4) 0.8 15.0 13.0 14.0 56.0 30.0
200.0 n.t. 200.0 n.t. wt.
(5) 0.25
(6) +
n.t. at. n.t.
+ _ _
17) +
+ at.
_ t
n.t. n.t. n.t.
4.40 2.75 4.05 n.t. n.t.
n.t. n.t. _ + n.t.
n.t. n.t. n.t. + n.t.
n.t. n.t.
n.t. n.t.
n.t. n.t.
ns. 33.0
Specific [IH] L-glutamatebinding displaced by 1.0 mM of several amino acids (expressed as percentage of total); lcw values (pM) for the same ligands and their physiological effect in insect CNS. not tested: n.t.; effect +; no effect Species and tissues from which data were obtained: (I) and (3) Pe?i~l~e~ff um~ri~anQ, nerve cords, Sepulveda and Sattelle, 1989a; (2) Locusta gregaria nervous system, Usoh et al., 1988; (4) Musco domestica heads, Sherby er al., 1987; (5) Drosophila mefanogaster heads, Fiedler et al., 1986; (6) Peripfanera americana D, motor neurone, Wafford and Sattelle, 1989; (7) Schisrocerca gregaria isolated neurone somata, Giles and Usherwood, 1985.
Insect amino acid receptors Motor
neurone
431
(Df)
DUM Neurone
L-Glutamate
L-Glutamate
v
v
L
-65 mV
lbotenate
lbotenate v
v
-65 mV
Guisqualate
ouisqualate
V
V
-65 mV
SmV
5mV
1 min
5 min _1
_I
Fig. 2. Bath application of excitatory amino acids to the cell body of cockroach motor neurone D, and to a dorsal unpaired median (DUM) neurone show different responses. Three types of responses are shown: L-glutamate (1.0 x 10e4 M) generates a hyperpolarizing response on motor neurone D, but depolarizes DUM neurones; ibolenate (1.0 x 10e4 M) elicits a hyperpolarizing response on both cells; quisqualate (1.0 x lo-‘) M induces a slow depolarization when applied to both cell types. From Wafford et al. (1991).
to reverse at I?,,_ has been detected. In some oocytes a slower, sometimes oscillatory, response has been detected in expression studies. Similar responses have been detected in RNA isolated from locust muscle (Harris et al., 1990). Further experiments are needed to characterize these receptors. For insects such as locust and cockroach, further expression studies using tissue-specific RNA will be of considerable interest.
Table
Patch clamp investigations of cockroach GABA-gated
Data are obtained in cell-attached patch recordings from dissociated adult neurons (20-25 PM in diameter) of the cockroach Periplanera americano. Pipette solution (in mM): tetraethylammonium235: I; HEPES, with 10.0: pH 7.3 adjusted Cl, tetramethylammonium-OH. External solution (in mM): sucrose 50.0; pH 7.4 adjusted with NaOH. EC,- is -88.8 mV at 20°C. assuming [Cl-], = 7.0 mM. GABA (2.0 x IO-‘M) is applied via the patch pipette. Based on Malecot and Sattelle (1990).
appears
Cl - channels Unitary conductance properties of insect GABAgated Cl- channels have been reported recently by several laboratories. Noise analysis has been used to estimate a conductance ( 19 pS) and a mean open time
2. Open
Open/closed of channel Gpe” Closed
and closed
state
lifetimes for insect GABA-gated channels
Cl-
Mean open and closed time for Periphnefa neuronal GABA-gated Cl channels (ms) I, 0.28 + 0.03 I, I .43 + 0.20 I, 0.25 + 0.07 fZ 2.22 + 0.21 I, 43.73 + 5.44
432
D.B.
SATTELLE
(a)
+ mRNA
(1.0~ IdeM)
L-Glutamate
5mV
Control
I
d
(1.0% ld”M)
(b)
L-Glutamate
Bicuculline
GABA
GABA
u y
lOnA&
Fig. 3. (a) Upper
trace shows the results of an experiment in which messenger RNA isolated from locust tissue was injected into the cytoplasm of an oocyte of Xenopus laezris. Functional L-glutamate receptors were expressed. As shown in the lower trace control oocytes injected with the equivalent volume of distilled water did not respond to L-glutamate. From Sattelle et al. (1991). (b) Messenger RNA isolated from the nervous system of the cockroach Periplanetaamericana results in
Schistocercu gregariu embryonic
expression of bicuculline-insensitive GABA receptors when injected into the cytoplasm of oocytes of Xenopus Inevis. From Lummis et al. (1991).
(11.8 ms) for the Cl channels activated by GABA (5.0 x 10e5 M) on cultured embryonic cockroach neurons (Shimahara et al., 1987). Recently, Malecot and Sattelle (1990) have obtained unitary conductances for Cl- channels operated by GABA in dissociated adult cockroach neurones. Conductances of 11 pS and 17 pS are detected in the cell-attached recording configuration. Two exponentials are required to fit the open-time distributions and three exponentials are needed to fit the closed-time distributions (Table 2). Channel currents reverse their direction close to the estimated Ec,_ . Openings are brief and occur in bursts. Channel activity is blocked by picrotoxin. The behaviour of GABA-gated Cl- channels in dissociated cockroach neurones resembles that of GABA-gated Cl- channels in chick cerebral neurons (Weiss, 1988; Weiss et al., 1988).
Work on single channel recordings is currently being extended to Drosophila melanogaster. Studies on dissociated neurones and cell lines are being pursued with the aim of characterizing in situ channels of Drosophila. Recently, Clchannels of 20-25 pS conductance which display multi-level conductances and remain in the open state for many seconds have been detected in dissociated Drosophila neurones (Yamamoto and Suzuki, 1988) and a Drosophila cell line 1182-4 (Miller et al., 1991). Further single channel work on GABA-gated Clchannels is needed to assist with classification of insect GABA receptor subtypes. Agonist actions on cockroach channels Studies depressor
on an identified motor neurone,
GABA-gazed
Cl-
neurone (the fast coxal Dr) in the cockroach.
Insect amino acid receptors
(6)
Control
433
+Picrotoxin
+Bkuculllne
1 /Fnfl 7 F
Tf-
GABA-induced 36CI- uptake (% con1roI)
(b)
0
40
80
120
160
ZOO
Control 7 +GABA (1.0 x lOaM) +GABA (1.0 x 10-6M) +Picrotoxin (1.0 x 10” M)
loglo
Fig. 4. Bicuculline fails to block GABA-gated Cl- channels in the cockroach Periplaneta americana. (a) Ionophoretic application of GABA (70 nA for 2 s) onto the cell body of the fast coxal depressor (D,) motor neurone. Input resistance changes are shown by injecting 2.0 nA current pulses of 250 ms duration delivered every 1.5 s. Responses to GABA are recorded in normal saline, following 30 min bath application of 10 JoM bicuculline, and after 30 min bath application of 1OpM picrotoxin. (b) 36C1- uptake in cockroach nerve cord membrane preparation as percentage increase of control (150-200cpm) after 4 s incubation with “Cl- and l.OpM GABA. From Sattelle er al. (1991). et al., 1988) have shown that GABA activates a Cl _ channel. The use of identified neurones such as D, has permitted a systematic analysis of the response to the natural transmitter and a range of ligands. Accumulating data in this way for a defined population of GABA-gated Cl - channels, on a cell of known function, may eventually assist in ascribing particular roles to GABA receptor subtypes. Hill coefficients greater than one suggest that at least two GABA molecules are required for activation of the insect receptors (Roberts et al., 1981; Sattelle et al., 1988) as has been previously determined for vertebrate GABA, receptors. Agonist profiles of several insect GABA receptors share some similarities with those described for vertebrate GABA, receptors. The potency of muscimol is equal to or greater than that of GABA (Roberts et al., 1981; Sattelle et al., 1988) and isoguvacine is also a potent agonist, but 3-aminopropane sulphonic acid (3-APS) is much less effective. Although there are reports describing bicuculline antagonism of insect GABAergic neuronal activity (Roberts et al., 1981; Waldrop et aI., 1987), many insect GABA responses are insensitive to bicuculline (Lees et al., 1987; Sattelle et al., 1988). Bicuculline-insensitive GABA-gated Cl -channels are abundant in the cockroach nervous system (Fig. 5). Also, of the potent GABA, receptor antagonists, including pitrazepin and the steroid derivative RU5 135, only picrotoxin will block GABA responses (Sattelle et al., 1988). Baclofen, the GABA, receptor agonist, has so far proved to be ineffective in insects. It remains to be seen if insects possess a second messenger-linked GABA receptor system. (Pinnock
Actions of receptors
benzodiazepines
2
I
+GABA (1.0 x lo+) +Bicuculline (1.0 x lo4 M)
on
cockroach
GABA
Actions of benzodiazepines and barbiturates have been reported in several insect preparations. Fluni-
GABA
3
dose (nc)
Fig. 5. Benzodiazepines enhance the responses to GABA recorded from the cell body of the cockroach fast coxal depressor motor neuron (Dr). Following a 60 min exposure to flunitrazepam (1.0 x 1O-6 M), the dose-response curve for ionophoretically-applied GABA is shifted to the left. Mean values f standard error are plotted, and the number of cells used for each data point is indicated. (nC = nanocoulombs). From Sattelle et al. (1988).
trazepam enhances GABA-evoked responses in both cockroach (Fig. 5) and locust neuronal preparations (Lees et al., 1987; Sattelle et al., 1988), and noise analysis reveals that its action appears to be mediated by an increased frequency of opening of GABAoperated channels (Shimahara et al., 1987), as in vertebrates. Although there have been few studies on the effects of barbiturates in insects, there is some evidence for enhancement by pentobarbital of GABA responses in locust somata (Lees et al., 1987). However, since some studies have failed to detect interactions of benzodiazepines and barbiturates with insect GABA receptors, it is not yet clear if these modulators interact with insect GABA receptors in precisely the same way in which they act to generate the observed potent enhancement of vertebrate GABAA receptor responses. The pharmacology of insect [ ‘HI-flunitrazepam binding sites differs from that of vertebrate GABA, receptor-linked benzodiazepine binding sites (Table 3). Indeed, in some respects insect receptors resemble more closely vertebrate peripheral benzodiazepine binding sites. Clonazepam and flumazenil (Ro 15-l 788), potent inhibitors of vertebrate GABA, receptor linked sites, have very little effect on specific insect [‘HI-flunitrazepam binding (Robinson et al., 1986), whereas Ro 5-4864, a ligand specific for vertebrate peripheral (non-GABA receptor-linked) benzodiazepine binding sites, is a potent inhibitor Table
3. Displacement by benzodiazepines of (‘HI-flunitrazepam binding to insect and vertebrate tissues
Liaand Ro 5-4864 Diazepam Flunitrazepam Clonazepam Flumazenil Reference n.t., not tested.
Cockroach nervous svstem 560 1000 1600 25,000
100,000 Lummis and Sattelle (1986)
lcso (nM) Human brain
Rat kidnev
> 100,000 30 3
I n.t. Speth er al. (1978)
2 19 16 2500 n.t. Regan et al. (1981)
D. B. SATIELLE
434
(Ozoe et al., 1984). However, the electrophysiological evidence discussed above indicates that at least some insect flunitrazepam binding sites are linked to GABA receptors.
One interpretation of these findings is that insects possess GABA receptor-linked benzodiazepine sites with a pharmacological profile distinctly different from that on the vertebrate receptor-linked sites.
Table 4. ICY values for steroid displacement of [“SJ-TBPS binding Rat membranes 1% (NW -GABA +GABA
5a-Pregnan-3a-ol-20-one
5P-Pregnan-3a&20-one
5a-Pregnan-3a,21
5
>lOO
Fly membranes tc, luW -GABA +GABA
0.04
>I00
0.08
>I00
36
>iO
-&of-2O-one
8
0.14
22
10
5a-Pregnan-Ba-ol-11.20-dione
13
0.52
29
47
inactive
77
5a-Pregnan-3P-ol-20-one
>I00
Inactive
Insect amino acid receptors
435
!5a-pregnan-3a-ob204me (SO)r M)
5a-pre1gnan-3a-ol-l1,2O-dione (100pM) 5a-pregnan3asl-20-one
(100~ M)
5mV
L2 min
Fig. 6. Pregnane steroids 5a-pregnan-3a-ol-20-one and 5a-pregnan-3a-01-11, 20-dione exhibit little or no effect on responses to GABA of the cell body membrane of cockroach motor neuron Dr. GABA application is indicated by the solid bars; steroid application is indicated by the cross-hatched bars. From Sattelle et al. (1991). Further support for this interpretation derives from the observation that insect benzodiazepine binding sites can be modulated by GABA, in a similar way to vertebrate receptor-linked sites, and in contrast
to vertebrate peripheral benzodiazepine sites (Le Fur, 1988). In the insect species examined, binding at this site is enhanced by GABA (Ozoe et al., 1989; Robinson et al., 1986). The enhancement appears to be biphasic, however, since it is reduced at high GABA concentrations (Abalis et al., 1983). An alternative explanation is that in insects, as there are receptor-linked and in vertebrates,
non-receptor-linked benzodiazepine sites which have different pharmacological profiles. Additional studies are needed to discriminate between these possibilities. Recently, the capacity of flunitrazepam to photoaffinity label its receptor has been exploited (Robinson et al., 1986). This work on the locust Schistocerca gregaria has revealed two binding proteins of M, 45,000 and M, 59,000. These results may indicate the existence of two protein subunits, each containing a benzodiazepine site, although later experiments using sarmazenil
HUM@J(l) PI BOVlNE(2) 11 82 RAT (2)
:: !:
SILITILSWVSF!m' ILQTYNPSTIITILSWVSFW ILQTYNPSIIITI ILQTYMPSIMITI moo""
DROSOPHlL4
HUMAN(l)
EMU
IDIYIJCCFVWFLALL ILX4YLMGCFVFVFtULL
TSARVALCITTVLlMlTIS -.."
BOVINE
SMRvALG1mm1 SAARvALGIm~I
rn"..
(3)a, a2 a3
01 n
Fig. 7. Amino acid alignment for (90) residues encompassing membrane spanning regions (MI, M2, M3) of GABA receptor subunits of mammalian brain and a putative GABA receptor of Drosophila melanogaster. The following mammalian GABA receptor subunit sequences are used in the alignment: human a,, /3,, y2; bovine a,.), B,.,; rat /3_,. Filled circles indicate identity of residues in the Drosophila sequence with human a,, /I, and y2. Some of the sequences used for alignment are as follows (1) Pritchett et al. (1989); (2) Ymer et al. (1989); (3) Levitan et al. (1988). From Sattelle et al. (1991).
D. B. SATTELLE
436
(Ro 15-45 13) suggest that the smaller band may be a breakdown product. Actions of steroids on insect and vertebrate GABA receptors Steroids can modulate vertebrate GABA, receptor function by acting at a distinct binding site (see, for example, Turner et al., 1989). Several naturally occurring steroids have been shown to enhance GABAactivated and barbiturate-activated %Zl~ uptake, and to inhibit [35S]-TBPS binding in vertebrate CNS preparations. A series of pregnane steroids are potent inhibitors of [%I-TBPS binding to rat brain membranes. and all are more effective in the presence of GABA (Table 4). However, when the same steroids are tested against insect [“S]-TBPS binding sites in the presence or absence of GABA they are two to three orders of magnitude less potent (Rauh et al., 1991; 1992). Thus, if a unique steroid-sensitive site is present on insect GABA receptor molecules, it possesses a quite different pharmacological profile from the corresponding vertebrate site. The insect-specific steroids ecdysone and 20hydroxyecdysone are also virtually without effect on [35S]-TBPS binding to housefly and rat brain membranes. Like the pregnane steroids, the insect steroids tested to date are also largely ineffective on electrophysiological responses to GABA recorded from the cockroach Dr motor neurone (Fig. 6). It is possible that other insect steroids or endogenous steroid metabolites may be found which are able to modulate insect receptors. Cloning and sequencing of putative melanogaster GABA receptor subunits
modulator neurones. L-glutamate receptors can be expressed in oocytes of Xenopus laevis. GABA-gated Cl- channels that are bicuculhneinsensitive are abundant in the cockroach nervous system. Bicuculline-sensitive GABA receptors have also been described, and these too probably gate Clchannels. GABA-gated Cl- channels are present on cell body and neuropile membranes in the insect nervous system. The sites of action of benzodiazepines, steroids and convulsants on a cockroach GABA-gated Cl- channel differ strikingly in their pharmacology from corresponding sites on vertebrate GABA, receptors. Block by picrotoxin, TBPS and many polychlorocycloalkane insecticides has been observed in the case of cockroach GABA-gated Cl - channels. Patch-clamp studies reveal single channel conductances of 11 pS and 17 pS for cockroach GABA-gated Cl- channels. The recent cloning of Drosophila melanogaster putative GABA receptor subunits offers the prospect of an enhanced understanding of structure-function relations of insect ion channels operated by amino acids. The combination of biochemistry, electrophysiology and molecular biology is enhancing our understanding of the properties and functions of GABA and L-glutamate receptors of the insect nervous system. Acknowledgements-The author is indebted to the following, all of whom contributed to aspects of the work reviewed here: K. A. Wafford. M.-I. Sepulveda, D. Bai. S. C. R. Lummis, N. M. Anthony, K. W. P. Miller, J. H. Wong and J. J. Rauh. The support of the Agricultural and Food Research Council of the U.K. and a grant from E. I. du Pont de Nemours is gratefully acknowledged.
Drosophila
Two putative GABA receptor subunits from Drosophila melanogaster have been reported. In our laboratory oligonucleotides have been prepared, based on the well-characterized mammalian GABA receptor sequences (Barnard et al., 1987; 1989). These synthetic oligonucleotides, designed using the highly conserved transmembrane regions. have been employed to probe a Drosophila melanogaster cDNA library from i sep (Stratagene). From the Drosophila nucleotide sequence an amino acid sequence has been deduced (Sattelle et al., 1991) and this is shown (Fig. 7) for the membrane spanning regions (MlM3). By extending this study, we aim to provide the complete sequence of a putative insect, p-like, GABA receptor subunit. Recently another laboratory has reported a Drosophila GABA receptor subunit. This subunit was discovered by identifying and sequencing a gene responsible for cyclodiene-resistance in the fruitfly (ffrench-Constant et al., 1991). CONCLUSIONS
L-glutamate binding sites have been characterized using cockroach nervous system membranes, and by means of radiation inactivation a MW of 77,800 has been established. L-glutamate gated Cl channels have been demonstrated in cockroach neurones, as well as several other distinct L-glutamate receptor responses. Cell-specific L-glutamate-gated channels are associated with functionally-distinct motor and
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