seminars in THE NEUROSCIENCES, Vol 3, 1991 : pp 2 5 1 -258

GABA and GABA receptors in invertebratesG. G. Lunt

GABA is the major inhibitory transmitter at invertebratesynapses in both the central and peripheral nervous systems .The receptors for GABA are well characterised electro-physiologically in a wide variety of invertebrate organismsbut their biochemical and pharmacological profiles are lesswell defined. In general invertebrate GABA receptors are lesssensitive to bicuculline than are vertebrate GA BAA receptors .There is much evidence that invertebrate GA BA A receptorshave benzodiazepine modulatory sites but their pharmacologicalprofile is distinct from that of vertebrate GA BAA receptors .The insect neuronal GA BAA receptor has been identified asthe site of action of the cyclodiene insecticides and theavermectins may also act on these receptors . Molecular cloningexperiments now in progress will reveal the detailed relation -ships between the invertebrate receptors and their mammaliancounterparts.

Key words: insect / nematode / GABA receptors / aver-mectins / pesticides

THE INHIBITORY actions of GABA were firstdemonstrated in experiments on invertebrate neurons .In 1954, Florey showed that a substance extractedfrom mammalian brain termed `Factor I' had apotent inhibitory effect on a stretch receptor neuronin the crayfish Astacus and he later showed that thiseffect was inhibited by picrotoxin . Contemporarystudies by Burgen and Kuffler demonstrated thatFactor I-now identified as GABA-also inhibitedthe isolated cardiac ganglion from the horseshoe crabLimulus polyphemus . Many other examples of theinhibitory influence of GABA on a wide variety ofinvertebrate systems followed these early landmarkexperiments and by the early 1970s it was believedthat GABA might be the universal transmitter ofjunctional neuromuscular inhibition in virtually allinvertebrate phyla from nematodes to arthropodsexcept for molluscs (note that, unlike mammals,many invertebrates have inhibitory neurons directlyinnervating skeletal muscles) (see ref 1 for review) .

From the Department of Biochemistry, University of Bath, BathBA 2 7A Y, UK

©1991 by W.B. Saunders Company1044-5765/90/0303-0009$5 .00/0

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Evidence continued to accumulate in support of thisview and by the 1980s there were data from a verywide phylogenetic range of organisms, embracingcrayfish, lobster, honeybee, cockroach, snails andannelid worms, showing that GABA was a majorinhibitory transmitter at both neuromuscular synapsesand within ganglia (reviewed in refs 2,3) .

It is somewhat ironical that invertebrates, havingprovided the experimental systems in which GABAachieved full neurotransmitter status, should sub-sequently have been relatively neglected . Thus, asis well documented elsewhere in this issue, the GABAreceptors of mammalian brain have been cloned,sequenced, expressed, characterised and mutatedwhereas their invertebrate counterparts are barelymore than partially characterised binding sites! Thetide, however, seems to be turning and currentlymuch attention is being focused on GABA and itsreceptor sites in several invertebrate species . It isbecoming clear that the invertebrate GABA receptors,although similar to the mammalian ones in somerespects, do not fit neatly into the classificationsystems that have developed from mammalian brainstudies . This should not really come as a surprisefor in evolutionary terms the mammalian nervoussystem represents a very small part of an enormousspectrum; the invertebrates cover an extremelydiverse range of organisms with correspondinglygreat scope for species variation . All too often itseems that when invertebrate data do not fit thecriteria established for mammalian systems theassumption is made that something is 'wrong' . Thereality is that the fundamental properties of systemsare constantly evolving and the invertebrates arebroadly repesentative of an earlier evolutionary stagethan the mammals . Differences of detail will,therefore, be observed, the objective comparativestudy of which can provide valuable informationabout the development and function of the systemsin question . So it is with GABA receptors : it isalready well established that invertebrate receptorsare different from their mammalian counterparts andan examination of the differences may help us tounderstand how the very complex and sophisticatedmammalian receptors function at the molecularlevel .

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Invertebrate GABA receptors have also becomethe focus of much attention from the pesticideindustry, for two reasons . First, there has been agrowing realisation that the GABA receptor complexis a major site of action of the cyclodiene class ofinsecticides . This class of pesticide represents animportant proportion of total world sales but alsosuffers from the problem of significant vertebratetoxicity and so is likely to decline in use . Second,there is an important difference between invertebrateand vertebrate GABA receptors that relates to theircellular and anatomical localisation . In vertebratesGABA receptors are restricted to the brain and thusenjoy the protection of the blood-brain barrier ;in contrast, the invertebrate GABA receptors,particularly those on muscles, are much moreexposed to exogeneous agents . Given that GABAreceptors are proven targets there is the potential todevelop new and more specific control agents ;compounds could be designed that are potentmodulators of GABA receptors but which would notcross the vertebrate blood-brain barrier . In somerespects the natural nematocides, the avermectins,have this property, although there is some doubt asto whether their effects are exclusively due tointeractions with GABA receptors .

GABA binding sites

The first invertebrate GABA receptor binding studieswere made in membrane fractions from crayfishmuscle, 4 ' 5 using two ligands, dihydropicrotoxinand muscimol, to identify putative GABA receptors .The binding characteristics of the picrotoxinin siteswere in accordance with those seen in physiologicalexperiments on crayfish muscle preparations . Themuscle membranes also showed specific, saturablebinding of [ 3H] muscimol that was independent ofthe Na + concentration ; this insensitivity to Na + isgenerally regarded as an important criterion thatidentifies a binding site as a receptor rather thanan uptake/transport component. An importantobservation in the crayfish experiments was thatsensitivity of the binding to bicuculline was greatlyreduced. Whereas µM concentrations of bicucullineeffectively inhibit both GABA and muscimol bindingto various vertebrate brain preparations, the crayfishsites required mM concentrations before inhibitionwas clearly demonstrated .

Further binding studies on housefly thoracicmuscle preparations6 indicated indirectly the presence

G. G . Lunt

of GABA receptor sites . The direct binding of[ 3H] GABA could not be detected but the binding of[ 3H ] flunitrazepam was demonstrated and further-more this benzodiazepine binding activity wasenhanced by GABA in a dose-dependent manner .The effects of GABA could be blocked by bicucullineat relatively low concentrations (1 .7 x 10 - 5M), so inthis respect the housefly sites more closely resembledmammalian GABAA receptors than the crayfishreceptor sites . This work was very important becauseit provided compelling evidence that an insect GABAreceptor may have the same allosteric modulatory sitesthat characterise the mammalian GABAA receptors,contrary to an earlier report 7 that benzodiazepinebinding sites were absent in the nervous systems ofearthworm, woodlouse, lobster, locust and squid . Theview that the intra-subunit allosteric modulation ofGABA receptor activity was a recent (in evolutionaryterms) development, confined to vertebrate brains, wasendorsed in 1983 in a review of GABA receptors, 8which concluded that invertebrates possess `simple'receptors devoid of multiple regulatory sites . Thisview is no longer tenable and many convincing piecesof evidence suggest that invertebrate GABA receptorsare complex proteins with multiple interacting sites(see below) .

A major difficulty that besets all invertebratestudies in this area is the source of the tissue. Thehousefly preparation 6 used whole thoraces ; theauthors attributed their binding activity to GABAreceptors on thoracic muscles but it would be difficultto provide unequivocal evidence for this . Thepossible contribution of thoracic ganglia membranesor of nerve cord cannot be completely discounted .There are reports in the literature of binding studiesmade using whole insect head homogenates or indeedof whole insects or nematodes ; clearly with suchpreparations it is extremely difficult to establishdefinitively that the observed binding is to neuronalmaterial . This may, in part, explain the considerablevariation in binding parameters from differentlaboratories . Species variation is also potentially amuch greater factor in invertebrate studies ; organismssuch as houseflies, locusts, cockroaches, crayfish andnematodes cover an enormous span of evolutionarytime yet there is a tendency to consider them as asingle group-the invertebrates .

Reports of binding studies in insect gangliapreparations followed, using both [ 3H] GABA and[ 3H]muscimol.2,3,9 Studies on locust, cockroach,housefly and honey bee all produced rather similardata showing specific, high affinity binding of

GABA receptors in invertebrates

muscimol and GABA that is sensitive to the agonistssuch as isoguvacine and to the antagonist picrotoxinbut insensitive to bicuculline . The insensitivity tobicuculline has been widely reported and seems torepresent a real difference between many insectGABAA receptors and their mammalian counter-parts . The in vitro binding data are supported byelectrophysiological studies on both intact neuronalpreparations 3,10 and cultured insect neurones ; 10 inall of these high concentrations of bicucullineconsistently fail to block GABA-evoked Cl - currents .Nevertheless there is GABAergic activity in theantennal lobes of the sphinx moth, Manduca sextallthat shows comparable sensitivity to bicuculline tomammalian brain receptors . At present it seemsreasonable to conclude that the moth antennal lobesystem may not be typical of insect GABA receptors,most of which are characterised by an insensitivityto bicuculline .

Detailed examination of the invertebrate GABA/muscimol binding literature reveals many discrepanciesbetween the findings of different groups . Many ofthese may derive from technical and proceduraldifferences, for example, a failure to fully appreciatethat when working with [ 3H]GABA it is vital toobviate binding to transport components, whereasthis problem does not arise if [ 3H] muscimol is theprobe. Such detailed analyses and arguments are wellreviewed elsewhere . 2,3

The main point to emerge from these studies is thatneural tissues from a wide variety of invertebrateshave specific binding sites that are closely similar tothe GABA/muscimol sites of mammalian GABAAreceptors. The invertebrate sites have a Kd forGABA/muscimol in the 20-100 nM range, regulatea Cl - channel and have an overall pharmacologylike that of the GABAA receptor with the importantexception of a markedly reduced sensitivity tobicuculline .

Benzodiazepine binding sites

Are these invertebrate sites allosterically linked toother sites on a multisubunit receptor complex? Asnoted above, there is compelling evidence 6 for thepresence of benzodiazepine binding sites in houseflythorax preparations and that the binding is modulatedby GABA . 6

Work from our laboratory3,12 has shown thepresence of specific, high affinity binding sites forbenzodiazepines in locust ganglia . In common with

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the corresponding sites on mammalian GABA Areceptors, GABA enhanced the binding of [ 3H]flunitrazepam to the site in locusts . Furthermore thelocust binding protein could be photolabelledto buttwo proteins of M r 45 kDa and Mr 59 kDa wereconsistently labelled, whereas in mammalian braina single band of Mr 49 kDa is most consistentlyfound .

Similar benzodiazepine binding activities incockroach have been reported 2 ' 9 and the modulationof GABA-evoked responses by benzodiazepines hasbeen convincingly shown in electrophysiologicalexperiments on both cultured locust neurons 13 andisolated neuron cell bodies . 14

Analyses of the various data on the insect benzodi-azepine site reveals some significant differences withrespect to mammalian brain . These can be generallysummarised by saying that in purely pharmacologicalterms the insect site more closely resembles themammalian peripheral (i .e . non-GABA receptor)benzodiazepine receptor than it does the GABA Areceptor site . Thus in insects the sites consistently showa higher affinity for Ro 5-4864 than for clonazepamof flumazenil . 2 ' 9 Apparently in contrast to this is theclear demonstration both in binding experimentsin vitro and in electrophysiological experiments onseveral different preparations that the insect benzodi-azepine receptor is functionally coupled to the GABA/muscimol binding site . 2,3,9,13,14 The photoaffinitylabelling experiments from our laboratory 12 lendfurther support to this conclusion as mammalianperipheral benzodiazepine receptors cannot bephotoaffinity labelled with [ 3H ] flunitrazepam .

In summary, insects have specific, high affinitybenzodiazepine receptors with K d values in therange 40-300 nM that can interact allostericallywith GABA/muscimol binding sites in a mannerreminiscent of mammalian GABAA receptors. Thepharmacological profile of the insect benzodiazepinereceptor sites is, however, clearly different from thatof their mammalian counterparts .

Picrotoxin, cage-convulsants, barbituratesand other modulators

What of the other modulatory sites for agents suchas the cage-convulsants and barbiturates thatcharacterise the mammalian GABAA receptor? Arethere corresponding sites on invertebrate receptors?As described elsewhere in this' issue, the mammalianGABAA receptor has, in addition to GABA and

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benzodiazepine recognition sites, sites for a varietyof ligands that can act as allosteric modulators of thebasic GABA-mediated Cl - channel . A diverse groupof agents encompassing the natural toxin picrotoxinand its analogues, trioxabicyclooctanes or cageconvulsants, barbiturates and steroids, all interactspecifically with the GABA A receptor complex andmodulate both the binding properties of other siteson the receptor and the functional activity of theCl - channel . The characterisation of these sites inthe various invertebrate systems is much less advancedthat that of either the GABA or benzodiazepine sites .

Important observations that provided the firstclues about a link between insect GABA receptorsand pesticide action were that [ 3H ] dihydropicro-toxinin binds to membranes from cockroach gangliaand that there are significant differences in thebinding properties for this ligand between cockroachesthat are resistant to cyclodiene insecticides andsusceptible cockroaches . 15,16 A steadily growingbody of evidence has since established that thecyclodiene insecticides are particularly effectiveblockers of insect GABA receptors . 2 Indeed somevery recent work' 7 provides strong evidence that themolecular origin of cyclodiene resistance in resistantstrains of Drosophila is an altered GABA receptorcomplex .In the mammalian GABAA receptor complex

the cage convulsants and picrotoxin show directcompetitive mutual inhibition of binding, stronglysuggesting that they interact with a common site .In invertebrates however the picture is far less clear .In general picrotoxin is a much weaker inhibitor ofcage-convulsant binding than in mammalian prep-arations but nevertheless relatively high affinitybinding of picrotoxin to both insect 15,16 and crayfish5preparations has been reported . In housefly prep-arations and locust ganglia the binding of the cageconvulsant [ 35S ] TBPS is reported to be relativelyinsensitive to picrotoxin . 18,19 In contrast, picrotoxinhas been found to be a reasonable inhibitor of TBPSbinding in housefly head preparations by anothergroup . 20,21

These inconsistencies persist when the interactionsof the TBPS binding site with other ligands areexamined . In both housefly thorax18 and locustganglia 19 an enhancement of TBPS binding byGABA is observed; in cockroach nerve corda marked inhibition of TBPS binding by µMconcentrations of GABA has been reported, 22 whereasin housefly heads 20,21 no effects on the binding ofTBPS by high concentrations of muscimol are seen .

G. G . Lunt

The effects of barbiturates on invertebrate GABAreceptors have not been extensively investigated .There is nevertheless some evidence for the presenceof a barbiturate site that is coupled to other siteson the receptor complex . In the locust, sodiumpentobarbital produces a modest enhancement ofGABA binding23 and also enhances TBPS binding . ' 9In contrast, in housefly head preparations TBPSbinding is inhibited by barbiturates . 20

The role of natural steroids of the pregnanoloneclass and of the synthetic anaesthetic steroids suchas alfaxalone as important modulators of mammalianGABAA receptors is well established (seeSimmonds, this issue 24) . There are very few data forthe effects of steroids on invertebrate preparations .A comparison of a series of pregnanolone derivativesin a TBPS binding assay in both housefly head andrat brain confirmed the well established picture ofpotent competitive inhibition in rat brain whereasthe steroids were largely ineffective on the houseflypreparation . 25 This lack of effect was confirmed inelectrophysiological studies on cockroach neurons .

The trioxabicyclooctanes (the cage convulsants)are potent convulsants and are GABAA receptorantagonists in mammals . Many of these compoundsalso have very potent insecticidal activity, an effectbelieved to have its origins in their interaction withinsect GABA receptors . 26 Some of the compoundsshow a marked differential toxicity between mammalsand insects and detailed examination of structure-activity relationships may provide valuable cluesabout the precise nature of the insect receptor site .It is also clear that several other insecticidal agentshave their main action through the picrotoxin/cageconvulsant/barbiturate part of the GABA A receptor .The cyclodienes (see above) and the polychlorocyclo-alkanes 9 ' 26 all seem to be active at this site althoughno precise mechanism of action has been formulated .From comparative binding studies it is clear that manyof these insecticides are more potent competitors forthe TBPS binding site in insect preparations thanthey are in mammalian brain GABAA receptorpreparations9-this must be a pointer to significantdifferences between the insect and the mammalianreceptor .

The picture of the invertebrate 'convulsant' siteis still somewhat confusing and incomplete . Someconclusions can, however, be drawn . First, there isclear evidence that a convulsant binding site ispresent. Second, there is evidence that in some insectsit is functionally linked to both the GABA and thebenzodiazepine sites . The full pharmacological

GABA receptors in invertebrates

profile of the site compared with its mammaliancounterpart is far less clear . It certainly plays a crucialpart in receptor function, as witnessed by thephysiological effects of insecticidal agents that seemto act here. At this stage it is thus reasonable toconclude that invertebrate GABA receptors have thesame basic functional organisation as mammalianGABAA receptors, namely a GABA-mediated Clchannel that is subject to modulation by benzo-diazepines and by convulsants of various chemicalclasses . Modulation of insect GABA receptors bysteroids seems much less certain but several classesof insecticides have greater potency at the insectreceptor than the mammalian one . The invertebrateand the mammalian receptors differ in that theformer are in general insensitive to bicuculline,they have a distinctly different benzodiazepinepharmacology and the relationship between thepicrotoxin site and other convulsant sites is different .

The avermectins

No account of invertebrate GABA systems iscomplete without mentioning the avermectins . Theseare macrocyclic lactones produced by strains ofStreptomyces avermitilis that are extremely potentanthelmintics and insecticides, and widely used ascommercial pest control agents. There are numerousreports of the effects of the avermectins on mammalianGABAA receptors . Earlier studies all seemed toindicate that they had a rather specific action inincreasing Cl - channel conductance, from bindingstudies, this was related to an enhancement of ligandbinding at the benzodiazepine site . 27 But closeexamination of a now very substantial literature leadsto the conclusion that this is a considerable over-simplification of their mechanism of action : there isno doubt that the avermectins act at GABAAreceptors but it is equally clear that several othersystems are affected . 2,27 In insects, several studieson both neuronal and muscle preparations stronglyindicate that the avermectins can act both onGABA-activated Cl - channels and on channels thatare unrelated to GABA receptors . 2 In both locust28and cockroach 2 neurons, electrophysiological experi-ments support the view that avermectins have aGABA-agonist-like effect that is reversible but thatother effects are seen that are unrelated to GABAreceptors and only partially reversible .

The apparent selectivity of the avermectins forinvertebrates compared to mammals is because the

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compounds do not readily cross the blood-brainbarrier in most mammals .27 Once this barrier isremoved, as in most in vitro biochemical orelectrophysiological experiments, selectivity is far lessclear-cut . In both vertebrates and invertebrates, itcan be concluded at present that GABA receptorsare certainly one site of action of the avermectinsbut it is much less certain what proportion of theoverall toxicity of these compounds derives from thisactivity .

Other invertebrate GABA receptors

So far I have discussed an invertebrate GABAreceptor complex that shares many basic featureswith the GABAA receptor of mammalian brain .Most of the data on this receptor in invertebratesderive from studies on insects and it is worthwhileonce more drawing attention to the enormousdiversity of species grouped under the heading`invertebrates' . There is growing evidence that someinvertebrates other than insects possess GABAreceptors that are clearly distinct from either theGABAA or GABA types known from mammalianstudies .The heart of the marine arachnid Limulus

polyphemus, the horseshoe crab, responds to lowconcentrations of GABA with a decreased beat fre-quency . 29 The GABA agonist profile of the Limulusheart looks rather like those of the mammalianGABAA receptor and the insect GABA receptors dis-cussed above : agonists such as muscimol, isoguvacineand 3-aminopropane sulphonic acid (SAPS) are alleffective. When antagonists are examined, however,some significant differences are seen ; bicuculline,picrotoxin and other potent GABA antagonists, suchas pitrazepine and SR 95103, have no effect .Furthermore, there are no responses to a wide rangeof benzodiazepines and barbiturates .

With such a drug profile, this receptor cannot beconsidered GABAA-like, so is it a GABAB receptor?The diagnostic ligands for a GABAB receptor inmammalian systems are baclofen and phaclofen (seeBowery and Maguire, this issue 30) both of whichhave no effect on the Limulus heart . The compound3-aminopropylphosphonous acid, a potent GABABagonist in vertebrate brain, does however, effectivelyinhibit the Limulus heart beat in the nanomolarrange, although it has no effect on locust neuronalGABA receptors. Thus the Limulus heart GABAreceptor29 clearly does not fit readily into either themammalian GABAA/GABAB receptor classification

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and is distinct from the GABA A-like receptors foundin many insect species. Interestingly, within thecentral ganglia of Limulus there are neuronal GABAreceptors that are rather similar to the insectGABAA receptors .The Limulus heart GABA receptor does, however,

in some respects resemble another interestinginvertebrate GABA receptor, that found on thesomatic muscle cells of the parasitic nematode Ascarissuum . A large body of evidence shows that GABAacts as an inhibitory neurotransmitter on thesecells 31 and that the inhibitory effects are mediatedby a Cl - channel. In a comprehensive analysis ofthe pharmacological characteristics of this receptor,several differences from both mammalian GABA Aand insect GABAA-like receptors were demon-strated . 32 The potencies of both muscimol andisogtivacine were about 25% that of GABA, whereasin rriammalian and insect preparations they are atleast equipotent and often more effective than GABA .The compound (z)-3- [ (aminoiminomethyl)thio ] -2-propenoic acid (ZAPA) was the only agent withcomparable activity to GABA . In vertebrate prep-arations ZAPA is a potent agonist at the GABAbinding site on GABA A receptors and it enhancesbenzodiazepine binding. In the Ascaris preparations,benzodiazepines do not enhance the effects ofGABA and bicuculline, picrotoxin and TBPSare similarly inactive. The GABAB agonist baclofenalso has no effect, indicating that the Ascaris receptoris not GABAB-like in its pharmacological profile .The receptor is blocked non-competitively byavermectin .Thus this receptor comprises a GABA-mediated

Cl - channel, a characteristic that is central to theGABAA receptor class, but which clearly differsfrom either mammalian or insect GABAA receptorsin almost all other respects . Further detailed analysisof agonist recognition by the Ascaris receptor, inparticular the stereoselectivity of the site, has led tothe conclusion33 that the receptor is GABAA-likewith respect to its agonist recognition site but thatits antagonist profile is distinct. Furthermore theseveral allosteric modulatory sites seen on bothmammalian and insect GABAA receptors are absent .The Ascaris receptor could, therefore, be closelyrelated to mammalian GABAA receptors but with asimpler, i .e. less heterogenous, subunit composition-could it in fact be a holo-oligemeric form composedonly of GABA-binding subunits? This can beanswered only by the application of molecularcloning techniques . At present it seems that there

are GABA receptors in some invertebrate species thatcan not be classified within the present A or B groupsof the vertebrate family . Such receptors may beattractive targets for the development of invertebrate-specific pest-control agents .The receptors from Limulus heart and Ascaris

muscle have already been sufficiently well characterisedto warrant designation as GABA receptors eventhough we do not yet have any substantial informationabout their molecular characteristics in terms ofsubunit composition and rr -)lecular size . Otherinvertebrate organisms show unusual responses toGABA that warrant similar detailed analysis . In themolluscs, responses to GABA that are both inhibitoryand excitatory have been reported and in the snailHelix aspersa both types of response are blocked bybicuculline and picrotoxin . 2 In Aplysia californicaGABA-mediated conductances of Na + , K + andCl - have been reported ; the Cl - conductance isblocked by both bicuculline and picrotoxin andenhanced by benzodiazepines but the Na + and K +conductances are not affected . 2 Several variations ofthe GABA receptor may therefore be present ininvertebrates and we have clearly only just begunthe task of identifying and characterising them .

Future directions

Although our understanding of invertebrate GABAreceptors lags well behind the mammalian field, thereis a growing realisation that these receptors representpotentially very important targets for species-specificpest control agents, which has already stimulatedresearch in the area .

Conventional methods of studying receptors,developed using mammalian tissues, are ofteninappropriate for invertebrate studies . The purificationof receptor proteins by procedures such as detergentsolubilisation and affinity chromatography invariablydepend on large quantities of starting tissue, whichfor most invertebrates is simply not available . Giventhe spectacular successes of molecular biologytechniques in mammalian receptor studies, it seemsobvious that this is the direction in which futureinvertebrate studies must be directed. Even thoughinvertebrate organisms are not a realistic source oftissue for the conventional biochemical isolation ofreceptors, the advent of such techniques as polymerasechain reaction (PC R) means that, at least so far asnucleic acids are concerned, availability of tissue isno longer a problem . Several groups have adopted

G. G . Lunt

GABA receptors in invertebrates

homology hybridisation strategies, using a vertebratecDNA probe for searching invertebrate libraries forhomologous sequences . In principle this approachhas much to commend it as our understanding ofprotein evolution leads us to believe that significanthomology is preserved over long evolutionary timescales .

In practice however the molecular biology approachhas failed to produce the hoped-for rapid advancesin the invertebrate receptor field . Nevertheless somesignificant progress is being made and within amatter of months cloned, expressed, functionalGABA receptors from invertebrate sources shouldbe reported . A clone isolated from a cyclodiene-resistant strain of Drosophila (see above) shows highhomology to a ß subunit of the mammalian brainGABAA receptor, 17 and other very promising clonesfrom Drosophila that resemble GABAA receptorsequences have recently been reported . 34 Clonesfrom the mollusc Lymnea stagnalis also show consider-able homology to GABAA receptor /3 subunits .35,36The availability of such sequences will permit thebeginnings of a real assessment of exactly how thevarious invertebrate GABA receptors relate to thewell-characterised mammalian receptor proteins .

One final feature of the work on invertebratereceptors that is intriguing is the growing body ofevidence for what might be called pharmacologicalcrossover . Drugs that have a very clear cut actionin mammalian preparations, restricted to oneparticular class of receptor, often cross over intoother receptor classes when applied to invertebratepreparations . A nicotinic cholinergic receptor onisolated locust neuronal cell bodies is reported to bealmost as sensitive to picrotoxin as the GABAreceptors on the same neuron .37 Bicuculline, whichhas no effect on the locust GABA receptor, effectivelyblocks the nicotinic response . In Ascaris the musclecells referred to previously also have nicotiniccholinergic receptors and again they are reported 38to be more sensitive to the GABA antagonistspicrotoxin and bicuculline than the GABA receptorson the same cells .

These intriguing observations emphasise again thedanger of attempting to classify invertebrate receptorson the basis of pharmacological profiles establishedin mammalian preparations . In evolutionary terms,GABAA receptors, nicotinic cholinergic receptors,glycine receptors and possibly other receptor classesbelong to a super-family of molecules that all formligand-gated ion channels . 39 It is clear that as thesuperfamily has evolved ligand recognition has

become increasingly specific, culminating in themany receptor types found in mammalian brain,each characterised by a distinct pharmacologicalprofile . The invertebrates are representative of anearlier stage in this evolutionary process, so theirreceptors may have ligand recognition sites that areless discriminatory than their mammalian counter-parts even though they share the same basic structuraland functional organisation .37 The comparativestudy of the invertebrate receptors will ultimatelyenable us to understand fully the evolutionaryprocesses that have led to the complex andsophisticated mammalian GABA receptors .

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16 . Tanaka K, Matsumura F (1985) Action of avermectin B1Aon the leg muscles and the nervous system of the americancockroach . Pestic Biochem Physiol 29 :124-135

17 . ffrench-Constant RH, Roush RT (1991) The cloning andtransformation of cyclodiene resistance in Drosophilamelanogaster : an invertebrate GABA receptor? in Neurotox'91, Molecular Basis of Drug and Pesticide Action (Duce IA,ed) . Elsevier, London, in press

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19. Brown MCS, Lunt GG, Stapleton A (1989) Furthercharacterisation of the [ 35S]TBPS binding site of theGABA receptor complex in locust (Schistocerca gregaria)ganglia membranes . Comp Biochem Physiol 92C :9-13

20 . Olsen RW, Szamraj O, Miller T (1989) [ 35 S]-t-butylbi-cyclophosphorothionate binding sites in invertebrate tissues .J Neurochem 52 :1311-1318

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