[3H]Muscimol binding to a putative GABA receptor in honey bee brain and its interaction with avermectin B1a

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

25, 279-287 (1986)

[3H]Muscimol Binding to a Putative GABA Receptor in Honey Bee Brain and Its Interaction with Avermectin B,, IBRAHIM M. ABALIS AND AMIRA T. ELDEFRAWI’ Department

oj’Pharmaco/ogy

and Experimental Baltimore.

Therapeutics, Maryland

University 21201

of

MarTland

School

of’Medic,itlcT.

Received February 14. 1985; accepted May 30. 1985 A putative GABA receptor was identified in honey bee brain by virtue of its specific binding of [iH]muscimol and its drug specificity. [3H]Muscimol bound with two affinities t&, of 3 nM and K+ of 144 nM), comparable to its affinities for binding to mammalian brain. The high-affinity binding was most sensitive to GABA agonists with the following decreasing order of potencies: muscimol > GABA > imidazole acetic acid > DL-GABOB > B-guanidine propionic acid. However, it was insensitive to the antagonist bicuculline, which is potent on [3H]muscimol binding to the mammalian GABA, receptor. It was also insensitive to baclofen, which is a potent agonist of mammalian GABA, receptor, as well as to picrotoxinin, pentobarbital, flunitrazepam, and ethyl-p-carboxylate, which bind to allosteric sites in mammalian GABA receptor. The low-affinity [3H]muscimol binding was inhibited with GABA agonists with the following decreasing order of potencies: imidazole acetic acid = B-guanidine propionic acid > DL-GABOB. The two muscimol binding affinities may represent binding to two sites on the same GABA receptor or to two kinds of GABA receptor. The most potent inhibitor of the high-affinity [3H]muscimol binding to honey bee brain was avermectin B,, (AVM), whose IC,, was 0.01 nM. AVM also inhibited the low-affinity [3H]muscimol binding with an IC, of 2 PM. 8 1986 Academic press. IK.

tration. These are GABA, receptors, which are located mostly postsynaptically, are activated by GABA and 3-amino-l-propanesulfonic acid and are inhibited by bicuculline. GABAn receptors occur presynaptically on non-GABAergic neurons, are activated by baclofen, are insensitive to bicuculline and their binding of GABA is dependent upon Ca’+, with the fluxing ion probably K+ (1, 2). Such subclassification has not been extended to insects. A GABA receptor has recently been purified from mammalian brains (3. 4) and found to be a single protein with a variety of drug-binding sites: A site that binds GABA, other agonists and competitive antagonists (e.g., bicuculline), another site that binds tranquilizers (e.g., benzodiazepine) and inverse agonists (e.g., p-carboline carboxylate). and different channel sites that bind convulsants (e.g., picrotoxinin, bicyclophosphates) and the sedative hypnotics barbiturates. These sites interact

INTRODUCTION

y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates and also in invertebrate skeletal muscles. There are at least three types of GABA receptors that differ in function, location, and/or drug sensitivity. Activation of most GABA receptors leads to an influx (hyperpolarization) or efflux (depolarization) of Cl-, causing in both cases inhibition, the type depending upon the neural location of the receptor (postsynaptic or presynaptic) (1). The direction in which Cl- fluxes depends upon the Cl- gradient across the membrane which results mostly from changes in intracellular Cl- conceni To whom requests for reprints should be addressed. ? Abbreviations used: GABA, y-aminobutyric acid; GABOB. y-amino-B-hydroxybutyric acid: AVM. avermectin B,,. 279

0048-3575186 $3.00 Copyrlghr 10 1986 by Academic Press. Inc. All rights of reprrniucrion in any fxm reserved.

280

ABALIS

AND

allosterically and affect each other’s affinities for drugs through conformational changes in the receptor molecule. Insect GABA receptors have long been studied in muscle and their electrophysiologic characteristics identified. A single GABA-channel current in locust muscle is 0.9 pA and the mean channel lifetime is 3-4 msec (5). Biochemical identification of GABA receptors in insects has lagged far behind those in mammals. [3H]Dihydropicrotoxinin was used to identify the Clchannel site on the GABA receptor in the nerve cord of the American cockroach (6). Also, a GABA receptor was identified in house fly thoracic muscles by means of its binding of [3H]flunitrazepam that was potentiated by GABA and agonists (7). Recently, three groups reported on interactions of cyclodiene insecticides and lindane with GABA receptors of rat brain and cockroach nerve cord and suggested that the GABA receptor may be the primary target for the toxic action of these insecticides (8- 10). Another study suggested that GABA receptors may be involved in the toxic action of type I pyrethroids (II). A few differences were also detected between insect muscle and mammalian brain GABA receptors (7). These findings increased the demand for developing new assays of GABA receptors in insect tissue to study their potential involvement in insecticidal action and to develop selective and environmentally safe insecticides. The present study was initiated to identify a GABA receptor in an insect brain by labeling its GABA binding site. We utilized muscimol (methylene-N-3-hydroxy-j-aminoethyl isoxazole) (which occurs naturally in the mushroom Amanita muscaria) to label the receptor because it is more potent than GABA in activating GABA receptors (12, 13), is less vulnerable to metabolism or uptake into presynaptic nerve endings and has been used successfully to identify GABA receptors in mammalian brains (14- 17). Avermectin B,, (AVM) is a macrocyclic

ELDEFRAWI

lactone anthelmintic drug that is also insecticidal (18). It was found to potentiate GABA release from rat brain synaptosomes (19) and also to interact with mammalian brain GABA receptors possibly at a site on the Cl- channel partially shared by picrotoxin and pentobarbital (20). Therefore, we studied the effect of AVM on [3H]muscimol binding to honey bee brain membranes so as to determine if an insect GABA receptor is a target for AVM action. MATERIALS

AND

METHODS

Tissue preparation. Workers of Italian honey bee (Apis melliferu L.), obtained from a local dealer, were knocked down by exposure to 4°C for =30 min then CO,. They were transferred with dry ice to a USA standard sieve assembly of No. 5 above a No. 7 and a collecting pan. The whole assembly was placed in a dry ice cabinet for 30 min, then shaken for about 30 sec. The heads collected in sieve No. 7, the abdomens and thoraces in the above sieve, and legs and wings in the pan. A few thoraces that contaminated the heads were removed manually in a few minutes. The heads were weighed and homogenized by Polytron in 10% (w/v> ice-cold solution of 0.32 M sucrose, 50 mM Na2HP04, pH 7, 1 mM EDTA, and 10 pg/lOO ml of the antiproteases pepstatin and trypsin inhibitor in Tris-HCI (1 mM). The homogenate was filtered over three layers of cheesecloth to remove debris then centrifuged at 1OOOgfor 5 min. The supernatant was centrifuged at 48,OOOg for 30 min then the light creamy colored top layer of the pellet was collected (the dark purple portion discarded) and homogenized in ice-cold solution of 5 mM Tris-HCl, 1 mM EDTA, pH 7.1 (10% w/v). It was centrifuged at 48,000g for 30 min and the pellet suspended in distilled water, lyophilized, and stored at -90°C. Specific [3H]muscimol binding was unchanged for at least 30 days under these storage conditions. When used, the lyophilized membranes

HONEY

BEE

BRAIN

were washed once with a solution of 50 mM Tris-HCl, pH 7.1, 1 mM EDTA, centrifuged at 48,OOOg for 30 min then suspended in same solution. Protein concentration was determined as described by Lowry et al. (21) and adjusted to 10 mg protein/ml. Binding assay. The membrane preparation (at =l mg protein) was added to polypropylene microcentrifuge tubes (1.5 ml capacity) containing a solution (final volume of 1 ml) of 50 mM Tris-HCl, 1 mM EDTA, pH 7.4, and [3H]muscimol (sp act 7.2 Gil mmol, New England Nuclear). After 30 min incubation over ice, the tubes were centrifuged at 12,500g for 6 min in a microfuge, the supernatants siphoned off, and the pellet surfaces rinsed carefully with 1 ml ice-cold same solution. The pellets were dissolved in the tubes overnight with 0.1 ml soluene-350 (Packard) each at 21°C. Beckman ReadySolv liquid scintillation cocktail was added to the tubes (1 ml/tube), mixed on a Vortex, the contents transferred to a mini-vial with an additional 3 ml cocktail and the radioactivity counted in a liquid scintillation spectrometer. Specific binding was calculated as the difference between total binding and the binding that occurred in the presence of 1 mM GABA. To study the effect of drugs or ligands that were insoluble in water 10 ~1 of ethanol solutions of the drugs were used, and in this case ethanol was also included in the controls. GABA, muscimol, DL-2,4-diaminobutyric acid, bicuculline. 3-aminopropane sulfonic acid, imidazoleacetic acid, isoguvacine, DL-GABOB, B-alanine, B-guanidine propionic acid, DL-nipecotic acid, L-pipecolic acid, picrotoxinin, and pentobarbital were purchased from Sigma. Baclofen was a gift from CIBA-Geigy, N.J. Data analysis. Binding data were analyzed by iterative nonlinear regression analysis using the Equilibrium Binding Data Analysis (EBDA) computer program on an IBM PC computer (22). The best fits of the data to receptor models, consisting of one, two or more binding sites, were

GABA

281

RECEPTOR

compared using the LIGAND program (23) adapted for use on IBM PC (22). RESULTS

Attempts to measure [3H]muscimol binding to honey bee brain membranes by filtration over Whatman GF/B filters were unsuccessful. The specific binding represented ~5% of total binding at 25 nM. On the other hand, the specific binding, measured by the centrifugal assay as described above, represented 30% of the total at 25 nM. Washing the lyophilized membranes once before assay increased the specific binding to 50% of total. Additional washings did not increase specific binding further. Specific [3H]muscimol binding to freshly prepared membranes without lyophilization was similar (30-50% of total) to that observed with lyophilized membranes. All subsequent experiments were performed on lyophilized membranes stored at -90°C. Binding of [‘Hlmuscimol to honey bee brain membranes increased linearly with protein concentration (Fig. 1); thus we consistently used 1 mg protein per assay.

0

0.4 protein

0.8 concentration

1.2

1.6

(mq)

FIG. I. Specific [3Hjmuscimo/ binding to membranes from honey bee heads as a function of protein concentration. Incubation volume wa.s 1 ml of 50 mM Tris-HCI, pH 7.1 I and [‘Hjmuscimol concentration was 25 nM. time was 30 mitt, and temperature 0-4°C. Specific binding was the difference between [3H]muscimol bound in absence and presence of I mM GABA. Symbols and bars are means and standard errors of two triplicate experiments.

ABALIS

282

AND ELDEFRAWI

Specific binding of [3H]muscimol exhibited a typical saturation isotherm (Fig. 2). However, Scatchard analysis of the binding data clearly indicated the presence of two affinities (Fig. 3): K,,, = 3 +- 2.4 nM, at a concentration of 0.05 & 0.03 pmol/mg protein and Kdz = 144 * 100 nM, at a concentration of 0.5 ? 0.16 pmol/mg protein. Hill 0.1 0.2 0.3 0.4 0.5 analysis of the binding data gives a slope of 0.796 ? 0.05. The rate of association was ‘H-Muscnnol bound (pmdlmg prole~nl FIG. 3. Scatchard plot of specijk [3HJmuscimol relatively fast (Tti < 2 min) (Fig. 4), and dissocation was almost complete in 1 min. No binding to honey bee head membranes. Concentration of [3H]muscimo/ OS-300 nM. Bound in pmollmg proattempt was made to determine the kinetic tein and free in nM. Analysis of computer-simulated rate constants because the centrifugal data of one-ligand, two-binding sites, corrected for assay, which takes 5 min, was too slow for nonspecific binding. Solid curve in best fit of the two classes of sites. Dashed lines are computer generated such determinations. Specific binding of [3H]muscimol was for the high- and low-affinity binding. Free concentration of [3H]muscimol is in nM. Inset is Hill plot oj optimal when measured at low temperatures (0-4”(Z), but decreased at higher temperatures. This may be due to proteolysis, despite the use of antiproteases during binding was stable in the pH range 6-8, but membrane preparation. Preincubation of was inhibited greatly at pH <6 or >8 (Fig. the membranes with chymotrypsin (1 5). These effects of pH, temperature, and mg/ml) for 30 min at 37°C inhibited specific chymotrypsin suggested that the binding [3H]muscimol binding at 5 nM by 71%, sites were proteins. while exposure of the membranes to denaThe high-affinity [3H]muscimol binding turing temperatures (95°C for 3 min) abol- to honey bee brain was similar in its affinity ished the binding. Specific [3H]muscimol to that reported to bovine brain (17) and close to values reported for rat and mouse brain (15, 16). Therefore, we studied the ef3.0 c E2 5

I

/

4 1.d

‘H-Muscimol

concentrotux

( nt.t )

FIG. 2. Saturation isotherm of binding of [)H]muscimol (0.5-300 r&f) to honey bee head membranes (1 mg protein). Total binding (O), nonspecific binding (A) measured in presence of I mM GABA. and specific [3H]muscimol binding (0). Results are the means of triplicates with SE < 10%.

Time

(mln)

4. Specific binding of 10 nM [3H]muscimol to honey bee head membranes (=I mg protein) as a function of time. Each symbol and vertical line is the mean L SD of six data points. FIG.

HONEY

BEE

BRAIN

GABA

283

RECEF’TOR

To determine whether the low-affinity [‘Hlmuscimol binding site was also a

GABA receptor,the effectof four GABA

PH

5. The effect of pH on specific binding of [3H]muscimol (5 nM) to honey bee head membranes. Different volumes of 50 mM monosodiam and disodium phosphate buffers tt,ere mixed to give the appropriate pH. Each point and vertical line is mean of triplicate determinations 2 SD. FIG.

fects of drugs on this high-affinity binding so as to determine the identity of the binding protein. Since binding of [3H]muscimol, measured at any concentration, is a composite of the binding of the two affinities, 5 nM [3H]muscimol was selected for the study to optimize the contribution of the high-affinity binding. Based on the for the two sites, it was calcuKd and hm lated that at this concentration -75% of total binding was due to the high-affinity site and -25% to the low-affinity site, and the apparent ICY, values obtained for drug inhibition of 5 nM [3H]muscimol binding were overestimates of the actual IC,, on the high-affinity site. Of the GABA agonists studied, muscimol had the highest potency followed by GABA, then imidazole acetic acid, DL-GABOB, p-guanidine propionic acid, and 3-aminopropane sulfonic acid (Table 1). Unlike mammalian brain receptors, bicuculline, the potent antagonist of mammalian brain GABA, receptor, had no effect on the high-affinity [3H]muscimol binding to the honey bee membrane. As expected, the GABA uptake inhibitors L-pipecolic, DL-nipecotic, and DL2,4,diaminobutyric acids were very poor. The GABA, agonist baclofen and the allosteric drugs picrotoxinin and pentobarbital were ineffective at 0.1 mM.

agonists was studied on [3H]muscimol binding at 90 nM, which represents 20% binding to the high-affinity site and 80% to the low-affinity site. All specific [3H]muscimol binding was totally inhibited by 100 p,M of these drugs (Fig. 6) with the following order of decreasing potency: imidazole acetic acid = P-guanidine propionic acid > DL-GABOB. Because of the insecticidal action of AVM and the suggestions that AVM acts on mammalian GABA synapses, we tested its effect on 5 nM [3H]muscimol binding to honey bee brain membranes. It was even more potent than muscimol (Table 1). Consequently, the effects of various concentrations of AVM were tested on both affinities, selecting 5 nM [3H]muscimol (where -75% of specific binding is calculated to be to the high-affinity site and 25% to the lowaffinity site) and 90 nM [3H]muscimol (where -80% of binding was calculated to be to the low-affinity site and 20% to the high-affinity site). AVM was much more potent on the high-affinity binding. with an XC,,, of 0.01 nM, while on the low-affinity binding its IC,, was 2 pit4 (Fig. 7). The flatness of the AVM inhibition curve on the high-affinity [3H]muscimol binding (Fig. 7) suggests that it may still represent binding to more than one site. DISCUSSION

Honey bee brain membranes bind [3H]muscimol with two affinities (Fig. 3): K,, of 3 nM and Kd2of 144 nM. These compare with the two affinities reported for [3H]muscimol binding to mammalian brains: 5.1 and 29.8 nM in rat (16), 9 and 70 nM in mouse (15), and 3 and 21 nM in cow (17). The curvilinear function in the Scatchard plot (Fig. 3) may also be due to negatively cooperative site-site interactions, two-step reactions with ternary complex formation, incorrect definition of specific

284

ABALIS

AND ELDEFRAWI TABLE

The Effects to Honey

of Various Bee Brain

GABAergic Membranes

1

Drugs on the High Affinity Binding in Comparison with Their Reported to Rat Bruin Membranes

of [‘HjMuscimol (Measured Effects on [3H]Muscimo/

at 5 nM) Binding

Apparent IC,, @Ml Mammalian brain Drugs

Honey bee brain”

Muscimol Avermectin B la GABA Imidazole acetic acid DL-GABOB B-Alanine B-Guanidine propionic acid 3-Aminopropane sulfonic acid Ethanol DL-Nipecotic acid L-Pipecolic acid Baclofen t + )Bicuculhne DL-2,4-Diaminobutyric acid Picrotoxinin Pentobarbital Flunitrazepam Ethyl-B-carboline-carboxylate

0.006 0.003 0.042 1.2 1.39 4.7 6.4 33 >I0 >I00 >lOO >I00 >I000 >I000 >I000 >I000 >I000 >I000

Mouseb 0.02 0.2 1.6

-

14 0.1 >I000 13 >I000 -

Rat’

Bovined

0.01 0.038 0.229 >lO 1.23 0.03 >I0 >I0 6.2 >I0 >lO >I0 >I0 -

0.003-0.021 -e 0.028-O. I5 0.9 42 0.07 >I00 >I00 -

(’ Data presently obtained using 5 nM [‘H]muscimol. Thus, apparent IC,, values, calculated from log doseresponse curves, are based on inhibition of binding that represented =75% high affinity and 25% low affinity binding. The actual IC,,, values are significantly lower. b Using 8.4 nM [3H]muscimol (15). r Using 2 nM [‘Hlmuscimol (16). d Using 15 nM [3H]muscimol (17). p AVM potentiates 13H]GABA binding (32).

binding, or ligand-ligand interactions (24). However, the complete inhibition of highand low-affinity [3H]muscimol binding to the honey bee membranes by the GABA agonists imidazole acetic acid, P-guanidine propionic acid, and DL-GABOB (Fig. 6) suggests that both affinities are GABA receptor specific. It is likely that binding is to two sites on a GABA receptor molecule or to two kinds of GABA receptors since they are inhibited by GABAergic drugs (Table 1; Figs. 5, 6). Also, the two affinities are still exhibited by the mammalian brain GABA receptor after purification (25). The high-affinity site is not a GABA uptake site because of the ineffectiveness of DL-nipecotic, L-pipecolic, or DL-2,4-dia-

minobutyric acids in inhibiting [3H]muscimol binding (Table 1). It is suggested that the high-affinity site is on a GABA receptor, based on the potencies of agonists in inhibiting it with the following decreasing order: muscimol > GABA > imidazole acetic acid > DL-GABOB > P-guanidine propionic acid > 3-aminopropane sulfonate (Table 1). It compares with relative efficacies in producing Cl- conductances in crayfish muscle of GABA > DL-GABOB > @alanine > p-guanidine propionic acid (26) or muscimol > GABA > P-guanidine propionic acid > DL-GABOB > 3-aminopropane sulfonic acid > p-alanine (27). The ineffectivenes of bicuculline in inhibiting 13H]muscimol binding to honey bee brain

HONEY

7

6

-Log

FIG. 6. Inhibition honey tions pionic

BRAIN

GABA

4 3 cont.(M)

of 90 nM [‘HJmuscimol binding to membrunes by different concenttaacetic acid (X), P-guanidine pro-

bee bruin of imidazole acid

5 drug

BEE

CO), DL-GABOB

(O),

and

methylisoguva-

CA,.

tine

membranes is in agreement with previous findings on binding to crayfish muscle (28) and house fly brain membranes (29). Furthermore, the ineffectiveness of picrotoxinin, pentobarbital, flunitrazepam, or ethylP-carboxylate to influence the high-affinity [3H]muscimol binding to honey bee brain high affinity

13

12

FIG. 7. Effect and

low

affinity

honey bee brain nM, respectively. data obtained dashed lines sults sites

1

'

1

;

'

1

'

1

II

IO

9

6

7

6

5

4

log

Avermectm

o

.

I

EIlo conc.( Ml

of uvermectin B,, on the high affinity specific binding of [3HJmuscimol to membranes. The straight

measured at 5 and 90 lines represent actual

at these tMjo concentrutions, connect points culculated from

to represent separately.

site

effect on binding On the high uf%ity

while these

the re-

to euch of the two binding, the d$

ference between the struight und d&led lines represents the 25% of total binding thut is of lo,\* affinity and is unaffected by AVM up to IO nM. On the other hand, afJnity finity

the difference between in binding represents binding which is almost

nM AVM. tions, SD

Each symbol < 10%.

the tHo lines of the 10~7 about 2070 of high uftotally inhibited by 10

is the mean

of six determina-

RECEPTOR

285

membranes is not unexpected because these drugs bind to allosteric sites on the mammalian GABA receptor, and in many cases even potentiate agonist binding. There are several differences in drug specificity between the high-affinity [3H]muscimol binding to honey bee and mammalian brains. @Alanine, an inhibitor of glial GABA uptake (30) and a poor GABA receptor agonist (26), is relatively potent on [3H]muscimol binding to the honey bee brain, more so than on mammalian brain binding (Table 1). The agonist 3aminopropane sulfonate is much less potent or ineffective, respectively, on the high-affinity [3H]muscimol binding to the honey bee receptor. A major difference is the ineffectiveness of bicuculline on the honey bee receptor, although it is a potent competitive antagonist of mammalian brain GABA, receptors (Table 1). It may be that because bicuculline is possibly a noncompetitive antagonist of invertebrate GABA receptors as shown in lobster muscle (31). it inhibits GABA potentiation of [3H]flunitrazepam binding to GABA receptors of house fly thoracic muscles (7), but does not inhibit [3H]muscimol binding to honey bee (Table 1) or house fly GABA receptors (29). Nevertheless, because of the effectiveness of other GABA agonists and the ineffectiveness of baclofen, the honey bee brain receptor is more like a mammalian GABA, than GABA, receptor (1). It is evident that AVM is very potent on the honey bee putative GABA receptor. with an IC,, of 0.01 nM in inhibiting the high-affinity [3H]muscimol binding (Fig. 6). Unlike previous reports on potentiation by AVM of [‘H]GABA binding to mammalian GABA receptor (32), we found AVM to inhibit [3H]GABA and/or [3H]muscimol binding to GABA receptors of both mammalian (33) and insect brains (Fig. 7). Yet AVM was at least lOO,OOO-fold less potent on binding of [3H]muscimol to rat brain than to honey bee brain receptor (Table I). Like previous reports (20, 34), we did find

ABALIS

286

AND ELDEFRAWI

AVM to potentiate binding of benzodiazepines to the mammalian GABA receptor (33). Furthermore, AVM inhibited binding of [35S]t-butylbicyclophosphorothionate to the channel site of the mammalian GABA receptor (33), which may be due to competitive or allosteric action. The inhibition by AVM of [3H]muscimol binding to the honey bee brain may result from its action as an agonist or antagonist. However, because of its GABA-like effects on binding [i.e., potentiating benzodiazepine binding and inhibiting [35S]t-butylbicyclophosphorothionate binding to mammalian GABA receptor (33)], AVM is probably acting as an agonist when it is binding to the GABA receptor’s [3H]muscimol binding site in honey bee brain. Furthermore, binding of [35S]t-butylbicyclophosphorothionate to a voltage-dependent Cl- channel in Torpedo electric organ (a tissue that is void of GABA, receptor) was not inhibited by AVM (35). ACKNOWLEDGMENTS We are grateful to Dr. Mohyee Eldefrawi for his valuable suggestions and criticisms and Dr. Richard Dybas of Merck, Sharp & Dohme Co. for kindly providing us with AVM. We thank Mr. Gordon Schweizer for his technical assistance and Ms. Evelyn Elizabeth for her excellent typing. This research was supported in part by NIH Grant ES 02594. REFERENCES 1. S. J. Enna, GABA receptors, in “The GABA Receptors” tS. J. Enna, Ed.), pp. l-23, The Humana Press, Clifton, NJ, 1983. 2. N. G. Bowery, D. R. Hill, and A. L. Hudson. Characteristics of GABA, receptor binding sites on rat whole brain synaptic membranes, Brit. J. Pharmacol. 78, 191 (1983). 3. R. W. Olsen, J. B. Fischer, R. G. King, J. Y. Ransom, and G. B. Stauber, Purification of the GABAibenzodiazepinelbarbiturate receptor complex, Neuropharmacology 23, 853 (1984). 4. E. Sigel and E. A. Barnard, A y-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with reservation of regulatory sites and their interactions, .I. Biol. Chem. 259, 7219 (1984). 5. S. G. Cull-Candy and R. Miledi, Junctional and extrajunctional membrane channels activated

by GABA in locust muscle fibres. Proc. R. Sot. Lord. B 211, 527 (1981). 6. S. M. Ghiasuddin and F. Matsumura, Inhibition of gamma-aminobutyric acid (GABA)-induced chloride uptake by gamma-BHC. Comp. Biothem. Physiol. C73, 141 (1982). 7. I. M. Abalis, M. E. Eldefrawi, and A. T. Eldefrawi, Biochemical identification of putative GABA/benzodiazepine receptors in housefly thorax muscles. Pestic. Biochem. Physiol. 20, 39 (1983). 8. L. J. Lawrence and J. E. Casida, Interactions of lindane, toxaphene and cyclodienes with brainspecific r-butylbicyclophosphorothionate receptor, Life Sci. 35, 171 (1984). 9. K. Tanaka, J. G. Scott, and E Matsumura, Picrotoxin receptor in the central nervous system of the American cockroach: Its role in the action of cyclodiene-type insecticides, Pestic. Biothem Physiol. 22, 117 (1984). 10. I. M. Abalis, M. E. Eldefrawi, and A. T. Eldefrawi. High affinity stereospecific binding of cyclodiene insecticides and CX-BHC to GABA receptors of rat brain, Pestic. Biochem. Physiol. 24, 95 (1985). 11. L. J. Lawrence and J. E. Casida, Stereospecific action of pyrethroid insecticides on the y-aminobutyric acid receptor-ionophore complex, Science (Washington, D.C.) 221, 1399 (1983). 12. N. Hori, K. Ikeda, and E. Roberts, Muscimol GABA and picrotoxin: Effects on membrane conductance of a crustacean neuron, Brain Res. 141, 364 (1978). 13. P. Krogsgaard-Larsen. G. A. R. Johnson, D. R. Curtis, C. J. A. Game, and R. M. McCulloch, Structure and biological activity of a series of conformationally restricted analogues of GABA. J. Neurochem. 25, 803 (1975). 14. K. Beaumont, W. S. Chilton. H. I. Yamamura, and S. J. Enna, Muscimol binding in rat brain: Association with synaptic GABA receptors, Brain Res. 148, 153 (1978). 15. Y.-J. Wang, P. Salvaterra, and E. Roberts, Characterization of [3H]-muscimol binding to mouse brain membranes, Biochem. Pharmacol. 28, 1123 (1979). 16. M. Williams and E. A. Risley, Characterization of the binding of [3H]-muscimol, a potent y-aminobutyric acid agonist, to rat brain synaptosomal membranes using a filtration assay, J. Neurochem. 32, 731 (1979). 17. D. V. Greenlee and R. W. Olsen, Solubilization of gamma-aminobutyric acid receptor protein from mammalian brain, Biochem. Biophys. Res. Commun. 88, 380 (1979). 18. D. A. Ostlind, S. Cifelli. and R. Lang, Insecticidal activity of the anti-parasitic avermectins, Vet. Re. 105, 168 (1979).

HONEY

BEE BRAIN GABA RECEPTOR

19. S.-S. Pong, C. C. Wang, and L. C. Fritz, Studies on the mechanism of action of avermectin B,,: Stimulation of release of y-aminobutyric acid from brain synaptosomes. J. Newwchem. 34, 351 (1980). 20. S.-S. Pong, R. Dehaven, and C. C. Wang, A comparative study of avermectin B,, and other modulators of the y-aminobutyric acid receptor . chloride ion channel complex, J. Newrosci. 21.

22.

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24.

25.

26.

(.o/ogy 27.

2, 966 (1982).

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