Jorirnal a/ Nciirochur~,i.~rr),1975. Vol. 25, pp 623-629. Pcrgamon Press. Printed in Grear Brltaln.

SUBCELLULAR LOCALIZATION AND PROPERTIES OF A CHOLINERGIC RECEPTOR ISOLATED FROM HOUSEFLY HEADS J. F. DONNELLAN, P. J. JEWESSand K. J. CATIZLL' Woodstock Laboratory, Shell Research Limited, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, U.K. and 'Department of Biological Sciences, The Polytechnic, Wolverhampton WVl ILY, U.K.

(Received 17 October 1974. Accepted 16 April 1975) Abstract-Protein(s) possessing the ligand-binding properties expected of the insect cholinergic receptor were demonstrated in both aqueous and chloroform-methanol extracts of an 80,000 g supernatant obtained from frozen housefly heads. There is reasonable correlation in both the properties and concentration of the housefly head cholinergic receptor in these radically different extracts. Binding constants for decamethonium, acetylcholine, nicotine and atropine have been determined with aqueous housefly head extracts using an ultrafiltration assay. These observations are taken as further evidence that the cholinergic receptor in the insect central nervous system differs in specificity to the more widely studied nicotinic and muscarinic cholinergic receptors from vertebrates.

We report here further studies aimed at the characWE HAVE previously reported the isolation from housefly heads of a proteolipid fraction which terization of the housefly head cholinergic receptor. appeared to possess some of the ligand-binding In particular we have examined the properties and properties expected of a cholinergic receptor (CAT- subcellular distribution of the cholinergic receptor'ELL & DONNELLAN, 1972). The fraction was extracted like material present in both aqueous and chlorowith a mixture of chloroform and methanol and was form-methanol extracts prepared from homogenates shown to bind acetylcholine and decamethonium with of flyheads in an attempt to determine whether these high affinity. The ligand-binding properties were mea- radically different approaches do in fact yield comparsured by incubating the chloroform-methanol able information on the nature of the insect cholinerextracts containing proteolipids with radiolabelled gic receptor. ligand and followed by chromatography on columns of Sephadex LH-20. This approach was bascd on MATERIALS AND METHODS comparable studies in which chloroform-methanol Preparation of housejy head extracts mixtures were used to extract similar proteolipid fracFly heads were obtained by manually shaking frozen tions with high-affinity binding for cholinergic ligands from vertebrate brain and muscle tissue and from the flies (Musca domestica L, 2-3 days old) and then using electroplax of the electric eel (DEROBERTIS& FISZER sieves to separate the heads from the debris. The heads were crushed in a chilled mortar and homogenised (10 DE PLAZAS, 1970; GOMEZ et Ul., 1970; DE ROBERTIS passes at 1150 rev/min) as a 10% (w/v) suspension in water et al., 1971; LUNTet al., 1971). at 4°C using a Teflon-glass homogeniser. After filtration The presence of a protein with cholinergic receptor- through nylon net (159 pm aperture) to remove gross parlike properties in aqueous extracts of insect central ticulate material, the filtrate was centrifuged at 20,000 g nervous tissue was reported by ELDEFRAWI & for 10min at 4°C. The pellet (P,) was suspended in water O'BRIEN(1970), ELDEFRAWI et al. (1971) and AZZI & prior to lyophilisation and the supernatant was further ELDEFRAWI (1973). Their results indicated that the centrifuged at 80,000 g for 1 h at 4°C. The supernatant (S,) as aqueous suspensions, from this step acetylcholine receptor in the insect CNS does not and the pellet (Pz), closely resemble the classical nicotinic or muscarinic were also lyophilised. The lyophilised materials were powreceptors present in other animals. The insect recep- dered and stored at -20°C until required. tor appears to be less specific since these workers Extraction of proteolipid and ligand binding assay observed a mixed binding profile for a variety of choProteolipids were routinely extracted from lyophilised linergic ligands. Electrophysiological studies on the material with chloroform--methanol (2: 1, v/v), concentrated insect CNS also support the existence of a less specia- under vacuum at' 30°C and then assayed for binding with lised receptor but at present it is not possible to radiolabelled ligands by chromatography on Sephadex exclude the hypothesis that a mixed population of LH-20 as described previously (CATTELL& DONNELLAN, cholinergic receptors is involved in transmission in 1972). In some cases the column eluant was monitored the insect CNS (SHANKLAND et al., 1971 ; FLAI-IUM directly at 280 nm for protein with a Cecil 212 Ultraviolet Monitor. Fractions (2-10 ml) were collected and analysed & SHANKLAND, 1971). 623

624

J. F. DONNELLAN, P. J. JEWESS and K. J. CATTELL

for protein and radioactivity by using the methods deet al. (1971). The relatively large differscribed by CATTELL ences in the refractive indices of chloroform (ngv' 1.444) and methanol ( n i v c 1.328) enabled changes in the polarity of the column eluant to be followed using an Abbe 60 refractometer. Control experiments, using radiolabelled ligand but no proteolipid, were always performed under identical conditions. Unless specified the recovery of radioactivity in the column eluant during control experiments was always less than 5% and was eluted with chloroform-methanol mixtures rather than with chloroform. The unrecovered radioactivity could not be eluted with methanol and was apparently firmly bound to the Sephadex LH-20 gel as measured by direct counting of the dried column material.

Sephadex LH-20 was purchased from Pharmacia (G.B.) Ltd., London. Acetyl [N-Me-3H]choline chloride (120 mCi/mmol), [Me-3H]decamethonium chloride (418 mCi/mmol), [G-3H]atropine (263 mCi/mmol), [G3H]nicotine dibitartrate (250 mCi/mmol), [Me-'4C]choline chloride (60 mCi/mmol) and d-tubocurarine di [MeI4C]ether iodide were obtained from the Radiochemical Centre, Amersham, Bucks. and used without further purification. Paraoxon was a gift from Dr. Shirley Webb and was periodically redistilled. Preparation of a-hungarotoxin

a-Bungarotoxin was isolated from the lyophilised venom of B. multicinctus by ion-exchange chromatography (BosMA", 1972; MEBSet d.,1972). Radiolabelled a-bungarotoxin was. prepared by acetylation using C3H]acetic anhyBinding studies with aqueous extracts et al. (1972). The [acetyldride as described by KARLSSON Lyophilised powders were suspended at a concentration 3H]a-bungarotoxin (27 pCi/mg) was lyophilised and stored of 5-10 mg/ml in the phosphate-free Ringer solution de- at - 20°C until required. Cation exchange chromatography et al. (1971). The binding of radioscribed by CHANCEAUX showed that the radiolabelled toxin was a mixture of the labelled ligands to fly extracts was measured using the mono- and di-acetylated derivatives. The Ca~etyl-~Hlamembrane ultrafiltration technique described by PAULUSbungarotoxin showed a similar level of activity to the (1969) and enabled 40 samples to be processed simul- native toxin on a rat phrenic nerve-diaphragm preparation taneously with a total filtration time of 3&60 min. (R. J. Dowson, personal communication). Reaction mixtures containing Ringer solution and fly extract (up to 1.4mg of protein) were initially incubated with0.1 pM-paraoxon for 10min at 25°C to inhibit the cataRESULTS lytic activity of the AChE present in the extract. RadiolabelStudies with aqueous extracts of the insect CNS led ligand was then added to make a final reaction volume of 0 5 ml. After a further incubation for 30 min at 25°C have shown that the cholinergic receptor is concenduplicate 200 pI samples were placed over Amicon UM-10 trated in a 100,ooO g supernatant fraction whereas the membranes (8mm dia) and filtered under 40psi of receptor from vertebrate tissues with nicotinic innernitrogen. When filtration was completed the undersides of vation appears predominately in membrane fractions the individual filters were flushed with 5 ml of ethanediol sedimenting at approximately 20,000 g (ELDEFRAWIet to remove any adhering filtrate. The filters were then sus- al., 1971; OBRIENet ul., 1972). We have previously pended in 1 ml portions of 1% (w/v) sodium dodecyl sul- reported the presence of a decamethonium- and acephate in scintillation vials and subjected for 5 min to the tylcholine-binding protein in a proteolipid fraction output of a Burndept BE 352 Ultrasonicator to detach the extracted from a 20,OOOg supernatant derived from protein from the filter. The amount of radioactivity in the aqueous homogenates of fly heads (CATTELL & DONsample was determined in an Intertechnique SL 30 Liquid 1972). In order to compare more closely the Scintillation Spectrometer and corrected for the radioacti- =LAN, vity that bound to filters in the absence of the fly extracts. properties of the insect cholinergic receptor in This amount varied with the ligand under study. With aqueous and chloroform-methanol extracts we have [Me-3H]decamethonium, for example, the control binding examined the subcellular distribution of the decawas in the order of 1%. The radioactivity of the unfiltered methonium-binding proteolipid fraction and the incubation mixture was determined using 20 p1 samples ligand-binding properties of aqueous suspensions of suspended in the scintillant cocktail, fly head extracts. AChE AChE, activity was assayed either by the pH stat method & KRUPKA(1970) or by the described by HELLENBRAND et al. (1961). colorimetric method of ELLMAN Protein content Aqueous and proteolipid extracts obtained from fly heads were analysed for their protein content by the modified Folin-Ciocalteau method described by CATTELLet al. (1971). Materials Chloroform and methanol were redistilled before use. Lyophilised venom of Bungarus multicinctus was obtained from Sigma Chemical Co., Kingston-Upon-Thames, Surrey. Diaflo UM-10 ultrafiltration membranes were obtained from Amicon Ltd., High Wycombe, Bucks.

Chloroform-methunol extracts Subcellular distribution of the decamethonium-binding proteolipid @action. These localization studies were performed with the 3 subcellular fractions obtained by the differential centrifugation of an aqueous homogenate of fly heads as described in the Methods Section. Proteolipids were then extracted from lyophilised samples of P,, P, and S2 and 5 m l aliquots of each were incubated with 0.39m-CMe3H]decamethonium for 1 h at room temperature prior to chromatographic analysis on identical Sephadex LH-20 columns using the batch elution sequence as described by CA-ITELL & DONNELLAN (1972). Figure 1 shows the respective elution profiles under conditions where the decamethonium-binding peaks would be expected t o be eluted with chloroform

Housefly head cholinergic receptor

C hloroform -

Chloroform Chloroform methanol ‘15: I 2:i ’ (b)

625

Chlmfwm Chloroform- methanol .I i 2 . 14.5

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t

_I

y- ;

, ,

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-

u ao

o Eluate volume,

ml

120 160 Eluate volume, rnl

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40

120 160

Eluate volume,

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FIG.1. Subcellular distribution of flyhead decamethonium binding proteolipid extract. Chloroformmethanol extracts from (a) PI (5.35 mg of proteolipid protein), (b) P, (3.4 mg of proteolipid protein), and (c) Sz (2.36 mg of proteolipid protein) were incubated in 5 ml aliquots with 0-39p~-[Me-~H]decamethonium for 1 h and applied to columns of Scphadex LH-20 (25 x 2 cm) equilibrated in chloroform. The samples were eluted with 5Oml of chloroform, 25ml of chloroform-methanol (15:1, v/v) and 100ml of chloroform-methanol (2: 1, v/v). Protein, -; Radioactivity, .~.-.-., Refractive Indcx, The recoveries of protein and radioactivity were respectively (a) PI - 30.8% and 13.7%, (b) P, - 47.9% and 28.8%, and (c) S, - 65% and 86%. ~~

(DONNELLAN & CATTELL, 1975). Therc was negligible ligand-binding by components of the proteolipid extracts from PI (the 20,000g pellet) and P, (the 80,OOOg pellet) and this is also reflected in the low recoveries of radioactivity (13.7% and 284% respectively) in the column eluants. However. the proteolipid fraction extracted from S2 (the 80,OOO g supernatant) showed the presence of a protein peak and associated decamethonium in the chloroform eluant showing that the 80,000 g supernatant fraction was enriched in a decamethonium-binding protein (see also Table 1). The recovery of radioactivity was correspondingly high-S6”/,. The refractive index traces in all 3 cases shows a distinct dip in the eluant immediately prior to the elution of the protein peak that co-chromatographed

with decamethonium. This is attributed to the methanol in the chloroform-methanol extract passing through the column as a discrete phasc. It is significant however, that the amount of ligand associated with the protein peaks is not the same with each of the proteolipid preparations and indicates that the observed co-chromatography of ligand and protein peak from S2 is a result of a specific interaction. It has been suggestcd that receptor-ligand interactions observed on Sephadex LH-20 are due to a nonspecific association fortuitously caused by the methanol present in the sample applied to the column (LEVINSON & KEYNES,1972). Using the Sz proteolipid extract and [Me-3H]decamethoniuq it can be demonstrated that variation in either the sample size or elution procedure can indeed change the resultant protein frac-

TABLE1. DISTRIBUTION OF THF

DECAMETHONILJM-BINDINGMATERIAL, PROTEIU AND AChE IN CHLOROFORWMETHANOL A h D AQlJtOUS EXTRACTS OBTAINED FROM A FLYHEAD HOMOGENATE

Content ~

PI

p2

s2

~

Lyophilised powder (mg) Protein (mg) Proteolipid protein (mg) Binding atudies (a) Chloroform-methanol extracts (Fig. 1) proteolipid protein (mg) Receptor peak eluted % distribution bound decamethonium with chloroform nmol decamcthonium pcr g protein (b) Aqueous extracts % distribution bound decamethonium nmol decamethonium per g protein AChE Specific activity (prnol per min per mg of protein) ”/, distribution

1098 I000 10.9

465 375 8.6

1710 1337 6.9

6.7 0.33

1.14 7.0 091

068 86.3 4.59

20.7 1.45

12.7 232

66.6 3.45

0.02 4.7

0684 59.2

0117 36.1

0.39

An aqueous homogenate of flyheads (22g) was fractionated to yield the fractions P I , P, and S,, as described in the Materials and Methods Section. The binding of decamethonium was measured (a) in chloroform-mcthanol extracts using the Sephadex LH-20 chromatographic assay, and (b) in aqueous extracts using thc ultrafiltration assay. AChE was assayed in aqueous extracts. N.C.

25/5--

F

J. F. DONNELLAN, P. J. JEWESS and K. J. CATTELL

626

tionation pattern. In all cases, however, there is a distinct protein peak associated with the radiolabelled & CATELL, 1975). ligand (DONNELLAN Binding constants for decamethonium. The saturation characteristics of the binding of decamethonium by the S, proteolipid extract were examined by measuring binding as a function of ligand concentration. Aliquots of the proteolipid extract (2.35 mg of protein) were incubated in 2.4 ml volumes of chloroform-methanol (21, v/v) with 0.1 to 2.5 pM (Me-'Hldecamethonium for 1 h at room temperature and then chromatographed on a series of Sephadex LH-20 columns (15 x 1.5 cm)equilibrated in chloroform. Analysis of the fractions for protein and radioactivity after batch elution showed similar profiles to that shown in Fig. lc. The decamethonium associated with the protein peak eluted with chloroform was in each case corrected for any radioactivity (