~'~Izimic~ ¢,!Biuphysica Actu. 1137(1902) 2 9 9 - 3 1 ~ © 1992Elsevier Science Publishers B.V. All rights reserved 016%4889/92/$05.00

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N i c o t i n i c a c e t y l c h o l i n e r e c e p t o r s o f t h e c e n t r a l n e r v o u s system o f Drosophila Eckart D. Gundelfinger and Norbert Hess ~ffN}~ t%.qme for Molecldar N~uro[~'clog~, IJnil ersiey of Hamburg, Hambl~tg ( Gertaa~y ) (Received 8 JUP.e 19921

Key words: Acet~lcholinereceptor; .~,.cltatory ncuTotransmlssJon;Central nervous system: CJen¢cloning;Evniution; ( D . mefalroga~fer)

Conlants 1.

tmroduclion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . "[~¢ chollnerglc systcm of l)romplti[a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . n. N i C O l i n i c r e c e p l o l ~ o t v e r t e b r a t c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A.

II. Bindin~sitcsforniootiniccholinergtcligaedsinthcDromidJilaCNS

......................

299 30{]

300

301

|ll. Eleclroph~iolo~ical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

302

IV. Moleculargenetic~of Drme#tzil~ nAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Gencs~ cDN.Asand the deduced proteins - implications for the cvolutlon of the nAChR gone family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. DO Ihe cloned genes encode functional rlAChR sub milts? . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Temlm~[ and spatla[ cxpl~slon of nAChR genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

302 30Z ]0S

V. Genetic apl)loaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3~

vI. CJor,.clndiagremarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

307

Acknowledgemcnts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

307

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

307

I. Introduction Chemical synapses play a central role in the transmission and processing of information in the nervous system, W h i l e in vertebrates glutamate is the prevalent excitatory neurotransmitter in the central ~ e ~ o u ~ syso

Corresimndcncc to: E.D. Gundellinger, ZMNH, Universit51skrankcnhaus Eppcndorf, Marlinistr, 52, ID-21~fl Hamhorg 20, Germany. Abbreviations: ACh, acctylcholine.; m~.ChR, niculini¢ acc~-lcho|in¢ lecsptor; ACHE, acctI/Icholinestera~e;CHAT, choline acetyllransferase; CNS, central nervous system; QNB, quinuclidil,.yl benzyMte; aBtx, a-b0ngaroto~in.

tern (CNS), many invertebrates including insects use acetylcholine ( A C h ) a t the majority of their C N S synapses. ,At the n c u r o m u ~ u l a r gynaps¢ t h e situation is reversed, as i~secm use g l u t a m a t e , a n d vertebrates ACh, as transmitter, This review concerns t h e structure and function of nicotinic A C h receptors ( n A C h R ) as synaptic components o f the nervous system of the fruitfly Dro~opkila. However, before discussing in detail the nicotinic receptors of the fly we will briefly examine the cho[iner$ic system in general (comprehensive information on the cholinergic and o t h e r neurotransmitter systems o f D r o s o p h i l a can be obtained from the excellent reviews by Restifo and White [1] and Buchner [2]) and the vertebrate nicotinic receptors.

30O

I-A. The cholinergic system of Drosophila The essential comooncnts of the cholincrgic system are: the ACh synthesizing enzyme choline acetyltransferase (CHAT), the degrading enzyme aeetyleholinesteruse (ACHE), a high-affinity uptake system for choline and two different receptor systems, i.e., the nicotinic receptor which is an ACh-gated inn channel and the G-protein-coupled muscarinie AChR. Both, ChAT and AChE are encoded by single copy genes, null mutations of which are recessive embryonic lethals [3,4]. In accordance with the assumed predominant role of ACh in the insect CNS [5] histochemical detection of esterase activity and immunohlstocbemical Idealization of ChAT revealed that both enzymes are widely expressed in the D~sophila nervous s~tem [6]. Anti-ChAT immunoreactivity conspicuously correlates with regions of high esterase activity. However, since some AChE activity was detected also in non-ebolinergie areas, ChAT is considered to he the most reliable marker for choIinergic areas [2]. Similarly, the uptake system for [~H]choline appears to be widely distr~uteo over the

Deo~c,p~,ila CI'qS [ 7 - 9 ] . The existence of both types of cholinergic receptor in Drosophila was first suggested by binding studies with nicotinic and muscarinie antagonists such as obungarntc.xin (aBtx) and quinuclidinyl benzylate {QNB~, respectively (for review see g e l 10). In comparison to vertebrates, the observed nicotinic binding sites are in large excess over mnscarinie sites in the Drosophila CNS [t0,H]. Access to genes of both receptor types was possible after suitable vertebrate probes became available for DNA homology screening. To date, five different genes with c~nsiderable similarity to those encoding ve~ehr2te nAChR subunits have been identified [12] (see below). The sole musearinic receptor homologae isola:.ed from Drosophila displays 60-70% similarity with the various vertebrate sub,pus [13,14]. When expressed in murine adrenal carcinoma cells this receptor stimulates phosphatidylinositol metabolism in the presence of a ebolinergie agonist [t4].

l-B, Nicotinic receptors of certebrates The most widely studied nAChRs are those of the ncuromuscular junction of vertebrates and the electric organ of certain fish species; the latter is a derivative of skeletal muscles and expresses nAChRs in extremely high amounts (for review see ReL 15). The muscle receptor consists of four different protein subunits assembled with a stoichiometry of a2~876. Binding of ACh to the two a-sobunits is thought to be the essential signal to transiently open the integral cation channel. Developmental ixoforms with different channel characteristics are created when the 'y-sobunil of the

N

us

h,=

O

e xll:aee I I_ula_¢

Fig. L SUXlClUI~dlm~el el nAChR subunil$based on hydrolmthy plot~. S-$: .'~r,~'ed :.~L~;l.rgl¢b.idge; MI to M4: putativt:turinbrahe-spanning a-helices: CHO: N-gh~us?lation site conserved among all neuronal nAChP, sulmnits.The two ;~dj~C~nl ¢y.~teines which are characteristic of all ligtmd-bindingnAChR a-subtlnilsare itxdicaledI~HS). embryonal extra~'naptic receptor is replaced by the e-subanit in the adult syaaptic nAChR [15]. All subunits are membrane-spanning glycoproteins. They display significant sequence similarity with each other and have a similar structural organization. The most popular structural model (Fig. 1) which applies not only to nAChR subunits but also to subunits of other iigandgated ion channels, i.e., GABA;, ~nd ~lyeine receptors [16], and 5-HT3 (serotonin) receptors [17] predlcts four hydrophobie membrane spanning domains (MI-M4). The M2 helices of the five subonits are presumed to line the pore of the ion channel [15,18]. The aminoterminal extraeeltular domain of about 220 amino acid residues harbors a coasmwed disulfide bridge. In nAChR a-subooits an essential element of the ligand binding site is located just upstream of MI and is characterized by two consecutive cystcinc residues [15,18]. The cytoplasmic loop between M3 and M4, and the short extracellular C-terminal tail are highly variable both in sequence and in length. In addition to the nAChRa expressed in muscle, a distinct group of receptors is found in the vertebrate peripheral and central nervous system, Sequences have boon deduced corresponding to many different neuronal nAChR subuoits from various organisms including goldfish, chick, rat and humans (for review see Ref. 19). According to their proven or postulated ligand binding characteristics they are divided into ligandbinding o-subanits ((=2-8; a l is the muscle a-suboni0 and structural non-a-, or ,8-subunits (/]2-5 in rat; non-~l to 4 in the avian system). The presence or absence of the t w o consecutive cystciaes (see above) is generally accepted for the classification of new subunits into o~-subunits and non-a- or ~-subunits, respectively. At least some subtFpes of the neuronal nAChRs appear to be composed of only two tlifferant suhunits

301 assembling into a peutameric receptor complex of l,.vo ~- and three ,O-subunits [:20-22]. II. Binding sites for nicotinic cholinergie ligands in the

Dresophila CNS The most uscful tool in biochemical and pharmacological studies of putative nAChRs from Drosophila wag ~-bougarotoxin (aBtx), a component of the venom of the snake Bungarus multicinctns, xvhich is a highly potent antagonist of vertebrate muscle nAChRs [15,18] and some subclasses of vertebrate neuronal nAChRs [23-25], High-affinity binding sites for IZSl-aBtx, with a dissociation constant (K o) in the range of 0,1-2 aM, "yore identified by several groups in membrane fractions from Drosophila heads [ 11,26-30], The reported abundance of these sitca ranges from 0.3 to 1.4 pmol/mg membrane protein. Binding of 12Sl-~Btxdigplays a pharmacological profile typical for nicotinic receptors, i.e.. it is specifically displaced by other nicotinic ligands including unlabeled aBtx, d-tubocurarine, nicotine or ACh, and insensitive to museariuic ligands, like atropine or QNB [10[. A more recent Scatehard analysis revealed at least two different classes of z~Btx binding sites in Drosoph#a head mcmbreaes with K D values of about 0.1 nM (class 1) and 4 aM (class 2) and abundances of about 025 and i.1 proof/rag protein, respectively [31]. ~Sl-aBtx also specifically binds to membrane preparations from embryonic Drosophila ncuron~ in c~1:t:rc [32] and late Drosophila embryos, where again both classes of binding sites have been observed [311. Autoradiographical and histcehemicel studies with iodinedabeled or horseradish pcroxidasc¢,~::jugatcd ¢~Btx demonstrated that the toxin binding sites are present in most synapfic neuropil regions of the Drosophila CNS in adult heads [26,29,33-35], 3rd instar larvae and pupae [29]. Only in the lamina region of the optic lobes was essentially no aBtx binding detected. Solubilized otBtx binding complexes have a mol¢¢0lar weight of 250000-300000 as estimated from centrifugatiou in sucrose density gradients [30,36,37]. The two different aBlx binding complexes cannot be separated on these gradients (discussod in Ref. 3g). Th~ observed molecular weight coincides with the sizes determined for solubilized nAChRs from vertebsates [15,18] as well as other insect species (see below). Preliminary reports on the purification of ~ t x binding complexes describe the detection of two to four different polvpoptides of 42, 57, 65 and 79 kDa in the finally purified fraction (L.M. Hall, personal communication to Ref. I, and Refs. 37,39). Chemical crosslinklng of l~l-aB~x to Drosopl~ila head membranes revealed a 42-kDa polypeptide as the major target for toxin binding [88], It should be noted that, in cLmtrast to other insect

species, there is currently no direct proof that @Btx acts on functional nAChRa in the fruitfiy. In the cockroach Periplanetaamericana, for example, ~Btx interfores with ACh-induccd icsponses on several identified CNS neurons in situ (reviewed in Refs. 1,40). An is~thiocyanate insecticide which suppresses the Wostsynaptic response at a defined cholincrgic synapse in the cockroach terminal abdominal ganglion, specifically inhibits binding of taSl-~Btx to extracts of both cockroach abdominal nerve cords and Drosophila whole flies [4l]. Insecticide concentrations that produced half-maximal inhibition of toxin binding (--10 -s M) were similar to those requited for a 50% reduction of the amplitude of the postsynaptic response, suggesting similar pharmacologica[ characteristics ['or soul= c~.kroach and Drosophila nAChRs. Another confirmation that ~Btx binding complexes of insects function as nAChRs derives from work on ganglionic recaptors of locusts. From the migratow locust (Locnsta mig~atoria) an aBtx-binding component has been purified to homogeneity. 11 migrates as a 250-300-kDa complex in native polyact~lamid¢ gels and as a single.. 65-kDa band under denaturing conditions [42]. Tire isolated protein was reconstituted in planar lipid bilaycrs and shown to form an ion channel that is ~ated by cholinergic agonists [431. More recently, the cloned o=suhanit of a nAChR from another locust species Schistoceroa gregaria was shown to form nicotine-gated ion channels in Xenopu~ oocytcs which are antagonized by o~Btx [44]. It should be mentioned that the subnnit composition of cBts-binding nAChRs is still matter o[ some debate (see Ref. 12). While the data described above are consistent with a homooligomcric quaternary structure of the locust ~Btxbinding receptor, there is evidence that at least or~e of the aBt'x-hinding components from Drosophila consists of two or more different types of subunits [38] (see below). Besides these aBtx-binding uAChRs several examples of nicotinic recoptor~, which are insensitive to the toxin have been described from various insect species [45-48[ including Drosophila [I,49]. This is reminiscent of the situatiop in vertebrates, where the majority of neuronal nAChP, s are not affected by ¢rBtx bu," are blocked by another Bangaros toxin called neuronal or fcBtx [15,19]. Neither the quantity nor the distribution within the Drosophila CHS of these ~BIx-insensitive nAChRs is currently known. The variability in the binding of nicotinic ligands to their r e , p l o t s in the insect CNS suggests a remarkable heterogeneity of nAChR subtypes. This diversity is also supported by immunohlstoehemical experiments ~sing a set of monoclonal antibodies against the nAChR from clcctroplax of the ray Torpedo californica, which detect cross-reacting cp[topes in the Drosophila CNS [50]. Only one subset of antibodies ~cognizes an anti-

302 s e n which shows a distribution in the synal~ti,2 neuropil o f the visual system, that tentatively coincides with the distribution of histochemically detected binding sites for HRP-eoniugated a B t x [35]. O t h e r antibodies stain distinct neural structures, e.g.. photoreceptors, mechanosensory bristle clcmenLs or certain fiber tracts [50]. However, there is no direct evidence that these epitopes arc part of functional Drosophila nAChRs. II!. ElectrolShysinlogical studies Unfortunately. the physiological characterization o f nicotinic receptors from Drosophila has not yet proceeded very far. While in other insect species several examples o f cholinergic responses recorded in site have established that nicotinie receptors have a crucial function in the CNS (for reviews see Refs. 1,40) only recently a paradigm of responses elicited by eholinergic agonists on a semi-intact preparation from Drosophila larvae has been deseribed [49]. Both nicotinic and musearini¢ receptors s e e m to control the rhythmic activity o f a CIqS-pharyngeal muscle preparation involved in larval feeding behavior. Since the excitatory transmitter at the neuromuscular synapse is glutamate, ACh is thought to act on the innervating motorneuron [49]. The nicotine-evoked response, a short bursting o r high-frequency tonic activity o f the muscle, was antagonized by relatively high concentrations (10 -4 M) mecamylamine but not by other nicotinic antagonists, such as curare o r ~Btx. It is not d e a r w h e t h e r this unusual pharmacology is due to the inaccessibility of the antagonists to the receptors o r whether a new, not previous!,,-" described type of n A C h R is involved. Based on their localization withip, the insect nervous ~ s t e m , synaptic and extrag!maptic nAChRs can be discriminated. Like the vertebrate muscle receptors, they may differ in their channel characteristics [51]. Patch-clamp recordings of individual ex'trasynaptie

n A C h R s on dissociated neurons of Drosophila larvae have been briefly described. ACh-gated single channels with a mean open time of about 2 to 3.5 ms and coaductanees of 9 to 25 pS were ohse~,ed at an ACh concentration of 100 n m o l / l [8]. T h e observed currents appear to ~ insensitive to a B t x (cited in Ref. D. Certainly additional studies are required to determine whether this channel is the homologue of the AChgated cation channels on neuronal cell bodies o f cockroach and locust, which have eonduetanees of about 40 pS a n d 35 pS, respectively [51,52]. While single channel data from synaptic receptors of Drosophila are not available, reconstituted receptor preparations from the locust indicate that synaptic n A C h R s o f insects may be characterized by shorter open times and higher conduetances than extrasynaptie receptors [43,51]. IV. Molecular geng/i~s o f Drosophila nAChRs

IV-A. Genes, cDNAs and the deduced proteins - implication8 for the et'ohaion of the aAChR gone family Homology screening with D N A probes from vertebrate nAC'hP,, subunits and subsequently with Dr6~sophilo receptor-derived probes has led to the isolation of a family of e D N A s and genes encoding five different putative snbunlts of Drosophila nAChRs (for review see Ref. 12). T h r e e of these subunits, called ALS [53], D o 2 or SAD [54-56] and D o 3 (Mfilhardt e t a l . , unpublished data; see T a b l e 1 for the definition o f abbreviationsL contain the two consecutive cysteines characteristic of ligand-binding a-subonits. T h e o t h e r two subunit.% named A R D or A C h R 6 4 B protein [57-59] and SBD [60], are missing the cysteines and thus most likely represent structural noa-a-subunits. T h e r e may be m o r e n A C h R suhunits which still await identification. In a preliminary report [61] the identification o f up t~ eight different n A C h R genes from the migratory

TABLE I ~bmnits of Dax~OldtflanMChRs Subttnit

Putative iuncUort a ligand hinding ligand hindin8

RefeteJlces [53] 154-5g]

~,;,D

De~ig~ation a_-h~esubunit _Dmsol~dl=a_-I~c subenR _2 Second a-like subunit

D~3

D~sop~//a o_-Iikesubunit _3

ligand binding

AnD

nAChR prolt:in t~om

struclnral

(Mfilhardl el at.. unpublished results) 157 ~q]

SBD

S_¢~ond_~-subunit from

structural

[¢~q]

ALS

Da2/

~roso~ita Based on tbe consctw',ai0n of two consecutive cystcin¢ residues which have been shown to be pan uf the ligand bisding site or a*suhunits [! ~]. h According to its chromosomal localization at position 64B the gone e,c0ding the ARD protein was also relined ACbR64B g:r.= ;S~],

3O3 locust has been described. Six of these (four or-like aud two non-t~-.',ubunits) have been fuLly or partially cloned and sequenced. The mRNAs encoding the D~2, ARD and SBD proteins are about 2-3 kb long [55-60] whereas ALS is translated from an unusually long transcript of more than l0 kb. The latter is characterized by a complex 5' leader of more than 1200 nucleotides (its full extension has not yet been determined) 1"53],which contains at least 6 upstream small open reading frames (OREs). Interestingly a similar complex leader was tbund in the transcript of the Drosophila gene encoding AChE [62]. It is noteworthy that the shorter leaders of D a 2 and ARD mRNAs also contain upstream A U O codons followed by short ORFs [54,56,57]. In the ease of the yeast GCN4 protein it has been demonstrated that upstream ORFs are involved in the control of translation from GCN4 transcripts [63]. Hence, the short upstream ORFs found in nAChR transcripts may be responsible for some of the difficulties found when expressing Dt~sophi£a nAChR subunits in cells of higher eukaryotes (personal a3mmunications from sev-

eral independent laboratories). These problems m i g h t be overo~me by the. removal of upstream A U G s . The deduced nAChR proteins of the fly share a similar predicted structural organization (Fig. l) and 33% to approx. 50% sequence similarity with nAC'hR subunits of vertebrates (Fig. 2). They have cleavable signal peptidcs at their N-termini and the putative mature proteins consist of 493 (SBD), 497 (AND), 535 (D~2) and 546 (ALS) amino acid residues. The sequence similarity is not evenly distributed along the polypeptide but is particularly high in t h e N-terminal extracellular domain and the first three hydrophobie regions ( M I to M3). Another region of relatively high similarity is located arotmd the putative transmembrant region M4, whereas the cytoplasmic loop between M3 and M4 and the C-terminal tail vary in length and display very low sequence conservation betxveen the various subunits (Fig. 2), The subunits contain between one and three potential N-glyeosylation sites one of which is conserved at amino acid position Asu-24 of air mature Drosophila nAChR subunits (it is also conserved in all subunits of vertebrate n e u r o n a l

c~

201

QI~DYDDSTgS NGIT~ r D~D~ IS DF PS~F ~ .S,~...D~....yD~GF&]N

300 ~VXi~ ~ I V ~ ~ a j C t j t ~ O f f i ~ B I ~ I ~ 3 293

.................

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.

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.

.

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.

~.00 ~cs~pFlmssL~la3~_t~rnY~?sp~mN~-Dxst~ecp~t,~l:TF~l~yalalL,m l e r z ~ s R ~ n ~ ~ ~ B

;01 ~ s o L v ~ I ~ LSGY~I~PSVLS~ . . . . . . . . . . . . 3S8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.........................................

DShSDV~pFEL57~I~mr~ .~nm~m,~xpRm,'rp,~vr.~:~,:~,v'~

~ ...............

~

~

DNBZ

:

~

M4

lVC~L

500 HD I 489 4#.0 461

Yr

F

C,LFI~O- LI~¢ISI.. ,.~

...................

"~ ....

¢ha2

ILQA H[:t~gQPIDILISKIA~NIC-~Esr~z,.,. BIbS D~t2 I YDTRS'~IDRO[~EIP~PDI~gT s1~o FE~'~DQDR[IEIYRGE. . . . . . . . . . . . . . . . . N~D

Fig, 2, Amino acid ~qucn¢~ alignmcm of the D r o ~ h i l u aAChR subunits wilh the chicker~ o 2 ~ub~nit Sequences ot tile rilature pmteias have been aligned. Gaps ( - ) were iauedueed tO maximize sequence idcn~qlz Black baekgrgund, residue~ conserved in all genes. Transraembrane domairl~ t M i , M2. MS. aod M4} ar~ indicated abut: l i l t sequences. Asterisks indieal¢ th~ i~vo adjacent cyst~ine residues ~ m m o a IO ligafid.binding rlAChR o-~uilanits. Putential phosphm~ialiml site~ were determined usirl.%the Pr~.~ite dalab~e [8~.83]. Pmteia kinase C, b ~ e d ; cAM P-dcpcndctn pio~cia kioa~¢, t~l}dcrlined; ty~sin¢ protein kina~c, dotted line. Thc first 7 untinu adds of the Ch a2 ~eqaence were omitted for simplicity. Sequences were taken from Re[~-: C h . 2 , [ST]: ALS, [53i; Da2.154]: SBD. 160]: A R D , 157].

304 "FABLE It Ft,Ken! seelllence identity ~etween l~so~haa IIAChl¢zub~aits The percentage identity wa~ oleul~ted on the basis of multiple sequence alignment of ltle gACIIR pmtein~ The d~iekeg =2 subtli~il(Cha2) of neuronal nAChRs wa~ included as it represents the vertebrate nAChR p~otein with the highest sequence similarity to Oro~pllNa gubunits [66]. The highly ~ariabl¢ pa~ts of the cytoplasmic region t~:twgun M3 and M4 t=orre.spo~ding to amino acid residues 329-421 of Cha2) were not taken into ~ccount for tile calf,Jlar.;ort e[ .~irnfiarity.

ALS Dot2 SBD ARD

Da2 66

SBD 6t 6.0

ARE) 4"/ 45 45

Drosophila n A C h R s u b u n i t s are m o r e similar t o subunits o f vertebrate n e u r o n a l n A C h R s t h a n to t h o s e receptors expressed in t h e vertebrate muscle. T h e t h r e e n A C h R s ubun l t s ALS, D a 2 a n d S B D display a significantly h i g h e r d e g r e e o f s e q u e n c e conservation to each o t h e r t h a n w i t h vertebrate subunits ( T a b l e II) suggesting that part o f the r e c e p t o r diversity in the fly m a y have evolved a f t e r the s e p a r a t i o n o f v c r l c b r a t e a n d invertebrate progenitors. T h e A R D protein, o n "he o t h e r h a n d , m a y have diverged from the o t h e r Drosophila s u b u n i t s v e ~ early in evolution [66]. T h e structures of the g e n e s e n c o d i n g the A R D , A L S a n d D a 2 p r o t e i n s have b e e n s t u d i e d [53,55,58,591. T h e g o n e sizes vary b e t w e e n a b o u t 5 a n d 8 k b for D a 2 a n d A R D , respectively a n d m o r e t h a n 5 4 kb for the g o n e e n c o d i n g A L S . W h e n c o m p a r e d with the. k n o w n structures o f v e r t e b r a t e n A C h R genes, the close relationship o f t h e Drosophila n A C h R subunits w i t h n e u ronal n A C h R s u b u n i t s rather t h a n t h o s e o f m u s c l e bcpam~s a p p a r e n t ( F i g . 3). W i t h o n a exception [23] all v e r t e b r a t e n e u r o n a l o A C h R g e n e s have the s a m e ¢ ~ o n / i n t r o n o r g a n i z a t i o n [67-69], c o n t a i n i n g five int rons within the p r o t e i n c o d i n g region. V e r t e b r a t e muscle g e n e s have all these i a t r o n s a n d from t h r e e t o

n A C h R s , b u t n o t in receptors expressed in the muscle [15]). A n o t h e r gLycosylation site in the region of the e o n s e ~ e d disulfide b r i d g e that is f o u n d in m a n y vcrtcbrat~ n A C h R s u b u n i t s [15] is not present in the DrosoFhita proteins. I n vertebrate n A C h R suhunits the regions b e t w e e n M 3 a n d M 4 h a r b o r phosphor~lation sites for at least t h r ee different types o f p r o t e i n kinase, i.e., c A M P - d e p e n d e n t protein kinase (PKA), protein kinase C ( P K C ) a n d tyrosine protein kinase ( p K T ) (for revlew see Ref. 64). P h o s p h o ~ l a t i o n o f this region h a s b e e n s hown to b e involved in the regulation o f the desensitization of n A C h R s [64] a n d o f t h e assembly o f the receptors f r o m individual ~ubunits [64,65], C o n s e n s u s signals for protein p h o s p h o r y l a t i o n c a n also be f o u n d in the corres p o n d i n g regions o f Drosophila n A C h R proteins. T h u s the A L S protein contains o n e potential phosphorylation site for P K A a n d two for P K C b e t w e e n M 3 a n d M4. T h e D a 2 protein contains o n e P K C a n d the A R D protein o n e PiCA c o n s e n s u s site. T h e 5 B D protein h a r b o r s a c o ~ s e n s u s signal for P K T in its cytoplasmic d o m a i n (Fig. 2), T h i s s u g g e s t s that p h o s p h o r y l a t i o n of subunits m a y play a role also in t h e regulation of insect nicotinic receptors. SP $ I[AL5

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Fig. 3. Cnmparhon or" the struclurc or nAChR genes. The arrows mark intron po~itions on a physical map depicting tee typical features of nAChR sllbunits. Th~ prt~sertce of an intron in a gone or in a group of genes is indicated by a circle (open: int¢on is ullique to a gent/group of genes; filled: intron L,~fouvd in at least two gcn=s/gmu[Js of gencsL SP: signal peptid~; ML M2, M3, and M4: putative Iransnvzmbran¢ regions; S-S: conserved disulfide b~idge. Tile gone structures were taken from the fotlowing Rots.: Drosol~tila genes, ALS [53]; D~2 [55]; ARD [~S,$g[. Verlcbrale genes, neuronal [6"7-69]; mus~te *r [67,B4]; muscle ~ [85]; m u.~cle ~"[S6,871;muscle ~ [87]: mn~le * [SSl.This figure was modified fr;xm Rats. 12,55.

305 six additional ones (Fig. 3). The Drosophila genes contain three or four of the original introns plus one to three in addition. However, none of the additional intron positions of th~ Drosophilu genes coincides with the ones found in genes expressed in the vertebrate muscle. Mapping of three nAChR genes encoding the subunits ALS, Da2 and SBD to the same chror.w0somal band in the 96A region of the 3R Drosophila chromosome suggested that these genes might form a gone cluster [53,55,60]. A tight linkage ot genes for two iigand-binding subunits (a3 and aS) and one structural subunit (~4 or non-~3) of neuronal nAChRs ha:, al.~o been observed in the rat and chicken genome [68,69]. A detailed anahlsis of the structure of the Drosophila gone cluster and a subse~ue."~t comparison with the vertebrate cluster may help to understand tbe evolution of the diversity of nAC'hR subunits in vertebrates and insects.

IV-B. Do the cloned genes encode fanetionM ~4ChR sebonits? The high similarity tff the Dtorophila nAChR genes with their vertebrate pendants strongly suggests (but of course does not prove) that they encode nAChR s,Jbunits. Furthermore, the a,lsembly of subunits identified by DNA homology screening into the various receptor types observed at the physiological and pharmaculogi. eal levels remains to be clarified. Two different approaches are being pr~rsued to establish the identity of the deduced proteint~ as functional nAChR subunits and to study their ass~mbly: (i) the expression of parts of eDNAs as bacterial fusion proteins to produce antibodies or to perform ligand binding studies, and (ii) the expression of cDNAs or synthetic RNAs in enkaryotic cells, such as Xenopus ooeytes, to test whether functional nicotine-gated ion channels can be formed. So far the two ligaqd-binding subunits ADS and Da2, as well as the structural subunit ARD, have been ineluded in these studies. Ba~erially expressed fusion proteins served as antigens for the production of antisera against ARD and ALS proteins [3t,38]. In particular antibodies against tile weakly conserved intraeellular loop between M3 and M4, which are expected to be specific for individual subunits, were useful tools to study subunit assembly. Immunoprecipitation experiments were performed to test whether the antisera recognize nAChR complexes with bound I~l~Btx. Atuisera against various regions of the ARD protein precipitated about 20 to 30% of all t2Sl~Blx binding sites solubilized from Drosophila head membranes [31]. A Scatchard analysis of the supernatant revealed that only one of the two classes of high-affinity aBtx binding sites (i.e., class 1, compare section ll) was removed specifically by anti-

ARD antibodies. These results suggested that the ARD protein which itself is not expected to bind the ligand is a structural subunit of the aBtx-hinding nAChR of class I. The first possible candidate Tot the orBtx-binding suhunit of class l receptors was the ALS polypeptide [53]. Bacterial fusion proteins including amino acids 134 to 224 [38] or 192 to 212 [70] of ALS were shown to bind 12Sl-aBtx, although with an affinity that was reduced by three to four orders of magnitude as compared to the binding sites from head membranes. Suhunit-specific antibodies against the cytoplasmic loop of the ALS protein also immnnoprceipitated approx. 25% of the hlgh-affinity ~xBtx bin~ing sites from head membrane extracts. The combination of antisera against ALS and ARD proteins did not precipitate additional toxin binding sites [3g]. These results are consistent with the assumption that both psoteins are subunits of the same hetero-oligomeric receptor complex with ALS as ligand binding subuuit and ARD protein as a structural subunit. Preliminary,' ceports on the co-expression of the two subt~!iL~ i~, ~le,~lopv,s ooeytes suppor~ this notion (discussed in Rcf. 38). The iujcctiun of a combination of synthetic ARD and ALS transcripts, but neither ARD nor ALS synthetic RNA alone, produced nicotineevoked currents in the frog oocyte. Similarly immunohistochemical studies have indicated that antigens recognized by several antisera against ALS and ARD proteins are co.localized in many netlropil regions of the Drosophila CNS (Schuster etal., unpublished data). Over wide regions ARD and ALS immunoreactivity is co-distributed with aBtx binding to brain sections as obsezwed by autorediogr;phy or histechemic-al studies [26,29,34,35]. However, from the present data it remains unclear whether class 1 receptors of Drosophila. like vertebrate neuronal nAChRs [20-22], consist of merely two different subunits, i.e., the ARD and ALS proteins, or whether one or more additional subunits contribute to the native receptor complexes, as is the case in the vertebrate muscle. Injection of RNA synthesized frctm the SAD eDNA into Xenap:f~" c,oc,ytes generated nicotine.regulated cation channels showing in principle that the Dtz2/SAD protein is a subunit of Drosophila nAChRs [56]. However, the extremely high nicotine concentration required to operate the channel (half-maximal response at 10 to 15 mM nicotine - usually nicotine concentrations in the micromolar range are sufficient to elicit half-maximal responses [19]) and the failure of nicotinic antagonizt~, such as D-tubocurarine or ~Btx, to block the observed nicotine-evoked currents indicate that a rather unphysiological receptor is formed in the ooeyte. One possible reason for this may be the lack of complementary subeoits [56]. From the locust Schi~toeerce gregaria a eDNA encoding a nAChR suhunit ( a L l ) has been cloned, and is vepy similar to

3O6 D a 2 / S A D [44]. In particular within the N-terminal part, including the large oxtracellular domain and the first three hydrophobic segments the two proteins are more than 9 5 ~ identical [12]- lnterestingly, the a l l snbunit behaves differently from D a 2 / S A D upon expression in Xenolms o0cytes. It forms cation channels that are gated by mieroatolar amounts of nicotine and that are blocked by aBtx, ~Btx, stpychnine and bledcollins and thus "¢e~ much resemble insect neuronal nAChRs recorded in situ [44]- For a L l receptors relatively small inward ¢u~cnts indicate that the synthesis, the assembly or the transport of the subunit to the cell sudacc may be not very efficient. Possibly the co-expression of an appropriate non-a-subunit would increase the amount of functional receptor reaching the C¢11surface. Although the described studies are far from being complete and in no ease has the complete quaternary structure of a Drosophila nAChR been established, the available data seem to confirm that the proteins encoded by the cloned genes are subunits of functional nAChRs.

IV-C. Temporal and spatial expression of m4ChR genes On Northern blots the transcripts from the genes encoding ALS. Do?.. ARD and SBD are detectable at all developmental stages of Drosophila starting from mid-aged embryos through adult flies [53,55-57.59,60}. Levels of ARD, Da2 and SBD transcripts are developmentally regulated. They are particularly high during late embr~ogenesis, in late pupae and in ~,oung adults and they significantly decrease during larval life and in aging flies. On the other hand the level of ALS transcript stays elevated in larvae [53]- The periods of high expression of the former three transcripts correlate well with periods of terminal neuronal differentiation and ~napse formation in the CNS [71,72]in late Drosophila embryos transcripts from all four anal~ed nAChR genes are expressed throughout the CNS [5b,59,6033], but there is no evidence for expression outside of the nervous system. Comprehensive in situ hybridization studies have only been performed for ARD transcripts [59,73] (see also Ref. II. Transcripts were first detected in neuronal cell bodies of 10- to I2-h embryos, i.e., during condensation of the ventral cord. Transcript levels in all parts of the CNS significantly increase during late embryogenesis dad then decrease to a relatively low level in larvae [59,73]. During metamorphosis in the pupa, A R D transcript levels rise again both in the cortical t~gions of the brain and in the thoracic ganglion. It is noteworthy thai the expression of ARD in the optic lobes appears to follow a different time schedule than in other parts of the CNS [39] (Wadsworth cited in Ref. 1). In the brain of newly eclosed arcs ARD transcripts are highly ex-

prcssed in all major part~ of the CNS except for the lamina of the optic lol~s where no or only very weak hybridization signals were observed [59,73], A t all developmental stages, i/l all hybridizing brain regions, neurons expressing ARD transcripts are intermingled with unla~ied neurons. Moreover, neurons expressing ARD do so to various degrees. In addition to the mature 3-kb transcript two classes of intron-containing ARD transcripts accumulate in late pupae and adult flies [66,73]. One class appears to represent unspliced transcripts, the second contains only the third and the fourth introns of the ard gent. In situ hybridization with an intron probe indicate that the partially or uaspliced transcripts accumulat~ in all ARD-expressing regions [73]- The functional significance Gf any) of these immature transcripts is comp|etely obscure. However, a high abundance of intron* containing transcripts has also been observed in various other cases including, for example, transcripts from the pard gene which encodes a voltage-gated sodium channel [74], the s/o gene encoding a component of calcium-activated potassium channels [75] and Da3 transcripts (Baumann and Gundeiflnger, unpublished observation). Regulation of gene expression at the level of splicing as reported for genes like suppressor-ofwhite-apricot, the P-clement transpo~ase and the tranSformer gene (for review see Rcf. 76) may provide an explanation for this phenomenon. V, Genetic apprtmehes Initial screens for nAChR mutants cmpioycd a protocol to identify nicotine-resistant flies [77]. A set of eight strains with increased resistance to nicotine hydrogen tartrare have been isolated. In three of these strains a chaldcteristie shift of the [soelectrle point of an ~Btx-hinding component has been observed. At least in the Hikon-R strain both the ahered p l of the aBtx-binding component and the nicotine resistance are linked to the X chromosome [36] (see also Ref. I). Unfortunately, there is as yet no further information about the nature of the affected gone. It will be interesting to see whether the Hikon-R mutation affects the Drt3 gene, which to date is the only identified nAChR gene that maps to the X chromosome at the 7E region (Mfilhatdt et al., unpublished observation). The availability of cloned genes offers new wa~s to create receptor mutants. Several laboratories have started to generate mutants by P-element mobilization (traasFosition). Thus P-elements inserted within, or close t,~s one of these nAChR genes can be detected by PeR technology [78-80]- Furthermore, the introduction of mutagenizad subunits into the fly, which may become assembled together with wild type subunits into inactive oligomeric receptor complexes may help to elucidate their function. A similar apprthaeh has been carried out

307 successfully for a n oligomerie potassium channel encoded i~ the shn~r complex [81]. VL C o n e l u d | n g r e m a r k s Both b i n d i n ~ sites for nicotinic ¢holinergi¢ ligands and transcripts for n A C h R subunils arc widely distributed throughout t h e Dro.vophila CNS. The d a t a are consistent wilh a pivotal role o f nicotinic :eceptors in excitatory neuroWaasmission in t h e insect nervous s y s tem. Like t h e i r vertebrate counterparts, insect , A C h R s

display a substantial diversity, which becomes apparent a t t h e pharmacological a n d physiological level as well as by the number of different genes encoding n A C h R proteins. However, both the final n u m t ~ r of n A C h R genes remains to b e determined and their assembly into the various receptor subtypes is Io be studied. So far we only have evidence t h a t two subunits, A L S and A R D , are p a r t o f the s a m e hetero-oligomcric I¢CCptor complex which constitutes one of the major n A C h R subtypes. It will b e interesting t o discover the fanctioas o f the various n A C h R subtypes in the insect nervous system. In particular the po~verful genetics o f the fruit fly system should help t o address these issues in the n e a r future, Aclmowh:dgemcots W e wish to t h a n k R J . Harvc.y, P. Jonas, and IL Sehnsler for t h e i r valuable c o m m e n l s on (he manuscript and M . Vkqgron for the provision of the va~¢as iu T a b l e I L T h e work in o u r laboratory is funded by the Bundesministerium £dr Forschung und Technologie and t h e Deutsche Forschungsgemeinschaf( (SFB 232). References I Resti[o, L L and White, K. (IbMI) Adv. lasc,:l Physiology. 22. 115-219. i 2 Buchner. ~, (IC-~ll J. Neu[ogenet. 7, 153-1o2. * 3 Greenspan, RJ. (19~) J. Comp. Physiol. 137,143-92. 4 Hall, J.C. and Ka~kL D.R. (1976) Genelics 83,517-535. Gerschenfeld, H.M. (1973) Ph~sml. Ray. ~3, f-119. ;I Buchner, E.. Bochner, S., C~wford. G., Mason. W.T.. Sal~atcrra, P.M. and S~ttelle, D.H. (198s5)Cell Tissue Res. 246,57-62. 7 Wu, C.-F-, Brrnekit~. J.M.a.rld Iladker, DE.. (1983) J, Neurochem. 40, 13~,-13~5, g Wu, C.-F.. :Suzuki. M. and Poe, M.-M. (1983) J. Neunv~i. 3, 1~1899. 9 Buchr~Jr, ~. and Rodrigues. V. 11983) Neu~c~.~ci.LetL 42, 25-31. 11) Dudai, Y. 0979) Tr~:nds Bioehem. Set. 4. ~1-44, II Salva;erra. P+M. and Faders, R.M. 119791J. Neumchem. 32, 1:5~9-1517. 12 ~3undclfinger. E.D. (1992) Tirends N c u ~ L 15, 2U6-21I. 13 OaaL T.. FitzGcra~a, M.G., Arakawa, S., G~zayl,~ I.D.+ Urquhan, D,~+ Hall+LA,+ Fraser, C,M., McCombie, W.R. and Ven,er, J.C. (1989) FEBS Lea. 255. 219-225. 14 Shapiro, R.A.. Waklmoto. B.T., Sober. ,~.M. amd Nathanyo.. N.M. 119891prec. Natl. Aead. Sd. LISASS. 9039-9943.

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