The EMBO Journal vol.9 no.9 pp.2671 -2677, 1990

Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a novel developmentally regulated a-subunit Erich Sawruk, Patrick Schloss, Heinrich Betz' and Bertram Schmitt' ZMBH, Universitait Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg and 'Max-Planck-Institut fur Himforschung, Deutschordenstrasse 46, D-6000 Frankfurt 71, FRG

Communicated by H.Betz

Two genes, ard and als, are known to encode subunits of the nicotinic acetylcholine receptor (nAChR) in Drosophila. Here we describe the isolation of cDNA clones encoding a novel member (SAD, or a2) of this receptor protein family. The deduced amino acid sequence displays high homology to the ALS protein and shares structural features with ligand binding nAChR a-subunits. Sad transcripts accumulate during major periods of neuronal differentiation and, in embryos, are localized in the central nervous system. Expression of SAD cRNA in Xenopus oocytes generates cation channels that are gated by nicotine. These data indicate heterogeneity of nAChRs in Drosophila. Key words: developmental regulation/Drosophila melanogaster/nicotinic acetylcholine receptor/receptor heterogeneity

Introduction Rapid synaptic signal transmission involves the activation of ligand-gated ion channels, which open upon neurotransmitter binding. Determination of the primary structures of excitatory nicotinic acetylcholine receptor (nAChR) (Noda et al., 1982; Boulter et al., 1986, 1990; Goldmann et al., 1987; Deneris et al., 1988; Nef et al., 1988) and inhibitory -y-aminobutyric acid (Schofield et al., 1987; Levitan et al., 1988) and glycine (Grenningloh et al., 1987, 1990) receptor subunits by cDNA cloning disclosed the existence of a large superfamily of genes encoding these membrane proteins. Common features of its members include a conserved transmembrane topology with four membrane spanning segments and a large N-terminal extracellular domain characterized by two cysteine residues spaced 14 amino acids apart. The recent determination of the primary structure of an excitatory glutamate receptor subunit of the kainate type (Hollmann et al., 1989) revealed similar structural features, however, only a low homology to other ligand-gated ion channel proteins. In vertebrates, nAChRs mediating neuromuscular transmission have been characterized extensively (for reviews, see Changeux et al., 1984, 1987; Stroud and Finer-Moore, 1985). These pentameric receptors contain two ligand binding a-subunits together with three other homologous polypeptides (subunit composition a21 b). The neuromuscular nAChRs are readily blocked by a-bungarotoxin (a-Btx), a basic snake venom protein. Related nAChRs are also found on neurons in different regions of the vertebrate Oxford University Press

nervous system. Here, however, a-Btx does not antagonize cholinergic responses although high-affinity binding sites for radioiodinated a-Btx are found on the plasma membrane of many neurons. Biochemical studies and expression of cloned DNAs indicate that these neuronal nAChRs are formed from only two types of subunit, a and : (Boulter et al., 1986, 1990; Whiting and Lindstrom, 1987; Ballivet et al., 1988; Deneris et al., 1988; Wada et al., 1988; Bertrand et al., 1990). Both exist in several variant forms (a2-a5; (32-fl4) in birds and rodents. In invertebrates including insects, cholinergic synapses are abundant in the central nervous system (CNS), whereas neuromuscular transmission is mediated by glutamate (Gration et al., 1981). Insect neuronal nAChRs are blocked by a-Btx (for a review see Breer and Satelle, 1987), and high levels of c-Btx binding sites are found throughout the ganglionic CNS (Dudai and Amsterdam, 1977; Sanes et al., 1977; Schmidt-Nielsen et al., 1977; Rudloff, 1978; Satelle, 1980; Breer, 1981). Purification of an a-Btx binding protein from locust ganglia (Breer et al., 1985) resulted in isolation of an apparently homo-oligomeric receptor composed of subunits of mol. wt 65 kd. Reconstitution of the purified protein in planar lipid bilayers generated functional acetylcholine-gated sodium channels, suggesting that the insect nAChR may correspond to an ancestral receptor composed of ligand-binding a-subunits only (Hanke and Breer, 1986). Cloning of nAChR homologues from Drosophila melanogaster identified cDNAs encoding a putative structural subunit, ARD (Hermans-Borgmeyer et al., 1986; Wadsworth et al., 1988), and an a-like subunit, ALS (Bossy et al., 1988). Both proteins show higher homology to vertebrate neuronal than to muscular nAChR polypeptides and are components of a high-affinity a-Btx binding site present in the head of fruitflies (Schloss et al., 1988; P.Schloss, H.Betz, C.Shroder and E.Gundelfinger, in preparation). Here, we describe the primary structure of a novel nAChR a-subunit (SAD, or a2) of D.melanogaster which shows remarkable homology to the ALS protein. The corresponding mRNA accumulates in the CNS during periods of neuronal differentiation in developing fruitflies. Furthermore, we demonstrate expression of nAChR-like channels in Xenopus oocytes after injection of SAD cRNA. Our data indicate that nAChRs in Drosophila are heterogeneous and likely to contain different types of subunits.

Results Isolation of SAD cDNA clones A stretch of ten amino acids preceding the fourth transmembrane segment is highly conserved in all neuronal nAChR subunits analysed currently. We used this conserved sequence to identify novel nAChR cDNAs in Drosophila. Screening of a Drosophila genomic library with an oligonucleotide mixture corresponding to this 'pre M4' region,

2671

E.Sawruk et al.

deduced from ARD and ALS cDNA sequences (see Materials and methods), resulted in the isolation of many hybridizing phage. Positive clones were verified further by hybridization to cDNA probes of ARD and ALS. The amino acid sequence deduced from the partial nucleotide sequence of one of the isolated genomic clones revealed significant homology to the fourth transmembrane segment of known nAChR polypeptides. This clone was therefore used to screen a lambda-ZAP cDNA library, prepared from late Drosophila embryos, under conditions of medium stringency. In addition to ARD and ALS specific cDNAs, which were identified by high stringency hybridization to corresponding cDNA 111

a

111

0.1

111

probes, two novel cDNA clones were isolated which encoded putative nAChR-related proteins. Neither of these clones, however, represented the cDNA equivalent of the genomic clone isolated originally. This and the second cDNA are still under investigation in our laboratory. Here we describe the analysis of cDNAs which code for a novel second alphalike subunit of a Drosophila neuronal nAChR (SAD). Overlapping clones covering the entire coding sequence for the SAD protein were isolated by rescreening the embryonic cDNA library with the parent clone, designated p37 (Figure la). Clone p37 contains the first 1391 nucleotides; clones p37-15 and p37-20 extend the sequence,.but differ 41.5

1.0

0.5

D

kb

RD D

R REV

R

2.0

II I

'LI _M1i41V" M2 M3 M4

SP

-

SAD cDNA

p37 p37-15

p37-20

b 1 TCAGTTAACAAGTTTGAATATTTTTTAGAATTTTTTAAGCACGAAATTGAGTTGGTGAAAATTAAAAAGACTTTTTAAATAAGTTAAGGCGGCCGGAGCAAAAACAAAATCAGCTGCCTG 121 GGCGCAGCGACTTCAGCTGCTCAAGCAGTGCGCCGAATCGGGAAACAGTTAGCAGCAGCGAGACTTGGAGACTTGTAAACACTTTTAGGCCATGTGCTAAAAGCCAGTTACTCGCTCTGC

241 CCCACGAACTGCGTCAGAGATTGAGCATCAAGGGGCAGCCAAGCACCCAAGGCGGGCAGAAAGCGTCAAGGTCCTTTTTATTTCTTTCCGCCGTGACGTCACCATGGCTCCTGGCTGCTG -41

M

A P

G

C

C

361 CACCACACGACCCCGCCCCATCGCCCTGCTCGCTCACATCTGGCGCCACTGCAAGCCACTCTGCCTGCTCCTGGTGCTCCTGCTCCTCTGCGAAACCGTTCAGGCGAATCCCGATGCCAA -35 T T R P R P I A L L A H I W R H C K P L C L L L V L L L L C E T V 0 A N P D A K 481

GCGACTCTATGACGATCTGCTAAGCAACTACAATCGCCTCATCCGCCCTGTGAGCAATAATACGGACACGGTGTTGGTCAAATTGGGCCTACGGCTCTCCCAACTCATCGATTTGAATCT

6

R

L

Y

D

D

L

L

S

N

Y

N R

L

I

R

P

V

S

[

N

T

D

T

V

L

V

K

L

G

L

R

L

S Q L

I

D

L

N

L

601 CAAAGATCAAATTCTAACGACCAACGTGTGGCTGGAGCACGAGTGGCAGGATCATAAATTCAAGTGGGATCCCTCGGAGTATGGCGGCGTCACAGAGCTTTATGTGCCATCCGAGCACAT 46 K D Q I L T T N V W L E H E W Q D H K F K W D P S E Y G G V T E L Y V P S E H I

721 CTGGCTGCCCGACATCGTGCTCTACAACAATGCCGATGGCGAGTACGTGGTCACCACCATGACGAAGGCCATCCTCCACTATACCGGCAAAGTGGTCTGGACTCCGCCGGCCATCTTCAA 86 W L P D I V L Y N N A D G E Y V V T T M T K A I L H Y T G K V V W T P P A I F K 841 GTCCAGCTGTGAGATTGATGTGCGCTACTTTCCCTTCGATCAGCAGACCT9CTTCATGAAGTTCGGCTCGTGGACCTACGACGGTGATCAGATCGATTTGAAGCACATCAGCCAGAAAAA 126 S S @ E I D V R Y F P F D Q Q T % F M K F G S W T Y D G D Q I D L K H I S Q K N A 961 CGACAAGGACAACAAGGTGGAGATAGGCATTGACCTGCGTGAGTACTATCCCAGTGTGGAGTGGGACATTCTCGGCGTTCCGGCTGAGCGGCACGAGAAGTACTATCCCTGCTGTGCCGA 166 D K D N K V E I G I D L R E Y Y P S V E N D I L G V P A E R H E K Y Y P @ A E I 1081 ACCGTATCCGGATATCTTCTTCAACATCACCCTGAGGCGAAAAACTCTCTTCTACACGGTTAACCTGATTATTCCATGTGTGGGCATCTCGTATCTTTCGGTGCTGGTCTTTTACCTGCC 206 I T L R R K T L F Y T V N L I I P C V G I S Y L S V L V F Y L P P Y P D I F F 1201 CGCCCGATTCTGGCGAGAAGATTGCTCTGTGCATCAGCATCCTGCTGTCGCAAACCATGTTCTTCCTGCTCATATCGGAGATTATACCATCGACTTCACTGGCATTGCCGCTACTGGGAAA 246 A D S G E K I A L C I S I L L S Q T M F F L L I S E I I P S T S L A L P L GK

[M

1321 ATATrCTACTGTTCACCATGTTGCTGGTTGGCCTGAGTGTTGTCATCACGATTATCATACTAAACATACACTACCGGAAGCCGAGTACGCACAAGATGCGACCCTGGATTAGGTCCTTCTT 286 Y L L F T M L L V G L S V V I T I I I L N I H Y R K P S T H K M R P W I R S F F 1-

I

-

-

..

.

..

-

.

-

..

..

-

-

-

-

-

-

1441 CATC.AAGAGATTGCCGAAACTCCTGCTGATGCGTGTGCCCAAGGACCTGCTGCGTGACCTGGCGGCCAACAAGATCAACTACGGCCTCrAAGTTCAGCAAGACCAUAGTnTCGGACAGGCGCT 326 I K R L P K L L L M R V P K D L L R D L A A N K I N Y G L K F S K T K F G Q A L

1561 GATG;GACGAGATGCAAATGAACTCGGGCGGCTCCAGTCCAGACTCCCTGCGGCGGATGCAAGGTCGTGTGGGTGCTGGTGGGTGCAATGGCATGCACGTGACCACGGCCACAAACAaAlrlr 366 M D E M Q M N S G G S S P D S L R R M Q G R V G A G G C N G M H V T T A T N R F 1681 CAGC:GGCTTGGTGGGGGCTTTGGGCGGCGGACTGAGCACCCTGAGCGGCTACAACGGACTGCCATCGGTGCTATCCGGATTGGACGACTCTTTGAGCGA7G7GGCCaCAr-arAAIRRnMI 406 S G L V G A L G G G L S T L S G Y N G L P S V L S G L D D S L S D V A A R K K Y 1801 TCCTrTTCGAGCTGGAAAAGGCCATCCACAACGTCATGTTCATACAGCACCACATGCAGCGGCAGGACGAGTTCAATGCGGAAGATCAGGACTGGGGCTTTGTGGCCATGGTCATGGATCG I 446 P F EL L E K A I H N VM F I VH H M Q R Q D E F N A E D D W G F V A M V M D R

1921 CCTAkTTCCTCTGGCTCTTTATGATCGCATCCTTGGTGGGCACATTTGTGATCCTAGGCGAGGCTCCGTCGCTATATGACGATACCAAGGCCATTGATGTTCAGCTATCCGATGTTGCCAA 486 L F L W L F M I A S L V G T F V I L G E A P S L Y D D T K A I D V Q L S D V A K 2041 526

GCAAATCTACAATCTAACCGAGAAGAAGAATTAAATTCATGAGAACGTAGGCTTAAGACAATTTTCCTAGTTTAATGCAGTGTTTCAGCAAATTGATGCGAATAAAAATTAAAAATACGC Q I Y S L T E K K N *

2161

TATAATAAAAACGTTGGCCCCAAAAGCAGCCAAGCAAAATGCTGAAATGCAAAAAAAAAAZAAAAA

Fig. 1. (a) Restriction map of the SAD cDNA and localization of cDNA clones p37, p37-15 and p37-20. The coding region is indicated by an open box. Transmembrane regions and restriction sites are shown. D, DraJI; R, RsaI; EV, EcoRV; MI -M4, transmembrane regions; SP, signal peptide. (b) Nucleotide and deduced amino acid sequence of the SAD cDNA. The predicted signal peptide, the bilayer-spanning regions MI -M4 and two potential polyadenylation signals are underlined. A putative amphiphilic helix extends from amino acid 449 to amino acid 466. Conserved cysteine residues are circled and possible N-linked glycosylation sites are boxed. A potential c-AMP/cGMP-dependent protein kinase phosphorylation site is found at position 313.

2672

Drosophila nAChR heterogeneity

slightly at their 5'- and 3'-ends. Clone p37-15 starts at nucleotide 978 and ends with a stretch of 17 adenosines at nucleotide 2227; p37-20 is very similar in size but lacks two 5' nucleotides and the short poly(A) tail seen in p37-15. The SAD protein shows significant homology to neuronal nAChR a-subunits Clones p37, p37-20 and p37-15 specify an open reading frame of 576 codons enclosed by 5'- and 3'-untranslated sequences with a stretch of several A residues at the 3'-end (Figure Ib). Two possible polyadenylation signal sequences are found in the 3'-untranslated region. The surroundings of the assigned start codon at position 344 of the sequenced cDNA fulfil the criteria of a eukaryotic translation initiation site (Kozak, 1986; Lutcke et al., 1987). An unusually long signal peptide (41 amino acid residues) is found at the N-terminus of the deduced protein sequence. After signal peptide cleavage, the presumptive mature SAD protein contains 535 amino acids with a calculated relative molecular mass of 60 955. Three possible N-linked glycosylation sites are located at positions 24, 213 and 529 of the mature SAD protein. Clone p37-15 contains an A at position 1037 instead of the G residue found in p37 and p37-20, resulting in an isofunctional amino acid exchange. We attribute this difference to allelic variation. Comparison of the deduced SAD sequence with known nAChR proteins reveals a high homology to neuronal a-subunits. An alignment of SAD with the Drosophila ALS and ARD proteins and neuronal (a2) and muscular (al) nAChR subunits from chicken is shown in Figure 2. Two 1 1 1 1 1

tandem cysteine residues in positions 202 and 203 located in close proximity to the agonist binding site of nAChRs (Mishina et al., 1985; Neumann et al., 1986; Kao and Karlin, 1986) characterize SAD as a putative a-subunit. The SAD and ALS proteins may therefore be classified as Drosophila a2 and al nAChR subunits. Furthermore, a remarkable similarity is found between both polypeptides, including stretches of identical amino acid sequences and the location of two putative N-linked glycosylation sites (compare Figures lb and 2). Calculated overall amino acid sequence identities of SAD to ALS are 56%, to ARD 39%, to the chick neuronal a2-subunit 41%, and to the chick muscular cal-subunit 35 %. As known from other members of the ligand-gated ion channel superfamily, the putative membrane spanning segments and a N-terminal extracellular domain, characterized by two cysteines separated by 14 amino acids (see Figure 2), are highly conserved. Accordingly, within the first three transmembrane regions (amino acids 217 to 310 in Figure 2) sequence identities of SAD to ALS, ARD, chick a2 and chick cal increase to 81 %, 63%, 68% and 57%, respectively. Expression of the sad gene during Drosphila development To define the appearance and size of sad transcripts, poly(A)+ RNA isolated at different stages of Drosophila development was analysed on Northern blots (Figure 3). No detectable amounts of sad transcript were found at early embryonic stages. A high level of expression, however, was seen during late embryogenesis. At larval stages, no sad

NPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKDQILTTNVWLEHEWDHKFKWDPSEYGGVTELYVPSEHIWLP SAD NPDAKRLYDDLLSNYNRLIRMVGNNSDRLTVKMGtRLSQIWDVNLKNQIMTTNVWVEQEWNDYKLKWNPDDYGGVDTLHVPSEHIWHP ALS SEDEERLVRDLFRGYNKLIRPVQNMTQKVGVRFCLAFV9INVNEKNQVMKSUVWLRLVWYDYQLQWDEADYGGIGVLRLPPDKVWKP ARD

REQKQPHGFAEDRLFKHLFTGYNRWSRWVPNTSDWIVKFGLSIAQZDDVDEKNQ4TTNVWLKQEWSDYKLRWNPEDFDNVTSIRVPSEMIWIP YEHETRLVDDLFREYSVVVHRDAVVVTVGLQLIQglNVDEVNQIVT7UVRLQQWTINLKWNPDDYGGVKQIRIPSDDIWRP

CHICK a2 CHICK al

89 DIVLYNIIDGEYVVTTMTKAILHYTGKVVTIPAIFKSSCEIDVRfDQTCUCSWZYDGDQIDLKHISQKNDKDNKVEIGIDLREYYPS 89 DIVLYlYUODANYEVTIMTKAILHHTGKVVWAIYKFCEIDVEDEQTCFCSWZYDGYMVDLRHLKQTADSDN IEVGIDLQDYYIS 89 DIVLFNDCGNYEVRYKSNVLIYPTGEVLWVPrAXYQSSCTIDVTI'FIDQQTCIFGSWIFNGDQVSL ALYN NKNFVDLSDYWKB 96 DIVLYItThDGEFAVTHMTKAHLFSNGKVKWV11IIYKSSCSXDVTUM1DQNCKNCFGSW YDKAKIDL EN MEHHVDIKDYWES 89 DLVLYNIDGCDFAIVKYTKVLLEHTGKITW?IAIFKSYCEIIVTfMf1DQQNCSaLGT YDGTMVVI NPESDRPDLSNFMES

SAD ALS ARD CHICK a2 CHICK al

184 183 176 181 174

*-0 M2 Ml VEWDILGVPAERHEKYYPCCAE PYPDXFFNITLRRxTLFYTVNLIIPCVGISYLSVLVmWLADSGEIIALCISILLSQTM!4FLLISEI IPSTS VEWDIMRVPAVRNEKFYSCCEE PYLDIVFNLTLRRKTLFYTVNLIIPCVGISFLSVLVV!LISDSCIISLCI8ILLSLTVIFLLLAEI IPPTS GTWDI IEVPAYLNVYEGDSNHPT ETDITFYI I IRRKTLFYTVNLILPTVLISFLCVLVWI.AEAC=VTLGISILLSLVVFLLLVSKILPPTS GEWAI INAIGRYNSKKYDCCTE I YPDITFYFVIRRLPLFYTINLIIPCLLISCLTVLWILSDCC=ITLCISVLLLTVFLLLITEI IPSTS

GEWVMKDYRGWKHWVYYACCPDTPYLDITYHFIMRLPQYFlIVNIPCLLFSFLTGSFLTWLPTDSGEIMTLSISVLLLTVFLLVIVELIPSTS

SAD ALS ARD CHICK a2 CHICK al

M3 278 LALPLLGKYLLFZMLLVGLSVVITI I ILIIHYRWPS RPWIRSFIRIKPKLLLMRVPKDLLRDLAANKINYGIKFSKTKFGQALMDEMQM 277 LTVPLLGKYLXITMMLVTL8VVVTIAVLNVNFRS1VU!IAPWVQRL7IQILIKLLCIERPKKEEPEEDQPPEVLTDVYHLPPDVDKFVNYDSKR 270 LVLPLIAKYLIZFTIMNTVSILVTVI I INWNFRPRUEMYIRSIFH YLPAFLFMKRPRKTRLRWMMEPQGMSMPAHPHPSYGSPAELPKHI 275 LVIPLIGEYLLYZMIFVTLSI IIITFVLNVHHRSSMPHWVRSFWLGFIRWLFMKRPPLLLPAEGTTGQYDPPGTRLSTSRCWLETDVDDK 269 SAVPLIGKYMLTZMVFVIASIIITVIVINTHHRSPSS!3flPWVRKIYIDTIPNIMFFSTMKRPSRDKPDKKIFAEDIDISEISGKQGPVPVNFY

SAD ALS ARD CHICK a2 CHICK al

373 372 365 370 346

KKYPFELEKAIHNVMFX SAD SGGSSPDSLRRMQGRVGAGGCNGMHVTTATNRFSGLVGALGGGLSTLSGYNGLPSVLSGLDDSLSDVAAR FSGDYGIPALPASHRFDLAAAGGISAHCFAEPPLPSSLPLPGADDDLFSPSGLNGDISPGCCPAAAAAAAADLSPTFEKPYAREMEKTIEGSRFI ALS

460 467 432 453 384

QHHMQRQDEFNAEDQDFIWG RLFLWL7MIASLVTFVILGEAPSLYDDTKAIDVQLSDVAKQIYNLTEKKN AQHVKNKDKFESVEEDWKYVMVIDRMFLWIFAIACVVGTALI ILQAPSLHDQSQP IDILYSKIAKKKFELLKMGSENTL AEHLRNEDLYIQTREDWKYVMWIDRLQLYIYFIVTTAGTVGILMDAPHIFEYVDQDRI IEIYRGK ADHLRAEDADFSVKEDWKYVMWIDRIFLWI IVCLIaTVGLFLPPYLAGMI AETMKSDQESSNAADEWIKFVMIDHLLLVIFMLVCI IGTLAVFAGRLIELNQQG

SAIGGKQSKMEVMELSDLHHPNCKINRKVNSGGELGLGDGCRRESESSDS WEEEEEEEEEEEEEEEEEKAYPSRVPSGGSQGTQCHYSCERQAGKASGGPAPQVPLKGEEVGSDQG SP

ILLSPEASKATEAVEFI

LTLSPSILRALEGVQYI LTKNPDVKNAIEGIKYI

ARD CHICK a2 CHICK al

M4 SAD ALS ARD CHICK a2 CHICK al

Fig. 2. Comparison of the SAD protein with the Drosophila a-like sequence ALS (Bossy et al., 1988), the Drosophila non-a-subunit ARD (Hermans-Borgmeyer et al., 1986), the chicken neuronal subunit a2 and the chicken neuromuscular subunit al (Nef et al., 1988). Amino acids identical in all subunits are shown in bold letters. Membrane spanning regions and conserved cysteine residues

to optimize the

are

indicated.

Gaps

were

introduced

alignment. 2673

E.Sawruk et al. EE LE 1L 2L 3L EP LP EA

a

'S

Spg

-9.5

.*.

-7.5 -4.4

0

=2.4 -1.4 .....

vc -Wir

:H::

Ti`

PA

x'..

Fig. 3. Northern blot analysis. The accumulation of sad mRNA during Drosophila development was investigated using poly(A)+ RNA isolated at different developmental stages: EE, early embryo (0-4 h); LE, late embryo (14-22 h); IL, first instar larvae; 2L, second instar larvae; 3L, third instar larvae; EP, early pupae; LP, late pupae; EA, early adult flies (1-2 days). Equal amounts of RNA (5 Ag/lane) were loaded onto the gel and, after transfer, visualized by staining with methylene blue. The 32P-labelled RsaI-EcoRI fragment of clone p37-20 (nucleotides 1405-2210) was used as hybridization probe. The sizes of RNA length standards are given in kb.

mRNA was detected. Expression of sad transcripts resumes at an early post-pupariation stage and increases in late pupae. In early adulthood, only low levels of sad transcripts were found. Throughout development, the size of the sad mRNA was invariant (about 2.6 kb). Localization of sad transcripts in the embryonic CNS As Northern blot analysis had revealed high levels of sad mRNA in the late embryo, we investigated its regional distribution by in situ hybridization on whole mount embryo preparations using a non-radioactively labelled probe (Tautz and Pfeifle, 1989). Control Southern blot experiments confirmed that our digoxigenin-labelled SAD cDNA probe did not cross hybridize to ARD and ALS cDNAs under similar conditions of stringency as used for the whole mount in situ hybridization (data not shown). As demonstrated in Figure 4a, staining with this SAD probe was restricted to the ventral cord and the sub- and supraoesophageal ganglia, which all are parts of the embryonic CNS. No specific staining outside these regions was observed; thus, expression of the sad gene seems to be restricted to nervous tissue in the embryo. Control hybridizations with a vector probe did not result in staining of the embryonic CNS (Figure 4b). Expression of SAD cRNA in Xenopus oocytes Co-expression of chicken and rat neuronal and fl-subunits generates functional nAChRs in the Xenopus oocyte expression system (Boulter et al., 1987; Ballivet et al., 1988; Deneris et al., 1988; Duvoisin et al., 1989; Bertrand et al., 1990). Therefore, in vitro synthesized SAD RNA was injected into oocytes, which 4-6 days later were analysed for agonist-elicited membrane currents in a voltage clamp set-up. At negative holding potentials, high concentrations of acetylcholine (in presence or absence of 10-4 M eserine) produced small inward currents of 10-50 nA (not shown). In some oocytes, these responses were followed by prolonged current fluctuations which were sensitive to atropine and reflect endogenous muscarinic acetylcholine receptor activation (Carpenter et al., 1990). The selective agonist a-

2674

b Spg I

sbg YC

Fig. 4. CNS-specific expression of sad mRNA in Drosophila embryos. Digoxigenin-labelled RsaI-EcoRI fragment of clone p37-20 (nucleotides 1405-2210) (a), or digoxigenin-labelled phagemid DNA (bluescript SK) (b), was hybridized to whole embryos as detailed in Materials and methods. spg, supraoesophageal ganglia; sbg, suboesophageal ganglia; vc, ventral cord. Note specific staining of the embyronic CNS in (a).

nicotine produced much larger inward currents in about 62% of all injected oocytes tested (n = 29; see Figure 5, inset). However, also here high agonist concentrations were required for current gating, with half-maximal responses seen at 10-15 mM agonist (Figure 5). Nicotine-elicited inward currents reversed at positive membrane potentials (about 30-60 mV), indicating cation-selective channels (not shown). No such response to nicotine (10 mM) was detected in control oocytes (see Materials and methods) or in oocytes injected with ARD cRNA. Attempts to pharmacologically characterize the nicotine response in oocytes injected with SAD cRNA proved unsuccessful; both d-tubocurarine (100 jtM) and a-bungarotoxin (10-6 M) had no major effect on the currents seen. We attribute this failure of current inhibition to the almost instantaneous dissociation of antagonists known to occur in an excess of competing ligand

(Wang et al., 1978). As structural fl-subunits

are essential for generating functional vertebrate neuronal nAChR in oocytes, we also performed co-injection of SAD and ARD cRNAs. This caused, however, no significant change of the responses seen. Therefore, other subunits may in addition be required to form a Drosophila nAChR of high agonist affinity.

Drosophila nAChR heterogeneity

200n

c

c *c

LM

20mM

Nlcon

--

Io 1

min

100

qo U

O1

10

.....,.0 100

Nicotine [mMJ Fig. 5. Nicotine-evoked membrane currents in oocytes injected with SAD cRNA. All measurements were done at a holding potential of -70 mV; the data are plotted in semilogarithmic coordinates. This experiment was repeated three times on different oocytes with reproducible results. The inset shows current responses to 2 and 20 mM nicotine. Perfusion times are indicated by bars.

Discussion The existence of two different nAChR subunits in Drosophila, the alpha-like-subunit ALS and the non-a subunit ARD, has been reported previously (Hermans-Borgmeyer et al., 1986; Wadsworth et al., 1988; Bossy et al., 1988). Here we describe the identification of cDNA clones encoding a third nAChR subunit in this organism, which we named SAD (second alpha-subunit of Drosophila). The deduced amino acid sequence of SAD shows high homology to other neuronal nAChR proteins, with a remarkable similarity to the ALS polypeptide. Furthermore, the SAD protein shares typical features with other members of the ligand-gated ion channel superfamily; this includes four membrane spanning domains and the two conserved extracellular cysteine residues. Like all nAChR subunits, the SAD polypeptide also contains a putative amphipathic helix preceding the fourth transmembrane region which is not seen in other members of the superfamily, e.g. the glycine and -y-aminobutyric acid receptor proteins (Grenningloh et al., 1987; Schofield et al., 1987). Two tandem cysteine residues in position 202/203 of the presumptive mature polypeptide classify SAD as a bona fide a-type subunit. Thus at least two a-subunit variants (a 1 = ALS, and a2 = SAD) exist in Drosophila. Northern blot analysis revealed that sad gene expression coincides with major periods of neural development during the life cycle of the fly. In the late embryo, i.e. when the CNS is formed (see Campos-Ortega and Hartenstein, 1985), high levels of sad transcripts are found. In larvae, no sad mRNA is detected. This is consistent with cells in the CNS being little affected by the histolysis characterizing larval development (White and Kankel, 1978). In the late pupa, sad transcript levels again are high, coinciding with formation of the adult fly brain during this period. Similar patterns of expression during development have been reported for ard (Hermans-Borgmeyer et al., 1986) and als genes, except that high levels of als mRNA are also seen in second instar larvae (Bossy et al., 1988). For both sad

and ard transcripts, in situ hybridization revealed their presence in cells of the embryonic and/or adult CNS (Wadsworth et al., 1988; Hermans-Borgmeyer et al., 1989; and this paper). In other words, these gene products constitute neural markers that may be employed in future studies on Drosophila brain differentiation. Our expression studies in Xenopus oocytes showed that the SAD polypeptide is principally capable of forming a nicotine-gated ion channel. The pharmacology of the expressed SAD receptors was, however, atypical in that channel gating required rather high agonist concentrations. This prevented a detailed analysis of the channels formed and probably reflects the lack of complementary subunits. In support of this view, vertebrate neuronal ca-subunits form functional nAChRs in the Xenopus oocyte system only upon co-expression of a structural (3-type subunit. In case of the rat a4 polypeptide, a modest response to acetylcholine has been observed after single subunit expression (Boulter et al., 1987). This is reminiscent of our results and argues for SAD in vivo being co-assembled with structural subunits. Coexpression of ARD cRNA, however, did not detectably affect the characteristics of the nicotine-evoked currents. Thus, the ARD protein either is not the adequate structural subunit, or additional subunits are needed to constitute a physiological insect nAChR. In favour of the former interpretation, ARD and ALS proteins have recently been shown to be components of a single a-Btx-binding Drosophila receptor (Schloss,P., Betz,H., Schr6der,C. and Gundelfinger,E., in preparation). Thus, other Drosophila non-cx nAChR subunits may co-exist with the SAD protein, a notion that is consistent with preliminary cDNA cloning data from our laboratory. In conclusion, the identification of the SAD cDNA indicates unexpected diversity of neuronal nAChR in invertebrates. Furthermore, the present and previous data strongly suggest insect nAChRs to contain more than one type of subunit. This conclusion contrasts with earlier data where an affinity-purified nAChR protein from locust, containing only a single polypeptide species, had been shown to form a functional ligand-gated cation channel upon reconstitution in planar lipid bilayers (Hanke and Breer, 1986). However, as there is a considerable evolutionary distance between holometabolic insects like Drosophila and hemimetabolic insects which include the grasshopper and the locust, nAChRs in the latter might indeed be simpler versions of this ligand-gated ion channel protein.

Materials and methods Isolation of genomic and cDNA clones A Drosophila genomic library (Maniatis et al., 1978) was screened with a 32P-labelled consensus oligonucleotide mixture 5'-GGTACCATGGCC/AACG/ATACTTCCAA/GTC-3', corresponding to a highly conserved region preceding the fourth transmembrane segment of ARD (HermansBorgmeyer et al., 1986) and ALS (Bossy et al., 1988). Hybridization conditions were 5 x SET (750 mM NaCI, 5 mM EDTA, 150 mM Tris-HCI, pH 8.0), 0.1% (w/v) SDS, 0. 1% (w/v) sodium pyrophosphate, 10 x Denhardt's solution, 100 ytg/ml denatured herring sperm DNA, at 42°C for 15 h. Filters were washed three times for 15 min, each, with 2 x SET, 0.1% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate. Positive clones were hybridized to ARD and ALS cDNAs under conditions of high stringency to identify corresponding genomic sequences. The remaining phage were purified and the inserts analysed on Southern blots. Some clones cross hybridized with ARD and ALS probes under hybridization conditions of moderate stringency (hybridization solution as above, temperature 55°C). EcoRI fragments hybridizing with the consensus oligonucleotide were

2675

E.Sawruk et al. subcloned into pSPT18 and partially sequenced (Sanger et al., 1977). Using this strategy, we identified one DNA with significant sequence homology to the fourth transmembrane region of nAChR subunits. Homology ends at a position where ard and als genes possess intervening sequences (Sawruk et al., 1988; Bossy et al., 1988; Wadsworth et al., 1988). Southern hybridization with 5'-sequences of ALS cDNA indicated the presence of additional coding sequence within this 13.6 kb genomic DNA. Screening of an embryonic lambda-ZAP cDNA library with the genomic clone at moderate stringency (see above) resulted in the isolation of several hybridizing cDNAs. By sequencing we identified a clone, p37 (see Figure la), and another phage which encodes parts of a novel nAChR subunit. Overlapping cDNAs were isolated by rescreening the same library with clone p37 at high stringency. The isolated phage were purified and insert containing Bluescript phagemids obtained by excision according to the manufacturer's protocol (Stratagene). RNA isolation and Northern blot analysis Drosophila at different developmental stages were homogenized in the presence of 4 M guanidinium isothiocyanate solution (Chirgwin et al., 1979), and RNA was isolated according to Cathala et al. (1983). Poly(A)+ RNA preparations (5 pg/lane) were separated electrophoretically on 1 % (w/v) agarose-formaldehyde gels, transferred onto Hybond-N membranes, stained with methylene blue solution [0.04% (w/v) methylene blue, 0.5 M sodium acetate, pH 5.2] and probed with a 32P-labelled RsaI-EcoRI fragment of clone p37-20 (nucleotides 1405-2210; the EcoRI site corresponds to the cDNA cloning site). Hybridization was performed in 5 x SET, 0.1% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate, 50% (v/v) formamide, 200 jig/ml denatured herring sperm DNA, at 42°C for 15 h. The membrane was washed three times for 20 min at 62°C in solutions of decreasing salt concentrations (2 x SET, 1 x SET, 0.1 x SET, with 0.1% (w/v) SDS and 0. 1% (w/v)

sodium pyrophosphate, each). In situ hybridization

Sad transcripts were localized by whole mount in situ hybridization with a nonradioactive probe as described by Tautz and Pfeifle (1989). The RsaI-EcoRI fragment (nucleotides 1405-2210) of clone p37-20 was labelled with a digoxigenin labelling kit according to the manufacturer's protocol (Boehringer, Mannheim, FRG).

Construction of SAD full length cDNAs and in vitro RNA synthesis The cDNA clones p37, p37-15 and p37-20 were used to construct different full length clones (FLCs). Clone p37 was digested with DraH and religated to delete the first 310 nucleotides of the 5'-untranslated region. This clone, named p37D, was used to substitute the nucleotides downstream from the EcoRV site with corresponding fragments of p37-15 (nucleotides 1093-2227) and p37-20 (nucleotides 1093-2210), resulting in clones FLC6 and FLC14. Substitution of the same nucleotides in the original clone p37 with the corresponding fragment of clone p37-20 yielded FLC26. In vitro synthesis of capped RNA was carried out using a transcription kit according to the manufacturer's instructions (Stratagene). Aliquots of RNA solutions (0.3 ug/ld) were stored at -70°C. No difference in functional expression was seen

with cRNA synthesized from the different FLCs.

Oocyte injection and electrophysiological recordings Preparation of oocytes and injection procedures were performed as described (Schmieden et al., 1989). Briefly, oocytes were isolated by collagenase treatment of freshly dissected ovaries, and the follicle cell layer was mechanically removed prior to injection of 50-70 nl RNA per oocyte. Oocytes were kept at 19°C with daily changes of the medium. Analysis for agonist responses was performed by voltage clamp 4-6 days after the injection under continuous superfusion (0.3 ml/s) with frog Ringer solution. Control recordings were performed on non-injected oocytes, or oocytes expressing in vitro transcribed RNA of the atl-subunit of the glycine receptor (Schmieden et al., 1989). In a few control oocytes, high concentrations of nicotine (2 20 mM) elicited a slow non-desensitizing membrane current that clearly differed from the channel current generated upon SAD expression.

Acknowledgements We thank Drs B.Bossy, M.Ballivet and P.Spierer for the

generous

gift of

ALS cDNA, and Drs T.Schwarz and R.Paro for providing DNA libraries. We are grateful to V.Schmieden for help with the electrophysiology, P.Prior

for support during screening procedures and C. Udri for excellent technical assistance. We are indebted to Drs C.Haas, P.-M.Kloetzel and R.Paro for helpful discussions concerning Drosophila. We also thank our colleagues

2676

A.Ultsch and D.Langosch for critical reading of the manuscript and I.Baro for help with its preparation. E.S. was a recipient of a Boehringer Ingelheim Foundation fellowship. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 317 and Leibniz-Program), the Bundesministerium fMr Forschung und Technologie (BCT 365/ 1) and the Fonds der Chemischen Industrie.

References Ballivet,M., Nef,P., Couturier,S., Rungger,D., Bader,C., Bertrand,D. and Cooper,E. (1988) Neuron, 1, 847-852. Bertrand,D., Ballivet,M. and Rungger,D. (1990) Proc. Natl. Acad. Sci. USA, 87, 1993-1997. Boulter,J., Evans,K., Goldman,D., Martin,R., Treco,D., Heinemann,S. and Patrick,J. (1986) Nature, 319, 368-374. Boulter,J., Connolly,J., Deneris,E., Goldman,D., Heinemann,S. and Patrick,J. (1987) Proc. Natl. Acad. Sci. USA, 84, 7763-7767. Boulter,J., O'Shea-Greenfield,A., Duvoisin,R.M., Connolly,J.G., Wada,E., Jensen,A., Gardner,P.D., Ballivet,M., Deneris,E.S., McKinnon,D., Heinemann,S. and Patrick,J. (1990) J. Biol. Chem., 265, 4472-4482. Bossy,B., Ballivet,M. and Spierer,P. (1988) EMBO J., 7, 611-618. Breer,H. (1981) Neurochem. Int., 3, 43-52. Breer,H., Kleene,R. and Hinz,G. (1985) J. Neurosci., 5, 3386-3392. Breer,H. and Sattelle,D.B. (1987) J. Insect Physiol., 33, 771-790. Campos-Ortega,J.A. and Hartenstein,V. (1985) The Embryonic Development of Drosophila melanogaster. Springer-Verlag, Berlin. Carpenter,M.K., Parker,I. and Miledi,R. (1990) Dev. Biol., 138, 313-323. Cathala,G., Savouret,I.-F., Mendez,B., West,B.L., Karin,M., Martial,J.A. and Baxter,J.D. (1983) DNA, 2, 329-335. Changeux,J.P., Devillers-Thiery,A. and Chemouilli,P. (1984) Science, 225, 1335-1345. Changeux,J.P., Giraudat,J. and Dennis,M. (1987) Trends Pharnacol. Sci., 8, 459-465. Chirgwin,J.M., Przybyla,A.E., MacDonald,R.S. and Rutter,W.J. (1979) Biochemistry, 18, 5294-5299. Deneris,E.S., Connolly,J., Boulter,J., Wada,E., Wada,K., Swanson,L.W., Patrick,J. and Heinemann,S. (1988) Neuron, 1, 45-54. Dudai,Y. and Amsterdam,A. (1977) Brain Res., 130, 551-555. Duvoisin,R.M., Deneris,E.S., Patrick,J. and Heinemann,S. (1989) Neuron, 3, 487-496. Goldman,D., Deneris,E., Luyten,W., Kochhar,A., Patrick,J. and Heinemann,S. (1987) Cell, 48, 965-973. Gration,K.A.F., Lambert,J.J., Ramsey,R. and Usherwood,P.N.R. (1981) Nature, 291, 423-425. Grenningloh,G., Rienitz,A., Schmitt,B., Methfessel,C., Zensen,M., Beyreuther,K., Gundelfinger,E.D. and Betz,H. (1987) Nature, 328, 215-220. Grenningloh,G., Schmieden,V., Schofield,P.R., Seeburg,P.H., Siddique,T., Mohandas,T.K., Becker,C.-M. and Betz,H. (1990) EMBO J., 9, 771-776. Hanke,W. and Breer,H. (1986) Nature, 321, 171-174. Hermans-Borgmeyer,I., Zopf,D., Ryseck,R.-P., Hovemann,B., Betz,H. and Gundelfinger,E.D. (1986) EMBO J., 5, 1503-1508. Hermans-Borgmeyer,I., Hoffmeister,S., Sawruk,E., Betz,H., Schmitt,B. and Gundelfinger,E.D. (1989) Neuron, 2, 1147-1156. Hollmann,M., O'Shea-Greenfield,A., Rogers,S.W. and Heinemann,S. (1989) Nature, 342, 643-648. Kao,P.N. and Karlin,A. (1986) J. Biol. Chem., 261, 8085-8088. Kozak,M. (1986) Cell, 44, 283-292. Levitan,E.S., Schofield,P.R., Burt,D.R., Rhee,L.M., Wisden,W., Kohler,M., Fujita,N., Rodriguez,H.F., Stephenson,A., Darlison,M.G., Barnard,E.A. and Seeburg,P.H. (1988) Nature, 335, 76-79. Lutcke,H.A., Chow,K.C., Mickel,F.S., Moss,K.A., Kern,H.F. and Scheele,G.A. (1987) EMBO J., 6, 43-48. Maniatis,T., Hardison,R.C., Lacy,E., Lauer,J., O'Connel,C., Quon,D., Sim,G.K. and Efstratiatis,A. (1978) Cell, 15, 687-701. Mishina,M., Tobimatsu,T., Imoto,K., Tanaka,K., Fujita,Y., Fukuda,K., Kurasaki,M., Takahashi,H., Morimoto,Y., Hirose,T., Inayama,S., Takahashi,T., Kuno,M. and Numa,S. (1985) Nature, 313, 364-369. Nef,P., Oneyser,C., Alliod,C., Couturier,S. and Ballivet,M. (1988) EMBO J., 7, 595-601. Neumann,D., Barchan,D., Safran,A., Gershoni,J.M. and Fuchs,S. (1986) Proc. Natl. Acad. Sci. USA, 83, 3008-3011. Noda,M., Takahashi,H., Tanabe,T., Toyosato,M., Furutani,Y., Hirose,T., Asai,M., Inayama,S., Miyata,T. and Numa,S. (1982) Nature, 299, 793 -797. Rudloff,E. (1978) Exp. Cell Res., 111, 185-190.

Drosophila nAChR heterogeneity Sanes,J.R., Prescott,D.J. and Hildebrand,J.G. (1977) Brain Res., 119, 389-402. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Satelle,D.B. (1980) Adv. Insect Physiol., 15, 215-3 15. Sawruk,E., Hermans-Borgmeyer,I., Betz,H. and Gundelfinger,E.D. (1988) FEBS Lett., 235, 40-46. Schloss,P., Hermans-Borgmeyer,I., Betz,H. and Gundelfinger,E.D. (1988) EMBO J., 7, 2889-2894. Schmidt-Nielsen,B.K., Gepner,J.I., Teng,N.N.H. and Hall,L.M. (1977) J. Neurochem., 29, 1013-1029. Schmieden,V., Grenningloh,G., Schofield,P.R. and Betz,H. (1989) EMBO J., 8, 695-700. Schofield,P.R., Darlison,M., Fujita,N., Burt,D., Stephenson,F., Rodriguez,H., Rhee,L., Ramachandran,J., Reale,V., Glencorse,T., Seeburg,P. and Barnard,E.A. (1987) Nature, 328, 221-227. Stroud,R.M. and Finer-Moore,J. (1985) Annu. Rev. Cell. Biol., 1, 317-351. Tautz,D. and Pfeifle,C. (1989) Chromosoma, 98, 81-85. Wada,K., Ballivet,M., Boulter,J., Connolly,J., Wada,E., Deneris,E., Swanson,L., Heinemann,S. and Patrick,J. (1988) Science, 240, 330-334. Wadsworth,S.C., Rosenthal,L.S., Kammermeyer,K.L., Potter,M.B. and Nelson,D.J. (1988) Mol. Cell. Biol., 8, 778-785. Wang,G.K., Molinaro,S. and Schmidt,J. (1978) J. Biol. Chem., 253, 8507-85 12. White,K. and Kankel,D.R. (1978) Dev. Biol., 65, 296-321. Whiting,P. and Lindstrom,J. (1987) Proc. Natl. Acad. Sci. USA, 84, 595-599. Received on May 11, 1990; revised on June 5, 1990

Note added in proof The sequence data for the D. melanogaster SAD cDNA have been deposited in the EMBL data library (accession number X53583).

2677