Neuronal Nicotinic Acetylcholine Receptors on Bovine Chromaffin Cells: Cloning, Expression, and...

Journal ofNeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Neuronal Nicotinic Acetyicholine Receptors on BovineChromaffin Cells: Cloning, Expression, and Genomic

Organization of Receptor Subunits

*A. Campos-Caro, *F. I. Smillie, *E. DomInguez del Toro, 1J. C. Rovira,*F. Vicente-Agulló, *~J.Chapuli, ~J. M. JuIz, §S. Sala, tF. Sala,

~J. J. Ballesta, and *M. Criado

Departments of * Neurochemistry, ~Histology, § Physiology, and tPharmacology,and Instituto de Neurociencias, University of Alicante, Alicante, Spain

Abstract: Neuronal nicotinic acetylcholine receptors frombovine adrenomedullary chromaffin cells play a primaryrole in triggering catecholamine secretion. In the presentstudy, their constituent subunits were characterized. Inaddition to the cx3 subunit, which we have previouslycloned, the presence of a5 and /94 but not of /32 subunitswas detected by reverse transcription—PCR analysis ofmRNA from adrenal medulla. In situ hybridization indi-cated that cr3, cr5, and ~34subunits are coexpressed inall chromaffin cells. The primary structure of a5 and /34subunits was determined and functional receptors wereobtained upon coinjection of subunit cRNAs into Xenopusoocytes. In contrast to other /34-containing nicotinic re-ceptors, the ones formed by the bovine /94 subunit areinsensitive to the agonist cytisine. Finally, we character-ized the intergenic region of cr3 and cr5 subunits, whichtogether with the /34 subunit, form a gene cluster in ratsand chickens. RNase assays and the existence of over-lapping cDNAs indicate that, in the bovine genome, thecr3 and cr5 genes overlap at their 3’ ends. This fact isprobably due to inefficient transcription termination, as aresult of weak polyadenylation signals. Key Words: Nico-tinic receptor—Subunits—Expression—Ion channel—Agonist sensitivity—Genes.J. Neurochem. 68, 488—497 (1997).

Adrenal chromaffin cells arise developmentallyfrom the neural crest together with other cells suchas postganglionic sympathetic neurons (Anderson,1993). They constitute a relatively homogeneous cellpopulation that perform many of the typical functionsof neurons and for this reason are very valuable forapproaching fundamental neural phenomena such asneurosecretion (Burgoyne et al., 1993) and neural dif-ferentiation (Unsicker, 1993). Chromaffln cells of thebovine adrenal medulla are cholinergically innervatedby the splanchnic nerve from the sympathetic nervoussystem. Acetylcholine (ACh), released upon stimula-tion of this nerve, activates nicotinic acetylcholine re-

ceptors (nAChRs) in chromaffin cells and the subse-quent depolarization triggers catecholamine secretion.nAChRs of neuronal and muscular origin are both pen-tameric oligomers (Cooper et al., 1991; Anand et al.,1991) composed of related subunits that, assembledin various combinations, determine different channelproperties (reviewed by Papke, 1993; Karlin and Aka-bas, 1995). Neuronal nAChR subunits are commonlyclassified as agonist-binding (designated cr2— cr9) andstructural (/32— /34) subunits. Functional receptors canbe formed by heterologous expressionof these subunitseither alone (cr7— cr9) or in pairwise combinations (cr2,cr3, or cr4 with either /32 or /34) (reviewed by Sargent,1993; McGehee and Role, 1995). On the other hand,immunoprecipitation studies have shown that some na-tive nAChRs can be composed of at least three kinds ofsubunits (Conroy et al., 1992; Vernallis et al., 1993),a fact recently corroborated by expression studies inXenopus oocytes (RamIrez-Latorre et al., 1996; Wanget al., 1996). Previously, we have cloned the bo-vine a3 (Criado et al., 1992) and cr7 subunits (GarcIa-Guzmán et al., 1995) of neuronal nAChRs, which areexpressed in chromaffin cells, probably as componentsof two different nAChR subtypes. Given that nAChRsassembled from bovine cr7 subunits are blocked bythe snake toxin cr-bungarotoxin (GarcIa-Guzmán et al.,1995) and that catecholamine secretion is toxin insen-sitive (Afar et al., 1994), it is reasonable to assume

Received June 24, 1996; revised manuscript received September20, 1996; accepted September 27, 1996.

Address correspondence and reprint requests to Dr. M. Criado atDepartment of Neurochemistry, University of Alicante, Ap. 374, E-03080 Alicante, Spain.

The first two authors contributed equally to this study.The present address of Dr. F. I. Smillie is Wythenshawe Hospital

Research Centre, Manchester M23 9LT, U.K.Abbreviations used: ACh, acetyicholine; nAChR, nicotinic acetyl-

choline receptor; NFR, normal frog Ringer solution; RT, reversetranscription; SSC, standard saline citrate.

488

NICOTINIC RECEPTOR FROM CHROMAFFIN CELLS 489

that the nAChR subtype involved in this process maybe formed by cr3 and other subunits not yet character-ized (Gu et al., 1996).

In an attempt to further characterize the nicotinicreceptor subtypes present in chromaffin cells, we showhere that as with cr3 subunits, cr5 and /34 subunits areexpressed in all chromaffin cells of the adrenal me-dulla. Moreover, we have cloned, sequenced, and coex-pressed these three subunits in Xenopus oocytes. Inaddition, we have characterized the intergenic regionof the cr3 and cr5 subunits. The cr3, cr5, and ,84 genesof the rat and chicken neuronal nicotinic receptors havebeen closely located (Boulter et al., 1990; Couturier etal., 1990). We provide evidence that a similar situationtakes place in the bovine genome, at least with respectto the cr3 and cr5 genes, which are not only tightlyclustered but have overlapping 3’ ends.

MATERIALS AND METHODS

Analysis of DNA librariesThe Xgtl0 eDNA libraries, constructed from poly(A)~

RNA from adrenal medulla, were a gift from Dr. P. Seeburg(University of Heidelberg, Germany). Screening was per-formed as previously described (Criado et al., 1992). TheEMBL-3 SP6/T7 bovine genomic library wasfrom Clontech(catalog no. BL1O15j). Screening was performed as pre-viously described (GarcIa-Guzmán et al., 1995). DNA frag-ments corresponding to different regions of the last exon ofcr3 and cr5 subunits were used as hybridization probes forthe screening. Positive clones were isolated and DNA pre-pared from plaque-pure phage. The inserts were subclonedinto the pBluescript vector (Stratagene) and the regions ofinterest further analyzed.

Isolation of cDNAs by PCRThe cr5 and /34 cloning general strategy by PCR was the

same as the one previously used for the cr7 subunit (seeGarcIa-Guzmán et al., 1995, for a detailed description of theprocedure). In brief, 2 ~.tgof purified mRNA was reversetranscribed. The first-strand cDNA was purified, polyadenyl-ated, and resuspended in a final volume of 50 ~l. This mate-rial, referenced below as cDNA product, was used in thedifferent PCRs.

A set of two degenerate oligonucleotides (synthesized onan Applied Biosystems 391 PCR-MATE synthesizer) wasused to amplify the central core of each subunit. Amplifica-tion wasperformed with 1.5 ~.tlof the eDNA product. Ampli-fication of the 3 ‘-cDNA ends, coding for the C-terminalregion of the cr5 and /34 subunits, was performed by twosuccessive PCRs. In the first PCR, 1.5 ~.tlof the cDNAproduct was amplified with a sense primer deduced fromthe core sequence previously determined and a degenerateantisense primer containing the stop codon. During the sec-ond PCR, material from the first PCR was amplified with asecond sense primer and the same antisense primer. The 5 ‘-

cDNA region was extended by two successive amplificationswith a set of two degenerate sense oligonucleotides and anantisense primer deduced from the core sequence previouslydetermined. Finally, the 5 ‘-cDNA end of each subunit, cod-ing for the initial ATG, and the signal peptide, which isnot conserved among previously sequenced subunits, wasobtained by using the RACE procedure (Frohman et al.,

1988; Harvey and Darlison, 1991). In a first PCR (in afinalvolume of 20 gil), 1.5 ~slof cDNA product was amplifiedwith primers oligo( dT) tail [CGGGATCTCGAGTATACG-C (T) 191 and an antisense primerdeduced from the sequenceof the 5 ‘-eDNA regionpreviously determined. One-tenth ofthis material was transferred to a fresh tube, and a newreaction wasperformed with the tail primer CGGGATCTC-GAGTATACGC], which contains part of the previouslyused oligo(dT)-tail primer, and a second antisense primer.PCRs were performed using Taq polymerase (Promega)un-der previously described conditions (GarcIa-Guzmán et al.,1995). Degenerate primers were designed considering thepreviously sequenced cr5 and /34 subunits (Cockcroft et al.,1992). (Sequences of the primers are not included for rea-sons of space but can be obtained, by request, from theauthors.)

The cDNAs coding for the complete cr5 and /34 proteincoding region were constructed from overlapping fragmentscontaining common restriction enzymesites. Two different,completely independent, final constructs were analyzed andsequenced. No differences were founcLbetween them.

Reverse transcription—PCR (RT-PCR) analysisTotal RNA was extracted from bovine adrenal medulla

by guanidinium isothiocyanate and acid phenol extraction(Promega). nAChR subunit transcripts were detected by acombined RT-PCR assay (Singer-Sam et al., 1990). In brief,samples of total RNA (1.2 ,ug) in a final volume of 30 ~slwere reverse transcribed with 1.25 U AMV reverse tran-scriptase (10 mm, 50°C),then PCR was performed for 35cycles (1 mm, 94°C; 2 mm, 58°C;2.5 mm, 72°C). Thefollowing primers were chosen so that fragments of similarsize were obtained for every subunit: cr3 (sense 5 ‘-CGG-TCATCATCCAGTTTGAGGT-3’; antisense 5 ‘-TCCTG-TATGCACGTTGAGCA-3’; PCR fragment has 815 bp,coding from amino acids 49 to 322); cr5 (sense 5’-GTT-CATGTACCATCGACGTCA-3’; antisense 5 ‘-TTCCAA-TCTTCAACAAGCTC-3’; PCR fragment has 759 bp,coding from amino acids 136 to 388); and /34 (sense 5’-TGAACTCGCTTCGGCTAC-3’; antisense 5 ‘-CATAAC-TGGGGTCCTGGTGG-3’; PCR fragment has 706 bp, fromthe 5 ‘-noncoding region to amino acid 219). The amplifiedDNA fragments expanded over at least an intron to rule outthe possibility of amplifying a potential contamination ofgenomic DNA. In fact, RT-PCR performed in the absenceof AMV reverse transcriptase (see Fig. 2) did not yieldfragments of the expected size.

In situ hybridizationSense and antisense digoxigenin-labeled riboprobes were

synthesized with SP6 and T7 polymerases from the corre-sponding linearized template (pSPT18) by using a Boeh-ringer Mannheim RNA labeling kit. Probes were directed toregions of the M3—M4 cytoplasmic loop unique for eachsubunit (amino acids 349—4 10 of the cr3 subunit, Criado etal., 1992; amino acids 318—380 of the cr5 subunit, see Fig.1A; amino acids 307—368 of the /34 subunit, see Fig. lB).Frozen adrenal gland tissue was cut into l0-~imsections,mounted in silanated slides, fixed in 4% formaldehyde (5mm at room temperature), deproteinated (10 mm in 0.2M HC1 at room temperature), and dehydrated in ethanol.Hybridization was for 16 h at 45°Cin a buffer containing50% formamide, 5>< standard saline citrate (SSC; 0.15 MNaCI, 0.015 M sodium citrate), lx Denhardt’s solution,10% dextran sulfate, 0.2% sodium dodecyl sulfate, 10 mM

J. Neurochem., Vol. 68, No. 2, 1997

490 A. CAMPOS-CARO ET AL.

dithiothreitol, 250 ~.tg/mlherring sperm DNA, 250 ~ig/mlyeast tRNA. Digoxigenin-labeled probes were diluted (inthe range 1:25 to 1:400) with hybridization buffer to obtaina suitable and equivalent concentration for each set of senseand antisense probes. This concentration was previously de-termined in dot blot assays. After hybridization, sectionswere washed twice for 30 mm in 0.lx SSC at 60°C.Visual-ization of the probe was performed with anti-digoxigeninantiserum as indicated by the manufacturer (BoehringerMannheim).

Oocyte expressionThe corresponding cDNAs were inserted into the pSP64T

vector (Krieg and Melton, 1984). At the time, the leaderpeptide of the cr5 subunit wasnot complete and, for unknownreasons, the cr3 subunit did not yield functional receptorswith its own leader peptide; therefore, both subunit proteincoding cDNAs were provided with the leaderpeptide of thecr7 subunit. Capped mRNA was synthesized in vitro usingSP6 RNA polymerase. Xenopus (Nasco) oocytes were in-jected with 25 ng RNA (equal proportions of each subunit)in 50 nl of sterile water.

Currents from oocytes were measured 2—6 days afterRNA injections with a two-microelectrode voltage-clampamplifier. The recording chamberwas continuously perfusedby gravity (8 ml/min) with normal frog Ringer solution(NFR), and solutions containing different concentrations ofnicotinic agonists were applied through a 1.5-mm-diameterpipette located close to the oocyte. The volume of solutionin the chamber was maintained at 0.4 ml with a suctionpipette. The NFR composition (in mM) was 115 NaC1, 2.5KC1, 1.8 CaCl2, 10 HEPES, pH 7.2. Agonist pulses anddata adquisition were controlled by a DigiData interface andpCLAMP software (Axon Instruments, Foster City, CA,U.S.A.). Currents were filtered at 50 Hz with an eight-poleBessel filter (Frequency Devices, Haverhill, MA, U.S.A.),digitized at 250 Hz, andstored on hard disk for later analysis.All experiments were performed at room temperature (20—24°C).

RNase protectionPoly(A)~ RNA was selected by oligo(dT)-cellulose

chromatography (Pharmacia) and used in the RNase protec-tion experiments. Probes were generated with SP6 and T7polymerases (Boehringer Mannheim), [cr-

32P I CTP (Am-ersham), and the corresponding linearized templates(pSPT18). The cr3 and cr5 polyadenylation sites were deter-mined using templates in which fragments corresponding tocertain regions of the last exon of each subunit were included(for cr3, the fragment corresponded to nucleotides 982—1,5 16 in Fig. SB, and for cr5, to nucleotides 3,787—4,450 inthe same figure). To analyze the cr3—crS overlap region, afragment whose sequence was detected in cr3 and cr5 eDNAclones (nucleotides 2,980—3,160 in Fig. SB) was cloned inpSPTl8 and probes were synthesized in both orientations.For calibration, several other RNAs of known size were alsosynthesized. RNase protection experiments were performedusing the RNase Protection Kit (Boehringer Mannheim) asindicated by the manufacturer. Protected fragments wereseparated on a 6% acrylamide/urea gel along with the la-beled RNAs used for calibration.

RESULTSCloning of the bovine ~5 and J34 subunits

To find which additional subunits could assemblewith the previously cloned cr3 subunit to form nAChRs

in bovine chromaffin cells, we considered earlier stud-ies that showed that cr5, /32, and /34 subunit mRNAswere present in the rat adrenal chromaffin tumor cellline PC12 (Boulter et al., 1990). Degenerate primerswere used for PCR amplification of eDNA synthesizedfrom bovine adrenal medulla RNA, to obtain the cen-tral region of each subunit, which is very conservedamong species. DNA fragments were obtained and,based on sequence identity, identified as coding for cr5and /34 subunits but not for /32. Several other attemptsto detect this subunit failed, and therefore, we concen-trated on the other two, whose protein coding regionswere obtained by PCR techniques.

Sequence analysis of the cr5 protein coding regionshowed a unique 1 ,308-bp open reading frame encod-ing 436 amino acids (Fig. 1A). By analogy with theN-terminus of the rat cr5 subunit, we deduced that onlya portion (11 amino acids) of the signal peptide hadbeen obtained. This would be cleaved at V_

1—A1. Sev-eral PCR strategies were used to obtain the completesignal peptide, but none was successful. Screening ofeDNA libraries from which the cr3 subunit eDNA wasobtained (Criado et al., 1992) was also performedwithout success. For this reason, we decided to linkthe cr7 signal peptide (GarcIa-Guzmán et al., 1995) tothe cr5 protein coding region, to functionally expressthis subunit. Two putative sites for N-glycosylation(N40 and N186) were detected in the N-terminal extra-cellular domain of the subunit. The following othergeneral features of ligand-gated neuroreceptor subunitswere present: four 22—27 amino acid hydrophobic seg-ments termed Ml —M4, a pair of cysteines separatedby 13 amino acids (127 and 141 in Fig. 1A), andanother pair thought to be close to the ACh bindingsite of AChR a subunits (191 and 192 in Fig. 1A).As expected, sequence comparison with rat nAChRsubunits showed the highest identity with the cr5 sub-unit (Fig. lC).

Sequence analysis of the complete /34 protein codingregion showed a l,488-bp open reading frame encod-ing 496 amino acids (Fig. 1B). By analogy with the N-terminus of the other /34 subunits, we predict a signalpeptide of 23 amino acids, which would be cleaved atV1—A1. Four putative sites for N-glycosylation (N13,N70, N115, and N143) were detected in the N-terminalextracellular domain of the subunit. They are alsofound in the human and rat ~34 subunits. The typicalfour 20—26 amino acid hydrophobic segments (Ml —

M4) and the pair of cysteines separated by 13 aminoacids (130 and 144 in Fig. 1B) were also present.Sequence comparison with rat nAChR subunitsshowed the highest identity with the /34 subunit(Fig. 1C).

Localization of a~3,aS, and ~34 transcriptsExpression of cr3, cr5, and /34 mRNAs in the bovine

adrenal medulla was first analyzed by a RT-PCR assay(Fig. 2). Although this assay was not quantitative,differences in expression levels between the three sub-

.1. Neurochem., Vol. 68, No. 2, 1997


FIG. 1. Primary structure of the bovine nAChR a5 (A) and /14(B) subunits. The deduced amino acid sequences of the cDNAs encodingthe nAChR a5 and /34 subunit from bovine adrenal medulla are designated B and aligned with their rat (R) and chicken (C) counterparts.Only different amino acids are indicated in the rat and chicken subunits, whereas a blank implies sequence identity. Deletions (*) andputative transmembrane segments (boxes) are indicated. The amino acids are numbered in the 5’ to 3’ direction beginning at thefirst amino acid after the leader peptide, as deduced by comparison with other cloned nAChR cr5 and /34 subunits. C: Comparisonbetween the cloned a5 and /34 sUbunits and rat nAChR subunits. The bovine cr5 subunit exhibits greater sequence identity to the ratcr5 subunit than with the other subunits, whereas the bovine /34 subunit shows highest identity to the rat /34 subunit.

J. Neurocheni., Vol. 68, No. 2, 1997


FIG. 2. RT-PCR analysis of nAChR subunits from the adrenalmedulla. Samples of total RNA from bovine adrenal medulla werereverse transcribed, and then PCR was performed as indicatedin Materials and Methods. To control specific amplification ofsubunit RNA, samples with (+) and without (—) AMV reversetranscriptase (Rev.T.) were used. Size standards are included(St.) and indicated at the right.

units were apparent; i.e., cr3 transcripts were moreabundant than those corresponding to the /34 subunit,cr5 mRNA being the less abundant. In situ hybridiza-tion experiments showed that transcripts encodingthese three subunits were found in all chromaffin cellsof the bovine adrenal medulla (Fig. 3). Hybridizationintensity differed among subunits, the cr3 signal beingstronger than the other two. This is in agreement withthe RT-PCR experiments (Fig. 2) in which the levelof cr3 transcripts was found to be the highest. Besidesthe difference in transcript levels, the three subunitswere coexpressed in all chromaffin cells, suggestingthat they may form part of the same receptor.

Functional expression of the cloned subunitsEvidence that the cDNAs cloned by PCR were cod-

ing for functional AChRs was obtained by injectingXenopus oocytes with the corresponding cRNAs andstudying the electrophysiological properties of the ex-pressed AChRs. Upon ACh or nicotine stimulation,voltage-clamped oocytes showed inward currents in aconcentration-dependent manner (Fig. 4A). As ob-served with other cr3/34 receptors expressed in oocytes,bovine receptors showed very little desensitizationwith low ACh concentrations (Cachelin and Jaggi,1991; Covernton et al., 1994). However, at very highagonist concentrations (not tested in the above re-ports), desensitization is more prominent, probablydue to open channel block (Sine and Steinbach, 1984).

No significant differences were observed betweenthe combinations cr3—/34 and cr3—/34—cr5. In contrastto ACh and nicotine, cytisine was ineffective at elic-iting currents at concentrations to 300

1tM (Fig. 4B).Currents evoked by nicotinic agonists were blocked by20 ,uM d-tubocurarine in a reversible manner, whereascr-bungarotoxin (20 nM) was unable to block (datanot shown). Figure 4C shows the voltage dependenceof nicotine-evoked currents. It can be seen that currentsshow inward rectification. An apparent reversal poten-tial of +5.5 mV was found.

The intergenic region of the a3 and aS subunitsDuring the screening of bovine adrenal medulla

eDNA libraries, several cr3 and cr5 eDNA clones wereobtained with, apparently, very lengthy 3 ‘-noncodingregions. Given that previous studies had shown thatthe genes encoding the cr3, cr5, and ~34subunits areclustered in both the rat and the chick (Boulter et al.,1990; Couturier et al., 1990) and that the cr3 and cr5genes were transcribed with convergent polarity, weconsidered the possibility that these two genes areoverlapping. If this were the case, interference at thelevel of gene transcription could exist. In fact, uponcharacterization and sequencing of the aforementionedcDNAs, it could be concluded that their 3’ ends werecomplementary (Fig. SB). The genomic region corre-sponding to the last exon of each subunit was isolatedand analyzed (Fig. 5A), and found to correlate withthe structure of the cDNAs, suggesting that the cr3 andcr5 genes appear to have overlapping 3’ ends. It isinteresting that a contiguous direct repeat of two 117-and 106-bp monomers was detected (Fig. SC), whichpartially extended over the a3—a5 overlap. Furthercomparison of this sequence with databases of nucleicacid sequences revealed close homology to Alu-likerepetitive sequences from Ruminantia.

The existence of overlapping cDNAs correspondingto the cr3 and aS subunits indicates that this region isbeing transcribed. If this is the case, transcripts corre-sponding to one subunit would specify endogenousantisense transcripts of the other, perhaps serving regu-latory functions in this way. Previous hybridization ofadrenal medulla mRNA with a bovine cr3 probe re-vealed the presence of two main cr3 transcripts of 3.2and 2.3 kb and two other less abundant bands of 6.9and 8.5 kb (Criado et al., 1992), which has been con-firmed by others (Gu et al., 1996). Although the startofthe cr3 mRNA has not yet been mapped, it is obviousthat the main transcripts are not long enough to reachthe cr3— cr5 overlap, whereas the larger transcriptswould be able to do so. RNase protection experimentswere performed (Fig. 6), to try to find the main poly-adenylation sites of the cr3 and aS transcripts and toconfirm that the a

3 —cr5 overlap region is present in,at least a fraction of, the transcripts. Thus, when usingan cr3 probe corresponding to the region where severalpolyadenylated cr3 cDNAs have been previously de-tected, two main protected bands of 350 and 250 baseswere observed (Fig. 6). Their size would agree withthe polyadenylation sites present in the cDNAs (nude-otides 1,233 and 1,331 in Fig. 5B). Transcripts withsuch a polyadenylation pattern may correspond to the‘~3.2-kbband detected in northern blots. Nevertheless,a typical polyadenylation signal (AATAAA) was notdetected in this region. In fact, such signals were onlypresent in areas close to the cr3— aS overlap (nucleo-tides 3,109—3,114, 3,168—3,173, and 3,768—3,773,Fig. SB). In addition to these two protected bands, aless prominent one of 530 bases was also observed(Fig. 6), which would arise from the protection of the



FIG. 3. Localization of a3, a5, and ~34transcripts in the bovine adrenal gland. In situ hybridization of nAChR subunits was performedwith antisense riboprobes corresponding to sequences specific for the a3 (A), a5 (C), and ~4 (E) subunits. Nonspecific labeling wastested with the same probes located in the sense orientation (B, D, and F for a3, a5, and ~4, respectively). Notice the absence oflabeling in the adrenal cortex (labeled as C) in contrast to the adrenal medulla (labeled M). Calibration bars: 50 tim.

whole a3 probe. This suggests that some a3 transcriptsextend over the main polyadenylation sites. In the caseof the a5 subunit, two bands of ~~468and 460 baseswere observed (Fig. 6), which correspond with poiy-adenylation sites at nucleotides 3,991 and 3,999 (Fig.5B), i.e., 304 and 296 bases downstream of the cr5 stopcodon. A polyadenylation signal in the a5 transcriptionorientation is present 227 bases downstream of the cr5stop codon (nucleotides 4,062—4,067). In addition, aband of 655 bases corresponding to protection of the

whole cr5 probe was detected. Again, this implies thata small fraction of cr5 transcripts extends over the mainpolyadenylation sites. Finally, when a probe corre-sponding to the cr3— cr5 overlap was used, protectionwas observed in both senses (Fig. 6), indicating thatsome cr3 and cr5 transcripts reach this region and evenmay extend beyond it, as the size of the protectedband corresponded approximately to the whole probe.Therefore, these experiments indicate that the mainpolyadenylation sites for both cr3 and cr5 subunits oc-



FIG. 4. Functional expression of bovine nicotinic receptorsformed by the cr3 and /34 subunits. A: Inward currents obtainedupon continuous stimulation with ACh. Traces represent currentsobtained at —40 mV with 10, 30, 100, 300, 1,000, and 3,000

1jMACh, fromtop to bottom, respectively. B: Dose—response curvesof the same receptors for three cholinergic agonists, ACh (0;13 oocytes, three donors), nicotine (U; 14 oocytes, four donors),and cytisine (•; five oocytes, two donors). Data points are nor-malized to the response obtained by 100 p~Mnicotine and repre-sent mean ±SEM values. Continuous line represents the fit ofdata to the Hill equation. EC50 (1.rM) and Hill coefficients were372 ±98and 1.4(ACh)and 113 ±l5and 1.5(nicotine), respec-tively. Cytisine data were too small to give reliable fits. C: Cur-rent—voltage relationship of currents evoked by 100 gM nicotine.A voltage ramp (120 mV, 80 ms) was applied in the presenceand the absence of agonist. Current records were subtractedand plotted against the membrane potential recorded by thevoltage electrode.

cur downstream of the region where the genes overlap,but also that a small but detectable fraction of tran-scripts reach and even pass this region.

DISCUSSION

The chromaffin cell of the bovine adrenal medullarepresents a relevant and accessible model in which tostudy a particular neuronal nAChR subtype with awell-defined function. Previous pharmacological (Hig-

gins and Berg, 1988), immunological (Higgins andBerg, 1987), and electrophysiological (Fenwick et al.,1982; Maconochie and Knight, 1992; Nooney et al.,1992) evidence suggested that the nAChRs from chro-maffin cells are of neuronal type. This was furtherconfirmed when we cloned the bovine a3 nAChR sub-unit (Criado et al., 1992) and revealed its expressionin chromaffin cells. The present study shows that thecr5 and /34 subunits are also expressed in the adrenalgland. Moreover, in situ hybridization experiments in-dicate that the three subunits are present in adrenergicand noradrenergic chromaffin cells. The coexpressionof cr3, ~34,and cr5 subunits in chromaffin cells suggeststhat these polypeptides may participate in the forma-tion of the neuronal nAChR involved in triggering cat-echolamine secretion. In fact, the same subunits appearto combine to produce nAChRs in the chick ciliaryganglion (Vernallis et al., 1993; Conroy and Berg,1995). In our case, we do not have direct evidence forthe assembly of the cr3, /34, and cr5 subunits, becausethe combination cr3— /34 produces functional channelsand no dramatic pharmacological differences were ob-served when the cr5 subunit was also present. A recentstudy with human cr3, cr5, and /34 subunits reportedno significant pharmacological differences betweenchannels containing or not containing the aS subunit(Wang et al., 1996).

A remarkable feature of bovine a3/34 nAChRs (withor without cr5) is their lack of sensitivity to the nico-tine-like alkaloid cytisine, which is a potent agonistfor receptors containing rat /34 in combination withany a subunit (Luetje and Patrick, 1991; FigI et al.,1992; Papke and Heinemann, 1994). Cytisine insensi-tivity is probably due to the bovine /34 subunit, as thecombination bovine cr3—rat /34 is cytisine sensitive(our unpublished results). Previous studies with chi-meric /32— /34 subunits have stressed the importanceof two amino acid residues of the rat /34 subunit (V108and T0, changed to F108 and S110 in /32) in determiningcytisine sensitivity (Figl et al., 1992). In the case ofthe bovine /34, T110 is conserved whereas V108 ischanged to L. This difference may be responsible forthe cytisine insensitivity of the bovine cr3/34 nAChR.It is interesting that, and in contrast to nicotine orACh, cytisine does not activate nicotinic receptors orcatecholamine secretion from bovine chromaffin cells(our unpublishedresults), which indicates that /34 maybe a component of the nAChR involved in triggeringexocytosis. Besides the different sensitivity to cytisine,the bovine cr3/34 nAChR shows physiological andpharmacological properties compatible with a gangli-onic nAChR, similar, for instance, to a recently charac-terized rat cr3/34 nAChR transfected in eukaryotic cells(Wong et al., 1995).

The cr3, cr5, and /34 subunits of nAChRs have beenfound tightly clustered in the avian (Couturier et al.,1990) and mammalian (Boulter et al., 1990) genomes,with the cr3 and cr5 genes contiguous and having op-posed transcription polarity. The present study shows



FIG. 5. The intergenic region of the cr3 and cr5 subunit genes. A: Structure and restriction map of the genomic region where the lastexons (represented by arrows of convergent directions) of the cr3 and aS subunits have been located. B: Nucleotide sequence of thelast exons of cr3 and cr5 subunits as deduced from cDNA clones. For simplification, only the sense strand corresponding to the cr3

subunit (and therefore the antisense strand for cr5) is shown. The amino acid sequence and stop codons (°)corresponding to the C-terminal regions of both subunits are located below the nucleic acid sequence. Proposed polyadenylation sites are underlined andboldfaced. The region where cr3 and cr5 cDNA5 overlap is underlined. The sequence of this region and adjacent areas (a total of ‘-~400bp) was also obtained from genomic clones and found to be identical to the cDNA sequence. C: Alignment of the 117- and 106-bpA/u-like repeats lying partially at the cr3—cr5 overlap.

that, at least in the bovine genome, these genes overlapat their 3’ ends. This previously unreported findingwas based on the existence of several cr3 and cr5cDNAs with overlapping 3’ ends, which matched thegenomic organization of the cr3— cr5 intergenic region,and it was confirmed by RNaseprotection experiments.Overlapping transcripts appear to constitute, however,only a minor fraction and probably arise from ineffi-

cient transcriptional termination. This process appearsto depend on several distinct cis-active elements, afunctional poly(A) signal, a downstream transcrip-tional pause site (Eggermont and Proudfoot, 1993),and some other sequence elements, especially a down-stream U-rich region (Chen et al., 1995). That cr3 andcr5 polyadenylation signals do not fulfill some of theserequisites may explain why transcriptional termination



and cr5 transcripts are found (Boulter et al., 1990)supports the possibility that in the rat there is also asimilar lack of strong poly(A) signals.

Note added in proof: In Fig. 5B the last amino acidcorresponding to the cr5 subunit has been erroneously quotedas S. It should be V.

Acknowledgment: This study was supported by grantsfrom the Ministries of Education (DGICYT, PB92-0346,and PB93-093l) and Health (Fis 95/1672) of Spain, theCommission of the European Economic Community(SC1 *CT9 1-0666), Generalitat Valenciana (GV-2535/94),and the Wellcome Trust (039284). A.C.-C. was a predoc-toral fellow of the European Economic Community. F.I.S.was a postdoctoral fellow of the Wellcome Trust. E.D. delT. and F.V.-A. were the recipientsof predoctoral fellowshipsfrom the Ministry of Education of Spain. J.C. was a predoc-toral fellow of FundaciOn CAM.

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FIG. 6. RNase protection analysis. The map below the autora-diograms shows the location of the strand-specific riboprobes(discontinuous line) with respect to the last exons of the cr3and cr5 subunits (convergent arrows). Thick continuous linesfollowed by (A), indicate major protected fragments with pro-posed poly(A) sites, which are observed in the correspondingautoradiograms (labeled as cr3 and cr5 polyA sites). Thin continu-ous lines represent protection of the whole riboprobe, whichusually yields weaker autoradiographic signals. In the autoradio-grams, Tcorresponds to the whole undigested riboprobe, includ-ing a small proportion of vector sequences; U is a negative con-trol digesting the probes in the presence of 30 ~tgof tRNA;and P corresponds to RNase digestions in the presence of thefollowing amounts (in /.1g) of adrenomedullary poly(A) * RNA: 7.6(cr3 polyA sites),30 (aS polyAsites), 10 (cr3 overlap), and 30 (cr5overlap). Signal intensity is not comparable among the differentautoradiograms, as they were performed in different experi-ments. Each experiment was repeated at least once with adrenalmedulla RNA from different donors.

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Neuronal Nicotinic Acetylcholine Receptors on Bovine Chromaffin Cells: Cloning, Expression, and...

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