Characterisation of 5-HT3 receptor subunits

*Molecular Discovery Research, GlaxoSmithKline, Harlow, Essex, UK

�Neurology and Gastrointestinal Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, UK

5-HT3 receptors are therapeutically important members ofthe superfamily of Cys-loop ligand-gated ion channels whichincludes the nicotinic acetylcholine receptors (nAChRs),GABAA, GABAC and glycine receptors (Reeves and Lum-mis 2002; Peters et al. 2005). The 5-HT3 receptor exists as apentameric ring of subunits that form an integral ion channelalong the central axis. To date, functional channels composedof a homomeric assembly of 5-HT3A (Maricq et al. 1991;Miyake et al. 1995); or heteromeric assembly of 5-HT3A and5-HT3B subunits (5-HT3AB) (Davies et al. 1999) have beendescribed. Each exhibits distinct channel characteristics(single channel conductance, rectification), but little differ-ence in pharmacology, at least in terms of responses to 5-hydroxytryptamine (5-HT) or 5-HT3 receptor antagonists(Reeves and Lummis 2002; Peters et al. 2005), although

small differences in responses to tubocurarine or picrotoxinare observed (Davies et al. 1999; Brady et al. 2001; Das andDillon 2003). These findings do not, however, explain theapparent pharmacological diversity of responses mediated bythe 5-HT3 receptor within native tissues.

Received August 18, 2008; revised manuscript received/acceptedOctober 28, 2008.Address correspondence and reprint requests to Martin Gunthorpe,

Neurology and Gastrointestinal Centre of Excellence for Drug Discov-ery, GlaxoSmithKline, New Frontiers Science Park (North), ThirdAvenue, Harlow, CM19 5AW, UK.E-mail: [email protected] used: 5-HT, 5-hydroxytryptamine; CFP, cyan fluorescent

protein; CHO, Chinese hamster ovary; DRG, Dorsal Root Ganglion; GI,gastrointestinal; HA,Hemagglutinin; TM, transmembrane;WT,wild type.

Abstract

The 5-HT3 receptor is a member of the ‘Cys-loop’ family of

ligand-gated ion channels that mediate fast excitatory and

inhibitory transmission in the nervous system. Current evi-

dence points towards native 5-HT3 receptors originating from

homomeric assemblies of 5-HT3A or heteromeric assembly of

5-HT3A and 5-HT3B. Novel genes encoding 5-HT3C, 5-HT3D,

and 5-HT3E have recently been described but the functional

importance of these proteins is unknown. In the present study,

in silico analysis (confirmed by partial cloning) indicated that 5-

HT3C, 5-HT3D, and 5-HT3E are not human–specific as previ-

ously reported: they are conserved in multiple mammalian

species but are absent in rodents. Expression profiles of the

novel human genes indicated high levels in the gastrointesti-

nal tract but also in the brain, Dorsal Root Ganglion (DRG)

and other tissues. Following the demonstration that these

subunits are expressed at the cell membrane, the functional

properties of the recombinant human subunits were investi-

gated using patch clamp electrophysiology. 5-HT3C, 5-HT3D,

and 5-HT3E were all non-functional when expressed alone.

Co-transfection studies to determine potential novel hetero-

meric receptor interactions with 5-HT3A demonstrated that the

expression or function of the receptor was modified by 5-HT3C

and 5-HT3E, but not 5-HT3D. The lack of distinct effects on

current rectification, kinetics or pharmacology of 5-HT3A

receptors does not however provide unequivocal evidence to

support a direct contribution of 5-HT3C or 5-HT3E to the lining

of the ion channel pore of novel heteromeric receptors. The

functional and pharmacological contributions of these novel

subunits to human biology and diseases such as irritable

bowel syndrome for which 5-HT3 receptor antagonists have

major clinical usage, therefore remains to be fully determined.

Keywords: 5-HT3, cloning, electrophysiology, ion channel,

pharmacology, serotonin.

J. Neurochem. (2009) 108, 384–396.

JOURNAL OF NEUROCHEMISTRY | 2009 | 108 | 384–396 doi: 10.1111/j.1471-4159.2008.05775.x

384 Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 384–396� 2008 The Authors

The term ‘5-HT3–like’ receptor was introduced to describecertain responses to 5-HT3 receptor antagonists which didnot fit the expected activity. For example, in one study, all 5-HT3 receptor antagonists tested potently inhibited the vonBezold-Jarisch cardiovascular reflex induced by 5-HT inanaesthetised rats, but some of these were able to reduce avisceral pain reflex in the same species (Banner et al. 1995).This and other differences in responses to 5-HT3 receptorantagonists, always described in native tissues (in vitro andin vivo), are still not adequately explained (Sanger 1995).Although there are many possible explanations, includingnon-selectivity of action, the discovery of further subunits ofthe 5-HT3 receptor now provides an opportunity to revisit theproposal that ‘5-HT3-like’ receptors might exist, withpharmacology distinct from the accepted 5-HT3A and5-HT3AB receptor forms that have been extensively charac-terized (Reeves and Lummis 2002).

In 2003, three human genes encoding novel 5-HT3C, 5-HT3D and 5-HT3E receptor subunits were reported (Karnov-sky et al. 2003; Niesler et al. 2003). The genes for thesesubunits are located on human chromosome 3q27 whilstthose for 5-HT3A and 5-HT3B are on chromosome 11q23.Further, the reported mRNA expression of the 5-HT3C-E

subunits tended to show a peripherally-restricted pattern,including high levels in the gastrointestinal (GI) tract (Niesleret al. 2003). Intriguingly, their orthologues seem to be absentin rodents (Karnovsky et al. 2003; Niesler et al. 2003).Together, these data suggest that 5-HT3C-E subunits areformed for a specific purpose within the peripheral nervoussystems in some species, including humans, which is bothdistinct from the more general functions of receptors formedby 5-HT3A and 5-HT3B.

To gain insight into their functional properties, andpossible physiological roles, we used RT-PCR from cDNAlibraries to further elucidate the distribution of the mRNA’sencoding 5-HT3C-E in human tissues. We discovered mam-malian orthologues of 5-HT3C, 5-HT3D and 5-HT3E byin silico searches of genomic sequence and PCR cloningfrom ferret and rabbit cDNA libraries. We report definitiveevidence that the genes are not human specific, as previouslysuggested (Niesler et al. 2003) but, rather, appear to be lostspecifically in the rodent lineage. Following the cloning of 5-HT3C-E from human tissue sources, we show that C-terminaltagged subunits localise to the plasma membrane and, usingwhole-cell patch clamp electrophysiology, we assessed thefunctional and pharmacological properties of the subunits ascandidate homomeric and heteromeric channels.

Experimental procedures

Sequence searches for subunit orthologuesTo explore species distribution NCBI databases were homology

searched using BLAST. Sequences predicted to encode HTR3C,

HTR3D and HTR3E were identified and peptide sequences predicted

using genewise (Birney et al. 2004). Phylogenetic analysis was

performed using neighbour joining methodology implemented in

PAUP (v4b10 for UNIX) (Wilgenbusch and Swofford 2003).

Cloning of ferret and rabbit orthologuescDNA encoding 5-HT3C and 5-HT3E was PCR-amplified from

libraries derived from ferret tissues using degenerate primers based

on dog sequence. Products from lung, spleen, liver and uterus were

cloned, sequenced and subjected to homology searches. A partial

fragment of rabbit HTR3C cDNAwas isolated from rabbit colon and

small intestine libraries by RT-PCR using degenerate primers based

on an alignment of human, dog and elephant HTR3C DNA

sequences. The 5¢end of the fragment was extended by 5¢-rapidamplification of cDNA ends using rabbit colon and small intestine

SMART rapid amplification of cDNA ends (Clontech, Mountain

View, CA, USA) cDNA libraries.

Cloning of human 5-HT3C, 5-HT3D and 5-HT3E cDNAsHTR3C, HTR3D and HTR3E were amplified by RT-PCR from

human intestine, kidney and colon Marathon-Ready cDNAs (BD

Transduction Laboratories, Lexington, KY, USA), respectively,

sequenced and subcloned into expression vectors. Appropriate

PCR primers were used for each variant to replace the native stop

codon with a 7–8 glycine spacer followed by Hemagglutinin (HA)

or FLAG tag and a newly created stop codon immediately after the

tag (Invitrogen, Carlsbad, CA, USA). Tagged subunits were

subcloned into the gateway destination vector pFastBacMam and

fully sequenced.

Distribution of mRNA in human tissuesThe distribution of the HTR3A–E subunits was assessed by TaqMan

RT-PCR (Holland et al. 1991) using transcript-specific primers and

probes (see Table S1). The mRNA based masterplates were derived

from human tissues obtained from multiple vendors and adhered to

GlaxoSmithKline (Brentford, UK) standards set for ethical use of

human tissue.

Tissue culture and transient transfectionsChinese Hamster Ovary (CHO)-K1 or Human Embryonic Kidney

293 (HEK293) cell lines were grown in D-MEM/F12 or D-MEM

media, respectively, supplemented with 10% foetal bovine serum

and 2 mM L-glutamine and maintained in a humidified incubator at

37�C with 5% CO2. Transient transfections were carried out using

lipofectamine 2000 (Invitrogen) according to the manufacturer’s

instructions. After 4 h in the presence of the lipofectamine/DNA

mix, the medium was replaced with the appropriate complete

medium and the cells were placed back in the incubator for another

40–48 h. For electrophysiological studies however, cells were

trypsinised and re-plated at a density of 4 · 104 cells/mL on sterile

polylysine-coated glass coverslips (BD Biosciences) prior to study

24–48 h post-transfection.

Electrophysiological investigation of subunit functionCHO or HEK293 cells were transiently transfected with 5-HT3

subunits, singly or in heteromeric combinations, with green

fluorescent protein (GFP) included as a visual marker of successful

transfection [in some experiments cyan fluorescent protein (CFP,

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 384–396

Characterisation of novel 5-HT3 receptor subunits | 385

Clontech) was included as a control for effects potentially resulting

from an additional translational burden on the cell], and the

properties of the resultant channels investigated using standard

whole-cell patch-clamp methods. Recordings were performed at

room temperature (20–24�C) using an Axopatch 200B amplifier

controlled via the pClamp9 software suite (Axon Instruments,

Union City, CA, USA). The intracellular and extracellular solutions

used were (mM): CsCl 140, MgCl2 4, EGTA 10, HEPES 10 and

NaCl 130, KCl 5, CaCl2, MgCl2 1, Glucose 30, HEPES 25,

respectively (both pH 7.3). Cells were voltage-clamped at )60 mV,

unless otherwise stated, and agonists and antagonists were applied

to cells via an automated fast-switching solution exchange system

(SF-77B, Warner Instruments, Hamden, CT, USA). Current-voltage

relationships were established using a voltage-ramp protocol ()60to +60 mV, 1.2 mV/s, 100 ms). Data were analysed using Clampfit

(Axon Instruments), Excel (Microsoft) and Origin (Microcal).

Unless otherwise stated, data are presented as mean ± SEM and

Student’s t-test was used to assess statistical significance (p < 0.05

being regarded as significant).

Indirect immunocytochemical analysis of tagged 5-HT3 subunitsCells were analysed in suspension 48 h post-transfection using

standard immunocytochemistry under non-permeabilized condi-

tions. All staining steps were carried out on ice to minimise

internalisation. Immunostaining was carried out using primary rabbit

polyclonal anti-HA or FLAG tag (Sigma, St Louis, MO, USA) and

secondary Alexa fluor488-coupled goat anti-rabbit IgG (Invitrogen)

antibodies. Cell nuclei were stained with Hoechst reagent according

to the manufacturer’s instructions (Invitrogen). Cells were mounted

in Immu-Mount (Thermo Shandon, Waltham, MA, USA) and

analyzed using epifluorescence (Zeiss-Axioskop 2, Maple Grove,

MN, USA) and laser scanning (Leica TCS SP, Bannockburn, IL,

USA) microscopes.

Results

Prediction and detection of species orthologuesCoding sequences for the putative orthologues of the 5-HT3C, and 5-HT3E subunits from dog and chimpanzeewere predicted from genomic DNA and their orthologyconfirmed by phylogenetic analysis (Fig. 1). The chimpan-zee and dog 5-HT3D orthologues were predicted to includestop codons in their open reading frames. This, togetherwith the long branch-lengths and incongruent position ofdog 5-HT3D in the phylogenetic analysis suggests that5-HT3D may be a pseudogene in these species. Mouse andrat genomic sequences were BLASTed for sequenceshomologous to 5-HT3C, 5-HT3D and 5-HT3E. No sequencemore homologous than the mouse and rat 5-HT3A and5-HT3B genes were found. Rodent orthologues of 5-HT3C-E

were also searched for in the syntenic region of the mouseand rat chromosomes (Using ENSEMBL AlignSplice-View http://www.ensembl.org/index.html) but no codingsequences were returned.

To explore the species distribution of the subunits,mRNA encoding 5-HT3C and 5-HT3E was RT-PCR ampli-fied from cDNA derived from ferret tissues. Products ofsimilar size were seen for colon and lung: the HTR3Cproduct was 78% identical to dog HTR3C whilst thesequence from the HTR3E product was 96% identical todog HTR3E (Fig. S1). In addition, HTR3C was partiallycloned from rabbit colon yielding a sequence 73% identicalto human HTR3C at the protein level (Fig. S1). Amplifi-cation of HTR3C from guinea pig tissues was attempted but

HTR3A humanHTR3A chimp

HTR3A dog

HTR3A rat

HTR3A mouseHTR3B ratHTR3B mouseHTR3B chimp

HTR3B dog

HTR3B human

HTR3D chimp

HTR3D human

HTR3E chimpHTR3E human

HTR3D dog

HTR3C dog

HTR3C humanHTR3C chimpHTR3E dog

Fig. 1 Neighbour-joining phylogenetic tree,

showing the relationships between the de-

duced peptide sequences of the novel hu-

man 5-HT3 subunits and their orthologues

in chimpanzee, mouse, rat and dog. Chim-

panzee and dog sequences were predicted

from genomic sequence whereas human,

mouse and rat were based on cDNA.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 384–396� 2008 The Authors

386 | J. D. Holbrook et al.

extensive efforts failed, suggesting the subunits are alsoabsent from this species.

Cloning and mRNA distribution of the novel subunits andtheir splice variantsIn human tissues, we confirmed the existence of the long andshort splice variants of HTR3A previously reported (Hopeet al. 1993; Downie et al. 1994; Belelli et al. 1995); thelonger variant (HTR3AL) has an insertion of 32 aa relative tothe shorter variant (HTR3AS; utilised in the functionalstudies in this paper) within the predicted transmembrane(TM) 3 region (Fig. S2). In addition a putative third variant

of HTR3A (HTR3Aext) was found, represented in thesequence databases (bt007204) with an alternative upstreamtranslation start site that would extend the N-terminus by sixamino acids (Fig. S2). The mRNA tissue distribution ofHTR3AS and HTR3AL were assayed by TaqMan (Figs 2and S3) indicating a relatively high level of expression inDRG, with both variants also clearly detectable in the GItract (colon, duodenum, ileum and jejunum), lymph nodesand tonsils.

There are three known splice variants of HTR3B (Tzvet-kov et al. 2007) which affect the translation initiation siteand therefore the N-terminus of the protein. One of these

HTR3A

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Fig. 2 Expression profiles of 5-HT3 receptor subunits in human tis-

sues assessed by TaqMan RT-PCR. Relatively high levels of the novel

subunits are detected in the DRG, GI tract and brain but subtype

differences are evident (see text & supplementary information). The

horizontal axis identifies the tissue from which the RNA was extracted

and the vertical axis indicates copies of mRNA detected per 50

nanogram of total RNA.



variants (HTR3B_V2) lacks the b1-b2 loop and therefore isunlikely to be functional and could operate as a dominant-negative. Taqman analysis of the longest variant (HTR3B)identified a relatively ubiquitous expression pattern withsubstantial expression in DRG, brain, kidney, liver, lung,testis and the gastrointestinal tract (Fig. 2) whereas theshorter variant (HTR3B_V1) resulted in a brain-specificexpression pattern (Fig. S3). The longest variant was usedfor the functional studies in this paper.

Initial attempts to clone human 5-HT3C resulted in theidentification of multiple PCR products of different sizesfrom a range of tissues (Fig. S4). In particular, products fromintestine and colon were larger than bands from liver andlung, sequence verification demonstrated that the only fulllength variant was from human intestine with other variantslikely to arise by exon deletion. We note that two of thevariants detected in human lung lack the Cys-loop region andhence are likely to be non-functional. TaqMan analysis ofHTR3C using primers to the Cys-loop containing exon 5,which should detect all functional variants, identified highestexpression levels in the DRG, lung and duodenum (Fig. S3 –HTR3C-2) whereas primers designed to exons 2 and 6uncovered more robust expression in the GI tract especiallythe cecum as well as DRG and lung. The longer ‘GI tractvariant’ was characterised further in this study (Fig. 2).

There are two known splice variants of 5-HT3D, the longervariant (HTR3DL, Fig. S2) has a 5¢ extension in the firstcoding exon, an inserted second exon and then a shorter exon3. Therefore, the two variants have very different peptidesequences at the N-terminus. The shorter variant (HTR3Ds,Fig. S2) seems to be the most frequently reported and wasused in this study. Its mRNA expression levels are low inmost tissues but expression is detectable in DRG and avariety of other tissues (Fig. 2). Primers designed to detectthe longer variant returned very low expression levels(Fig. S3). Other known variants lack the Cys-loop regionand so are predicted to be non-functional. 5-HT3E also hasthree known variants that differ in coding exons 1, 2 and 3(Fig. S2). Variant 3 is not represented in the public sequencedatabases but was cloned within this study. Given that it wasthe most readily cloned and contains all functional motifs itwas used in the further studies described in this paper.TaqMan analysis demonstrated that it was ubiquitouslyexpressed across a wide range of tissues including the GItract and was present at especially high levels in the DRG,brain and pituitary gland (Fig. 2).

Functional characterisation of novel 5-HT3 receptorsubunitsTo assess whether the newly identified subunits formreceptors at the plasma membrane, C-term HA- or FLAG-tagged constructs were generated. Based on the structure ofthe homologous nicotinic acetylcholine receptor (Unwin2005) and the 3D model of the 5-HT3 receptor (Lummis

et al. 2005), we hypothesized that tags positioned at theC-term, rather than N-term, would cause least interferencewith receptor function and provide greater accessibility toantibodies for immunocytochemical analysis. Indeed, inindependent experiments, a 5-HT3A-lumio C-term taggedconstruct gave rise to robust 5-HT-gated responses indistin-guishable from the wild type (WT) receptor (C. Gill,unpublished observations). Mammalian expression vectorsfor 5-HT3B-HA, 5-HT3C-FLAG, 5-HT3D-FLAG and 5-HT3E-FLAG were therefore generated with an extra seven or eightglycine residues included between the receptor sequence andtag to allow for maximum flexibility and accessibility toantibodies at the extracellular surface (no additional aminoacids were introduced within the 5-HT3 coding sequence).

Immunocytochemical analysis of the various taggedsubunits using standard fluorescent microscopy approachesindicated expression at the cell periphery in a subset of cells(Fig. 3a). This staining pattern is as expected for thetransient transfection approach taken and is consistent withplasma membrane localisation of each of the tagged variants.This result was confirmed by confocal microscopy showinga circumferential staining (although not continuous incertain instances), consistent with membrane-bound expres-sion for each variant (Fig. 3b). Only background stainingwas seen in neighbouring untransfected cells (Fig. 3a), or incells transfected with the empty vector alone (data notshown). These data suggest that at least a proportion of thesubunits assemble as receptors at the plasma membrane.

(a) (b)

5-HT3C

5-HT3D

5-HT3E

5-HT3B

Fig. 3 Immunocytochemical analysis of the tagged 5-HT3 receptor

subunits 5-HT3B-HA, 5-HT3C-FLAG, 5-HT3D-FLAG and 5-HT3E-FLAG

in CHO cells. (a) Conventional fluorescence microscopy. Panels show

representative fields of view identifying the respective 5-HT3 receptors

(green fluorescence; left panels) and Hoechst nucleic acid staining

(blue fluorescence; right panels) overlaid to visualise neighbouring

non-transfected cells. (b) Laser scanning confocal microscopy. Rep-

resentative confocal images showing positive staining for HA or FLAG

tag. Arrows in (a) indicate plasma membrane-bound immunoreactive

signals.



Attempts to further characterise the different homomeric5-HT3 receptors at the protein level were made by westernblotting using corresponding anti-tag antibodies (seeFig. S5). 5-HT3B-HA, 5-HT3C-FLAG and 5-HT3E-FLAGall showed abnormal electrophoretic profiles, consisting ofmultiple distinctive bands with molecular weights lower thantheir predicted molecular weights of 50–60 kDa, potentiallyindicating that additional mechanisms or accessory proteinsmay be required for achieving or maintaining receptorintegrity.

To determine if the novel 5-HT3 subunits were capable offorming functional homomeric receptors, we expressed eachsubunit individually in CHO cells using green fluorescentprotein as a marker of transfection and used whole-cell patchclamp electrophysiology to examine 5-HT-evoked responses.In all experiments, a comparatively high test concentration of5-HT was applied (�50-fold > EC50 at 5-HT3A) and main-tained for at least 20 s, and parallel experiments with 5-HT3A

were used as a positive control. Robust responses werereadily identifiable in 5-HT3A transfected cells in response to30 lM 5-HT. In contrast, no functional responses to 100 lM5-HT were detected in WT cells or those transfected with 5-HT3B or any of the novel subunits (Fig. 4) indicating that thenovel genes do not form functional 5-HT-gated homomericreceptors.

To begin to assess the ability of the novel subunits to formheteromeric receptors we first compared the 5-HT responsesof cells transiently transfected with either 5-HT3A or 5-HT3A + 5-HT3B to define suitable conditions for the clearidentification of known heteromeric receptors. Transfectionof cells with 5-HT3A + 5-HT3B led to currents with a similaroverall magnitude and appearance to 5-HT3A receptors(Fig. 5), however, these responses activated significantlymore rapidly (Rise time 1758 ± 139 compared to899 ± 38 ms, for 5-HT3A versus 5-HT3AB receptors, respec-tively) and showed linear rather than inward rectification[Rectification ratio (I+60mV/I)60 mV) = 1.02 ± 0.04 (n = 5)rather than 0.34 ± 0.01(n = 4); reversal potentials were

unchanged; Fig. 5] indicative of the formation of functionalheteromeric 5-HT3AB receptors (Davies et al. 1999; Das andDillon 2003). Following a recent report demonstrating thatmouse 5-HT3AB receptors were less sensitive to picrotoxinblockade than 5-HT3A (Das and Dillon 2003), we alsoexamined this aspect of human receptor pharmacology. Wefound that picrotoxin inhibits the human 5-HT3A receptormaximally at 300 lM (99 ± 0.3% block) yet only resulted in84 ± 2% block in cells transfected with 5-HT3A + 5-HT3B

(Fig. 5d). Overall, these data are consistent with the expres-sion of functional human 5-HT3AB receptors providingconfidence in the methods employed to detect novel 5-HT3

receptor heteromerisation; our results also suggest thatpicrotoxin is a less potent inhibitor at the human comparedto mouse 5-HT3 receptors (Das and Dillon 2003, 2005).Thus, we next compared recordings from cells transientlytransfected with either 5-HT3A or 5-HT3A + 5-HT3C. 5-HT-evoked responses in cells transfected with 5-HT3A + 5-HT3C

were similar in appearance but significantly smaller thancurrents recorded in cells transfected with 5-HT3A alone(Fig. 6); this difference was apparent in a comparison of allcells studied or the subset of responding cells only. Furtherexamination of the properties of the 5-HT responsesincluding the response rise-time, current-voltage relationshipor degree of blockade by picrotoxin did not, however,indicate any other clear signature suggestive of the occur-rence of a novel heteromeric receptor with distinct biophys-ical or pharmacological properties (Fig. 6).

The 5-HT3C experiments contained a DNA control,namely inclusion of an equivalent amount of emptypcDNA3.1 vector in the 5-HT3A transfection to equate tothe amount of 5-HT3C used in the co-transfection experiment(see legend to Fig. 6). To provide additional confidence inthe conclusion that the reduction in 5-HT3A function by 5-HT3C was a specific effect of this subunit we also incorpo-rated a further ‘translational control’ in parallel with ourexperiments with 5-HT3D. In these studies, conducted inHEK293 cells, CFP was included in place of the test subunit

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Fig. 4 Functional assessment of 5-HT3 receptors expressed in CHO

cells. Whole-cell patch clamp recordings of cells transiently trans-

fected with the subunit of interest were visually identified using GFP

and test responses to 30 or 100 lM 5-HT were recorded to assess

functionality. (a) robust 5-HT3A responses were readily identifiable

upon application of 30 lM 5-HT. Seven of eight cells responded with a

mean inward current of 1677 ± 1166 pA (Range 164–4086 pA). (b) No

functional responses were detected in cells transfected with 5-HT3B or

any of the novel subunits. Mean currents were 1 ± 5, 0 ± 1, 1 ± 4, and

2 ± 2 pA for 5-HT3B, 5-HT3C, 5-HT3D, and 5-HT3E, respectively (Range

was £ 10 pA for all recordings; n ‡ 4 for each subunit).



to determine if an increased translational load on the cellbecause of the additionally expressed protein may impact 5-HT3A expression. In each case, the responses to 5-HT werenot significantly different and there were no apparentdifferences in current kinetics or current-voltage relationships(Fig. 7). Responses detected in cells from all three groupsalso showed similar sensitivity to 30 lM picrotoxin (Fig. 7).We therefore conclude that 5-HT3D has no detectable effecton 5-HT3A function and, based on the CFP data, any effectsdetectable on peak current with any of the novel test subunitsare unlikely to be because of the effects of altered DNA orprotein load on the cell.

Our final set of heteromer experiments conducted with 5-HT3E (Fig. 8) indicated a clear trend towards a decrease inpeak current on co-transfection of the 5-HT3E subunit.Comparison of the current responses indicated that therewere no apparent difference in kinetics and current-voltagerelationships did not indicate any alteration in rectificationratio or reversal potential. Furthermore, no change inpicrotoxin sensitivity was detected (Fig. 8).

Discussion

In this study we have used a range of in silico and in vitroapproaches to assess the roles of the novel 5-HT3C-E

subunits, with particular emphasis on human physiology.All three genes encode the four TM domains found in the 5-HT3A and 5-HT3B subunits whereas the cysteine loop region(a defining motif of the mammalian ‘Cys-Loop’ family) isconserved in 5-HT3C and 5-HT3E but absent from 5-HT3D

likely rendering this subunit non-functional. Similarly, theorthologous 5-HT3D sequences from the chimpanzee and dogwere predicted to code for open reading frames includingstop codons, which suggests that in these species 5-HT3D is apseudogene.

Contrary to Niesler et al. (2003) but in line with theSouthern Zooblot finding of Karnovsky et al. (2003), wefound that 5-HT3C and 5-HT3E are not human specific.However, we confirm the apparent loss of these genes fromthe rodent lineage. This is particularly interesting becausethe rodent digestive system differs dramatically from that

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5-HT 3A

5-HT 3A + 3B

V h mV

[Picrotoxin]

0

20

40

60

80

100

% r

espo

nse

inse

nsiti

ve

to b

lock

ade

by p

icro

toxi

n

5-HT

*

*

**

** **

3A

5-HT 3A + 3B

300 µM 30 µM 100 µM

P < 0.05 P < 0.01

(a) (b)

(c) (d)

Fig. 5 Characterisation of heteromeric 5-HT3AB receptors. Whole-cell

patch clamp recordings of CHO cells transiently transfected with either

5-HT3A or 5-HT3A + 5-HT3B (1 : 1 ratio; GFP included as a positive

marker of transfection) were used to ascertain hallmarks of receptor

heteromerisation. (a) Current-voltage relationships were established

using a ramp protocol as shown, which was timed to coincide with the

peak of the 5-HT-gated current. (b) Current-voltage relationships

established in cells transfected with 5-HT3A were inwardly rectifying

[I+60 mV/I)60 mV = 0.34 ± 0.01 (n = 4)], whereas those transfected with

5-HT3A + 5-HT3B were linear [I+60mV/I)60 mV = 1.02 ± 0.04 (n = 5)].

Reversal potentials were not significantly different: )2.7 ± 0.6 for

5-HT3A and )5.3 ± 0.5 mV for 5-HT3B. (c) 5-HT responses showed

differential sensitivity to picrotoxin: % block was near maximal with

300 lM picrotoxin versus 5-HT3A receptors (99 ± 0.3%) yet only

resulted in 84 ± 2% (n ‡ 3; p < 0.01) block in cells transfected with

5-HT3A + 5-HT3B. (d) The occurrence of a significant (p < 0.05)

picrotoxin-insensitive component of 5-HT activated-currents, over the

concentration range 30–300 lM, is consistent with the expression of

heteromeric 5-HT3AB receptors (Das and Dillon 2003).



of human. Rodents are unable to vomit (a mechanismactivated via 5-HT3 receptors in other species; (Malik et al.2006) and excrete faeces as dry pellets, in contrast to mostother mammals; rabbits excrete faecal pellets but exhibitcoprophagia to fully digest and absorb the water contente.g. (Ebino et al. 1993). These differences in GI physiologycould be related to the loss of the 5-HT3C and 5-HT3E

genes within the rodent lineage. Clearly, the lack of afull compliment of 5-HT3 receptor subunits suggeststhat conclusions drawn from studies on 5-HT3 receptorfunction in these species will not necessarily translate tohumans.

We were unable to PCR-amplify guinea-pig orthologues of5-HT3C and 5-HT3E subunits, however, a small amount ofguinea-pig genomic sequence (AAKN01387346), which isclosely homologous to 5-HT3C does exist in public databasesand hence may represent a pseudogene. It therefore seemslikely that functional 5-HT3C and 5-HT3E subunits were lostbefore separation of the guinea-pig and muroid rodentlineages. It is noteworthy that our identification of coding

sequences for 5-HT3C and 5-HT3E in rabbits, ferrets and dogidentifies potential laboratory species for future studies intothe functions of these genes.

In humans, we confirmed that 5-HT3C and 5-HT3E mRNAis expressed in tissues of the GI tract. However, we also notethat the distribution of 5-HT3C transcripts may be moreskewed in favour of the gut than previously suggested(Niesler et al. 2003) indicating a potentially prominent roleof 5-HT3C and 5-HT3E here. Such an hypothesis is supportedby a recent report of high and approximately equal levels of5-HT3C and 5-HT3E mRNA in human duodenum mucosa,compared with low levels of 5-HT3A and 5-HT3B expression;the authors argued that, in this tissue, the 5-HT3CE heteromerrepresents the predominant 5-HT3 receptor (van Lelyveldet al. 2008). Selective 5-HT3 receptor antagonists are used totreat emesis (Costall and Naylor 2004; Thompson andLummis 2006) and irritable bowel syndrome (De and Tonini2001; Spiller 2004). Fasching et al. (2008) showed asignificant association between a coding polymorphism in5-HT3C and response to anti-emetic 5-HT3 antagonists

5-HT3A 5-HT3A + 3C

10 µM 5-HT 10 µM 5-HT

2 s

100 pA

2 s

100 pA

5-HT3A

10 µM 5-HT

Control 30 µMpicrotoxin

Wash

100 pA

2 s

5-HT3A + 3C

Control 30 µMpicrotoxin

Wash

2 s

50 pA

*+

Vh mV

I Nor

mal

ised 5-HT3A

5-HT3A + 3C

–60 –40 –20 20 40 60

–1.0

–0.5

0.5

1.0

0

100

200

300

400

500

600

Pea

k cu

rrre

nt (

pA)

All cells Responding cells

5-HT3A

5-HT3A + 3C

n = 23 n = 20n = 27 n = 28

(a) (b)

(c) (d)

Fig. 6 Functional assessment of the potential for heteromerisation

between 5-HT3A and 5-HT3C. Whole-cell patch clamp recordings of

CHO cells transiently transfected with either 5-HT3A (1 : 3 ratio with

1 lg 5-HT3A cDNA + 3 lg empty pcDNA3.1 vector + 0.4 lg GFP) or

5-HT3A + 5-HT3C (1 : 3 ratio; 1 lg 5-HT3A + 3 lg 5-HT3C + 0.4 lg

GFP) were conducted to investigate the occurrence of potential sig-

natures of heteromeric channel assembly. (a,b) Currents recorded in

cells transfected with 5-HT3A + 5-HT3C were similar in appearance but

significantly smaller than currents recorded in cells transfected with 5-

HT3A alone [significance was apparent between a pairwise compari-

son of all cells (+, black bars) and responding cells only (*, grey bars)].

(c) Current-voltage relationships did not indicate any significant dif-

ference between responses detected in cells transfected with 5-HT3A

[Erev = )4.5 ± 0.6 mV (n = 9) I+60 mV/I)60 mV = 0.50 ± 0.04 (n = 9)] or

5-HT3A + 5-HT3C [Erev = )3.9 ± 1.8 mV (n = 7); I+60 mV/I)60 mV = 0.45

0.07 (n = 7)]. (d) Responses detected in cells transfected with 5-HT3A

and 5-HT3A + 5-HT3C showed similar sensitivity to 30 lM picrotoxin: %

Block was 52 ± 3 (n = 4) 58 ± 4 (n = 3), respectively.



during chemotherapy (Fasching et al. 2008). As the 5-HT3C,

5-HT3D and 5-HT3E genes are closely situated on chromo-some 3 it is also possible that polymorphisms in 5-HT3D or5-HT3E, contribute to the genetic association. It is of interestthat the expression of each of the 5-HT3 subunits is also highin the DRG, providing a functional link between the gut andthe spinal cord. Potentially, therefore, the existence of novel5-HT3 receptor subunits within these tissues offers thepossibility of identifying improved therapies for GI condi-tions.

Using immunocytochemical approaches to study C-termi-nally tagged subunits we demonstrated that each of the5-HT3B-E variants can independently reach the plasmamembrane. This finding contrasts with reports from others(Boyd et al. 2002, 2003; Niesler et al. 2007) using morecommonly applied N-terminal tagging approaches (Boydet al. 2002, 2003) that indicate that with the exception of5HT3A, the other variants do not appear to reach the plasma

membrane unless co-expressed with 5-HT3A. Based on ourconfocal microscopy data, functional validation of a 5HT3A-lumio C-term tagged construct and the evaluated structuraldata leading us to take this novel approach (Lummis et al.2005; Unwin 2005), we therefore suggest that the C-terminusis more accessible to antibodies at the cell surface and faraway from the pore region enabling efficient HA and FLAGtag labelling with minimal effects on receptor function.Although other possible explanations for these differingresults exist, such as endogenous expression of 5HT3A

receptors (Boyd et al. 2002, 2003; Sun et al. 2003; Quirket al. 2004; Niesler et al. 2007); N.B. we did not detectfunctional expression of 5HT3A in WT cells), differentialexpression of chaperones such as RIC-3 (Castillo et al. 2005;Cheng et al. 2005, 2007), or components of receptorglycosylation (Simon and Massoulie 1997; Quirk et al.2004) or Protein Kinase C pathways (Sun et al. 2003) in thedifference host systems used, direct comparative studies to

(a) (b)

(c) (d)

0

100

200

300

400

500

600

5-HT 3A + 3D

5-HT 3A

+ CFP

Pea

k cu

rren

t (pA

)

5-HT 3A

n = 20 n = 24 n = 20 n = 18 n = 23 n = 18

10 µ M 5-HT

5-HT 3A 5-HT 3A + 3D 5-HT 3A + CFP

10 µ M 5-HT 10 µ M 5-HT

200 pA

4 s

200 pA

4 s

5-HT 3A + 3D

5-HT 3A + CFP

5-HT 3A

Control 30 µ M picrotoxin Wash



–60 –40 –20 2 0 4 0 6 0

–1.0

–0.5

0.5

1.0

V h mV

I Nor

mal

ised

5-HT 3A

5-HT 3A + 3D

5-HT 3A + CFP

10 µ M5-HT



between 5-HT3A and 5-HT3D. Whole-cell patch clamp recordings of

HEK cells transiently transfected with either 5-HT3A or 5-HT3A + 5-

HT3D (1 : 3 ratio; 1 lg 5-HT3A cDNA and 3 lg 5-HT3C cDNA; GFP also

included) were conducted to investigate the occurrence of potential

signatures of heteromeric channel. In a third transfection group CFP

was included in place of 5-HT3D to act as a translational control (1 : 3

ratio; 1 lg 5-HT3 and 3 lg CFP; GFP also included). (a,b) Comparison

between peak amplitude currents from all cells (black bars) or

responding cells only (grey bars) were not significantly different be-

tween those cells transfected with 5-HT3A + 5-HT3D to those trans-

fected with 5-HT3A alone. There were no differences in current

kinetics. Inclusion of CFP in place of 5-HT3D also failed to alter current

kinetics or peak amplitude. (c) Current-voltage relationships did not

indicate any significant difference between responses detected in cells

from the three transfection groups: 5-HT3A [Erev = )2.8 ± 0.9 mV

(n = 5) I+60 mV/I)60 mV = )0.44 ± 0.04 (n = 5)] or 5-HT3A + 5-HT3D

[Erev = )4.6 ± 2.0 mV (n = 4) I+60 mV/I)60 mV = )0.50 ± 0.06 (n = 4)]

or 5-HT3D + CFP [Erev = )1.0 ± 1.9 mV (n = 5) I+60 mV/I)60 mV =

)0.50 ± 0.03 (n = 5)]. (d) Responses detected in cells from all three

groups showed similar sensitivity to picrotoxin: % block was 70 ± 4%,

70 ± 4% and 69 ± 5%, respectively (n = 5).



ascertain if N-terminal tagging approaches have a directeffect on 5-HT3 receptor translocation are now warranted.

We used whole-cell patch clamp electrophysiology todetermine if 5-HT3C, 5-HT3D and 5-HT3E can form func-tional homomers or distinct heteromeric receptors with5-HT3A. Our deduced human 5-HT3A receptor propertiesare consistent with previous work (Maricq et al. 1991;Miyake et al. 1995; Reeves and Lummis 2002). The sameconditions did not provide evidence for functional expressionof 5-HT3B homomers, as expected, and additionally demon-strate that 5-HT3C, 5-HT3D and 5-HT3E are incapable offorming functional 5-HT-gated ion channels. These findingsare in agreement with (Niesler et al. 2007) who employed anon-direct aequorin Ca2+-based cellular assay to assessfunctionality, and we suggest are most likely because of theimpact of differences in key amino acids in the TM2 channellining domains of 5-HT3A and the other subunits. Forexample, the ‘polar ring’ (2¢ position in TM2 (Peters et al.2005) is normally occupied by a serine or threonine residueby members of the Cys-loop family and yet the novel

subunits bear a proline in this position, a non-conservativechange that may have a large impact on the structure of thispart of the channel, altering ionic conduction and/or selec-tivity. Similarly, the -1¢ ‘intracellular ring’ normally bears anacidic residue such as glutamate in 5-HT3A and the majorityof cationic Cys-loop receptors yet 5-HT3C, 5-HT3D and5-HT3E all have asparagines at this position (Fig. S2).Interestingly, however, the 5¢ lysine residue, a feature of the5-HT3 TM2 domain implicated in desensitisation, is retainedin all of the subunits (Gunthorpe et al. 2000). Given that ourimmunocytochemistry studies defined localization of the 5-HT3C, 5-HT3D and 5-HT3E subunits at the plasma membrane,the possibility that other (novel) agonists or co-agonists maybe required for activation requires further consideration.Other possibilities are that the particular splice variants weassayed are non-functional or accessory subunits may berequired for these novel subunits to attain functionality.

Studies to unambiguously define the occurrence of novelheteromeric 5-HT3 receptors are not straightforward. Therequired co-expression of the test subunit with 5-HT3A

(a) (b)

(c) (d)

0

400

800

1200

1600

5HT 3A + 3E

Pea

k cu

rren

t (pA

)

5-HT 3A

n = 26 n = 23 n = 21 n = 20

10 µ M 5-HT 10 µ M 5-HT

5-HT 3A + 3E 5-HT 3A

200 pA

2 s

–60 –40 –20 2 0 4 0 6 0

–1.0

–0.5

0.5

1.0

I Nor

mal

ised

V h mV

5-HT 3A

5-HT 3A + 3E

5-HT 3A +3 E



5-HT 3A

200 pA

2 s

10 µ M 5-HT



between 5-HT3A and 5-HT3E. Whole-cell patch clamp recordings of

CHO cells transiently transfected with either 5-HT3A or 5-HT3A + 5-

HT3E (1 : 3 ratio; 1 lg 5-HT3A + 3 lg 5-HT3E + 0.4 lg GFP) were

conducted to investigate the occurrence of potential signatures of

heteromeric channel assembly. (a,b) Currents recorded in cells

transfected with 5-HT3A + 5-HT3E showed a trend towards smaller

peak amplitudes compared to those recorded from cells transfected

with 5-HT3A alone. This difference was not significant following com-

parison between all cells (black bars) or responding cells only (grey

bars). There were no apparent differences in current kinetics. (c)

Current-voltage relationships did not indicate any significant difference

between responses detected in cells transfected with 5-HT3A

[Erev =)0.6 ± 0.6 mV (n = 12) I+60 mV/I)60 mV = )0.42 ± 0.04 (n = 12)]

or 5-HT3A + 5-HT3E [Erev = )1.7 ± 1.2 mV (n = 9) I+60 mV/I)60 mV =

)0.47 ± 0.08 (n = 9)]. (d) Responses detected in cells transfected

with 5-HT3A and 5-HT3A + 5-HT3E showed similar sensitivity to

picrotoxin: % block was 79 ± 5% and 74 ± 4%, respectively (n = 9).



means that there is a background level of homomeric 5-HT3A

receptor activity to deal with before the contributions of anyadditional heteromeric receptors can be resolved. The designof our comparative studies was therefore carefully controlledwith the same amount of 5-HT3A DNA used in single ordouble transfections, such that the effects of addition of thesecond subunit were tested upon a uniform background.Parallel assessments of 5-HT3Awere also made to control forany variation in transfection efficiency or expression and thestudies employed high 5-HT concentrations to minimisepotential contributions of changes in agonist affinity to theresults obtained. Furthermore, our DNA (empty vector) and‘translational’ (protein load) control experiments providedconfidence that any effects on 5-HT3A function werespecifically because of an interaction with 5-HT3A. Usingthis approach we successfully validated the known hetero-meric interaction between 5-HT3B and 5-HT3A (Davies et al.1999; Das and Dillon 2003). Next we used this approach tocharacterise the effects of 5-HT3C, 5-HT3D and 5-HT3E on 5-HT3A receptor properties. Like 5-HT3B, the predictedchannel lining TM2 and ‘Membrane Associated’ (MA)stretch (Kelley et al. 2003) regions of the novel subunitsdiffered markedly to those of 5-HT3A so we hypothesizedthat incorporation of these subunits into 5-HT3 receptorswould yield channels with markedly altered properties.However, with the exception of a significant decrease inthe mean current detectable for 5-HT3C, the profiles of 5-HT3

receptor responses were not altered by expression of thenovel subunits. The effect of 5-HT3C on the mean 5-HTcurrent suggests a specific effect on 5-HT3 receptor conduc-tance or expression. In this respect, it is noteworthy that ofthe novel subunits, 5-HT3C exhibits the greatest disruption ofthe Membrane Associated stretch with a number of the keyarginine residues identified in 5-HT3A (Kelley et al. 2003)not being represented in the 5-HT3C sequence (Fig. S2). Ourfindings therefore provide a potential explanation for the�30% decrease in apparent maximum efficacy (Emax) of 5-HT mediated Ca2+ responses in 5-HT3AC expressing cellsnoted by (Niesler et al. 2007). These authors also describeeffects on Emax for the contribution of the 5-HT3E

(described in Fig. S2 as HTR3E_V2) (44% increase) and5-HT3Ea (Fig. S2 as HTR3E_V1) (57% decrease) variants.Our studies utilised a further 5-HT3E variant, V3, and hencealthough they also indicate a potential interaction, thedirection and magnitude of effects differ and cannot bereconciled based on the available information. Furtherstudies are needed to understand this apparently divergentregulation by 5-HT3E variants.

Overall, the lack of any overt change in any measuredreceptor properties beyond peak current response in theheteromer experiments means that the present data do notprovide definitive evidence for the contribution of 5-HT3C and5-HT3E as channel lining subunits. Niesler et al. (2007)reported changes in Emax and Bmax as well as immunopre-

cipitation evidence that the novel subunits form novelheteromeric receptors. Clearly, in the absence of the directevidence of changes in receptor properties sought here,whether or not the novel subunits contribute to 5-HT3 receptordiversity remains to be determined. The effects of novelsubunits on Emax, Bmax and peak current are of coursesuggestive of an interaction between the proteins in therecombinant host cell and further studies are now warranted toexamine these effects in greater detail and understand theirrelevance to native tissue biology. We also note that thepotential for subunit heteromerisation and interaction need notbe bounded by receptor subfamilies (van Hooft et al. 1998),hence many additional possibilities need to be addressed toenable the full potential for the novel 5-HT3 subunits tocontribute to receptor diversity to be appreciated. Suchconsiderations need to pay close attention to the agonistemployed and the potential requirement for additional subunitsand/or accessory proteins to reconstitute or regulate function.

In conclusion, we have provided compelling data demon-strating the conservation of 5-HT3C and 5-HT3E throughmammalian evolution. Indeed, the maintenance of theirmRNA expression in human GI tissue is suggestive of a rolein moderating the gut’s response to 5-HT. Our demonstrationthat these subunits are non-functional when expressed aloneindicates that their primary role may be tomodulate or regulatethe responses of other 5-HTor more distant Cys-loop receptorrelatives. Indeed, recent discoveries of single nucleotidepolymorphisms in HTR3B linked to major depressive disor-der, in particular, highlight the key roles of such subunits asmodulators of receptor function and neurotransmitter signal-ling (Krzywkowski et al. 2008). Further appreciation of thecontributions of these subunits to 5-HT receptor biology andhuman physiology, may allow us to improve upon currenttherapeutics targeting this receptor system.

Acknowledgement

We are grateful to David Murray and Daniel Hoston for construction

of the masterplates used for the TaqMan expression analysis.

Supporting information

Additional Supporting Information may be found in the online

version of this article:

Figure S1 Alignment of the novel HTR3 subunits found in

different species.

Figure S2 Multiple alignment of deduced peptide sequences of

human HTR3 subunit splice variants.

Figure S3 Additional TaqMan profiles of 5-HT splice variants.

Figure S4 RT-PCR and schematic of HTR3C cloning.

Figure S5 Expression analysis of the tagged 5-HT3 receptor

subunits 5-HT3B-HA, 5-HT3C-FLAG and 5-HT3E-FLAG in CHO

cells.

Table S1 Transcript-specific primers and probes used for

TaqMan analysis of 5-HT3 receptor subunits.



Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

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Characterisation of 5-HT3 receptor subunits

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