RESEARCH ARTICLE

Physiological responses of ionotropic histamine receptorsPxHCLA and PxHCLB to neurotransmitter candidates in abutterfly Papilio xuthusHiroshi D Akashi1 Pei-Ju Chen1 Tokiho Akiyama1 Yohey Terai1 Motohiro Wakakuwa1 Yasunori Takayama23Makoto Tominaga23 and Kentaro Arikawa1

ABSTRACTHistamine is the only known neurotransmitter released by arthropodphotoreceptors Synaptic transmission from photoreceptors tosecond-order neurons is mediated by the activation of histamine-gated chloride channels (HCLs) These histaminergic synapses havebeen assumed to be conserved among insect visual systemsHowever our understanding of the channels in question has thus farbeen based on studies in flies In the butterfly Papilio xuthus we haveidentified two candidate histamine-gated chloride channels PxHCLAand PxHCLB and studied their physiological properties using awhole-cell patch-clamp technique We studied the responses ofchannels expressed in cultured cells to histamine as well as to otherneurotransmitter candidates namely GABA tyramine serotoninD-L-glutamate and glycine We found that histamine and GABAactivated both PxHCLA and PxHCLB while the other molecules didnot The sensitivity to histamine and GABAwas consistently higher inPxHCLB than in PxHCLA Interestingly simultaneous application ofhistamine and GABA activated both PxHCLA and PxHCLB morestrongly than either neurotransmitter individually histamine andGABA may have synergistic effects on PxHCLs in the regionswhere they co-localize Our results suggest that the physiologicalproperties of the histamine receptors are basically conserved amonginsects but that the response to GABA differs between butterflies andflies implying variation in early visual processing among species

KEY WORDS Visual system GABA Chloride channelVisual processing Photoreceptor

INTRODUCTIONInsects perceive the visual world via photoreceptors in theircompound eyes Upon light stimulation the photoreceptorsdepolarize through activation of TRP channels (Hardie and Franze2012) which in turn leads to the hyperpolarization of postsynapticlamina (or large) monopolar cells (LMCs) in the first optic ganglionthe lamina In flies it has beenwell established that this sign-invertedsynaptic transmission is mediated by histamine-gated chloride

channels (HCLs) (Hardie 1987 1989 Sarthy 1991) Histaminehas been found in the photoreceptors of a number of speciesincluding horseshoe crabs barnacles cockroaches locusts mothsand butterflies (Battelle et al 1991 Callaway and Stuart 1989Hamanaka et al 2012 Naumlssel et al 1988 Simmons and Hardie1988 Skingsley et al 1995) suggesting that it is evolutionarilyconserved as the major transmitter of arthropod photoreceptors

Two genes hclA and hclB respectively encoding HCL subunitsHCLA and HCLB have been identified in the fruit fly Drosophilamelanogaster (Gengs et al 2002 Gisselmann et al 2002 Zhenget al 2002) These channels belong to a superfamily of Cys-loopreceptors which are pentameric ligand-gated channels (Thompsonet al 2010) Each receptor is composed of five subunits whichform homomeric or possibly heteromeric receptors In flies HCLAand HCLB subunits appear to form a heteromer in co-transfectedcells but it is unclear whether this occurs naturally in the visualsystem (Gisselmann et al 2004 Pantazis et al 2008) In contrastthe expression of HCLA and HCLB homomers has been confirmedHCLA exists in neurons postsynaptic to photoreceptors whileHCLB exists in glial cells in the lamina (Pantazis et al 2008) and inthe region of the medulla (the second optic ganglion) where longaxons of some photoreceptors terminate (Schnaitmann et al 2018)Electrophysiological analysis has demonstrated that HCLAhomomers expressed on cultured cells exhibit similar channelproperties to the histamine-gated chloride channels on isolatedLMCs (Pantazis et al 2008 Skingsley et al 1995) Furthermoreelectroretinography has revealed that hclA null mutants fail toproduce fast transient components derived from LMCs (Pantaziset al 2008) These studies suggest that the channels on the LMCsare HCLA homomers in D melanogaster

Although considerable evidence has accumulated for thefunctional role of HCLs in visual processing in flies their visualsystem is in fact peculiar in many aspects For example theprojection pattern of photoreceptor axons is unique In the laminaeight photoreceptors originating from seven neighboringommatidia are bundled together with five LMCs to form alamina cartridge in a connection scheme known as neuralsuperposition (Hardie 1985) Six of the eight photoreceptors areshort visual fibers (svfs) which terminate in the lamina andare presynaptic to LMCs The other two photoreceptors make nosynaptic contacts in the lamina and instead send their long axonsdirectly to the medulla where they terminate on their postsynaptictargets these photoreceptors are termed long visual fibers (lvfs)(Hardie 1985) No photoreceptors have lateral processes extendinginto neighboring lamina cartridges in flies (Rivera-Alba et al2011) which also appears to be a characteristic unique to this insectorder In moths and bees for example a single cartridge comprisesphotoreceptor axons from a single ommatidium and these haveReceived 27 April 2018 Accepted 3 September 2018

1Department of Evolutionary Studies of Biosystems SOKENDAI (The GraduateUniversity for Advanced Studies) Hayama Kanagawa 240-0193 Japan 2Divisionof Cell Signaling Okazaki Institute for Integrative Bioscience (National Institute forPhysiological Sciences) National Institutes of Natural Sciences 5-1 HigashiyamaMyodaiji Okazaki Aichi 444-8787 Japan 3Department of Physiological SciencesSOKENDAI (The Graduate University for Advanced Studies) 5-1 HigashiyamaMyodaiji Okazaki Aichi 444-8787 Japan

Author for correspondence (arikawasokenacjp)

KA 0000-0002-4365-0762

1

copy 2018 Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

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long lateral processes penetrating into neighboring cartridgeswhere they form specific neuronal circuits (Ribi 1976 Stoumlckl et al2016) Such variations in the lamina circuitry strongly suggest thatthe visual processing in the lamina is variable between speciesprobably reflecting differences in their visual ecology (Greineret al 2005)Accumulated evidence indicates that butterflies have spectrally

complex eyes and sophisticated color vision We have initiated acomprehensive investigation of the lamina of the Japanese yellowswallowtail butterfly Papilio xuthus which has a tetrachromaticvisual system (Koshitaka et al 2008) in order to understand howvisual information processing has diverged among insects In thepresent study we focused on the physiological function of HCLchannels inP xuthusWe first identified and cloned cDNAs encodingPxHCLA and PxHCLB subunits and analyzed their physiologicalproperties by performing whole-cell patch-clamp recordings Asseveral neurotransmitter candidates have been detected in the laminaofP xuthus (Hamanaka et al 2012) and other insects (Kolodziejczyket al 2008 Raghu and Borst 2011 Sinakevitch and Strausfeld2004) in addition to histamine we also studied HCL responses tothese molecules namely GABA (γ-aminobutyric acid) tyramineserotonin D-L-glutamate and glycine

MATERIALS AND METHODSAnimalsWe used spring-form adult Japanese yellow swallowtail butterfliesPapilio xuthus Linnaeus of both sexes The butterflies were taken

from a laboratory culture derived from eggs laid by females caughtaround the SOKENDAI campus in Hayama Kanagawa Japan

Identification of PxhclA and PxhclB cDNA sequencesWhole-head tissues were dissected and preserved in RNAlatersolution (Thermo Fisher Scientific Waltham MA USA) TotalRNAwas extracted from head tissue using TRIzol reagent (ThermoFisher Scientific) and used for first-strand cDNA synthesis byPrimeScript II 1st Strand Synthesis Kit (TaKaRa Shiga Japan) Weretrieved the P xuthus PxhclA and PxhclB predicted cDNAsequences from GenBank (httpwwwncbinlmnihgovgenbank)by BLASTN search (Altschul et al 1990) using D melanogasterhclA and hclB as query sequences PxhclA XM_013313248 andPxhclB XM_013319237 respectively We then retrieved the 5prime-and 3prime-untranslated region (UTR) of these homolog sequences fromPapilioBase (httppapiliobiotitechacjppapiliohtml) (Nishikawaet al 2015) We designed primers for PCR on both UTR sequencesas follows PxhclA_F1 (5rsquo-taaggcaaaaaacttcccgcgaac-3prime) andPxhclA_R1 (5rsquo-tgctaaatcgagactggaagag-3prime) and PxhclB_F1 (5prime-ct-tgtgtgcgcgattgtacagtgac-3prime) and PxhclB_R1 (5prime-gttccacgag-tattggttttcatc-3prime) We used these primer pairs to amplify thePxhclA and PxhclB full-length cDNAs by PCR using Ex Taq HSpolymerase (TaKaRa) Reactions for the cDNA amplifications werecarried out in a Mastercycler (Eppendorf Hamburg Germany)using the following protocol an initial denaturing step at 93degC for3 min 30 cycles of denaturation at 93degC for 1 min annealing at 60degC for 1 min extension at 72degC for 1 min 35 s and a final extension

HCLA Amel (DQ6671871)

HCLA Nvit (FJ8510891)

HCLA Pxut (LC373508)

HCLA Dmel (AY0497741)

HCLA Mdom (XM0051834833)

HCLB Amel (DQ6671881)

HCLB Nvit (FJ8510901)

HCLB Pxut (LC373509)

HCLB Dmel (AY0497751)

HCLB Mdom XM0051914862)

RDl Nvit (FJ8510971)

RDl Amel (KJ4857101)

RDl Pxut (XM0133236021)

RDl Dmel (M690572)

RDl Mdom (AB1775472)

GluCla Cele (AAA507851)

GluCl Amel (DQ6671851)

GluCl Nvit (FJ8510991)

GluCl Pxut (XM 0133181571)

GluCl Dmel (AF2975001)

GluCl Mdom (AB1775461)

NtR Dmel (FR6897501)

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Fig 1 Maximum likelihood treeshowing relationships betweenCys-loop chloride channel subunitnucleotide sequences of Apismellifera Nasonia vitripennisDrosophila melanogaster Muscadomestica and Papilio xuthus Grayboxes highlight PxHCLA and PxHCLBwhich were determined in this study andthe tree indicates that these sequencesare likely to be HCLA and HCLBhomologs Bootstrap values with 1000replicates are shown at each node andthe scale bar indicates two changes pernucleotide Caenorhabditis elegansglutamate-gated chloride channel isincluded in the tree for reference todeduce the domains and structuresparticular to Cys-loop chloride channelsDrosophila melanogaster nicotinicacetylcholine receptor is included as anoutgroup Accession numbers for eachsequence are shown in parenthesesHCLA histamine-gated chloridechannel subunit A HCLB histamine-gated chloride channel subunit B RDlresistant-to-dieldrin or GABA (gamma-aminobutyric acid)-activated chloride-selective receptor GluCla glutamate-gated chloride channel alpha GluClglutamate-gated chloride channel NtRnicotinic acetylcholine receptor AmelApis mellifera NvitNasonia vitripennisDmel Drosophila melanogasterMdomMusca domestica Pxut PapilioXuthus Cele Caenorhabditis elegans

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step at 72degC for 1 min DNA sequencing was performed for thepurified PCR products using BigDye Terminator v31 CycleSequencing Kit (Applied Biosystems Foster City CA USA)with the PCR primers or inner primers PxhclA_seqF1 (5prime-gacattt-tcttcgcccaaacgtg-3prime) and PxhclA_seqR1 (5prime-caggacatgatgacgatga-gac-3prime) and PxhclB_seqF1 (5prime-actttccacgagatgagcatacc-3prime) andPxhclB_seqR1 (5prime-ccaaagtaagcaacgatgtgac-3prime) The sequenceswere analyzed using an ABI3130xl DNA Sequencer (AppliedBiosystems) The PxhclA and PxhclB sequences determined in thisstudy were deposited in the international nucleotide sequencedatabase DDBJ (accession numbers LC373508 and LC373509respectively)

Sequence analysis of PxhclA and PxhclBWe obtained coding sequences of the following molecules fromGenBank Cys-loop chloride channel subunits in Apis melliferaNasonia vitripennis D melanogaster Musca domestica and Pxuthus glutamate-gated chloride channel in Caenorhabditiselegans nicotinic acetylcholine receptor of D melanogaster Theaccession numbers of these sequences are shown in Figs 1 and 2Wetranslated these sequences and constructed the multiple proteinsequence alignments with ClustalW (Thompson et al 1994) usinga gap-opening penalty and a gap-extension penalty of 3 and 18respectively with the Gonnet 250 protein weight matrix Forphylogenetic analysis we converted the aligned amino acidsequences back to nucleotide sequences In MEGA7 (Kumaret al 2016) we inferred a phylogenetic tree from the alignment byusing the Maximum Likelihood method based on the General TimeReversible model of sequence evolution with gamma distributedrates and 1000 bootstrap replicates All positions containing gapswere eliminated resulting in a total of 990 positions in the finaldataset for the analysis The tree is scaled with branch lengthsmeasured in the number of substitutions per site Nicotinicacetylcholine receptor ofD melanogasterwas used as the outgroup

Vector constructionWe designed primers PxhclA_F1_IF (5prime-taccgagctcggatctaagg-caaaaaacttcccgcgaac-3prime) and PxhclA_R1_IF (5prime-cacactggactagtgtgc-taaatcgagactggaagag-3prime) andPxhclB_F1_IF (5prime-taccgagctcggatccttgtgtgcgcgattgtacagtgac-3prime) and PxhclB_R1_IF (5prime-cacactggactagtggttccacgagtattggttttcatc-3prime) the 15 bases at 5prime end of these primersoverlapped with the ends of the cloning site of the linearized vectorfor cloning We used these primers to amplify the cDNA containingthe entire coding region of PxhclA or PxhclB by PCR usingPrimeSTAR Max DNA polymerase (TaKaRa) Reactions for thecDNA amplifications were carried out in Mastercycler (Eppendorf)using the following protocol an initial denaturing step at 98degC for3 min 30 cycles of denaturation at 98degC for 10 s annealing at 55degCfor 15 s extension at 72degC for 2 min and a final extension step at 72degCfor 1 min After purification of the PCR products we independentlysubcloned each cDNA (PxhclA and PxhclB) into the pcDNA31(+)vector (Invitrogen Carlsbad CA USA) using the In-Fusion HDCloning Kit (TaKaRa) The plasmids were transformed into ECOScompetent E coliDH5α cells (Nippon Gene Tokyo Japan) Each Ecoli cell with plasmid clone was grown in 2 ml of LB mediumsupplemented with ampicillin overnight The plasmid DNAs werepurified from the culture using QIAprep Spin Miniprep Kit (QiagenGermantown MD USA) We confirmed the target insert sequencesby PCR and sequencing

Cell transfectionWe transfected HEK293 cells with pGL1 vector (01 microg) and thepcDNA31(+)-PxhclA vector (1 microg) the pcDNA31(+)-PxhclBvector (1 microg) both vectors (05 microg each) or neither (as the control)in OPTI-MEM medium (Life Technologies) using LipofectaminePlus reagents (Life Technologies) After 3 h of incubation at 37degCwe replaced the OPTI-MEM medium with D-MEM medium(Life Technologies) and the cells were settled and cultured on12 mm-diameter coverslips at 37degC We thus obtained four types

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GluCla Cele (AAA507851)GluCl Amel (DQ6671851)GluCl Dmel (AF2975001)GluCl Mdom (AB1775461)GluCl Nvit (FJ8510991)GluCl Pxut (XM0133181571)

HCLA Amel (DQ6671871)HCLA Dmel (AY0497741)HCLA Mdom (XM0051834833)HCLA Nvit (FJ8510891)HCLA Pxut (LC373508)

HCLB Amel (DQ6671881)HCLB Dmel (AY0497751)HCLB Mdom (XM0051914862)HCLB Nvit (FJ8510901)HCLB Pxut (LC373509)

RDl Amel (KJ4857101)RDl Dmel (M690572)RDl Mdom (AB1775472)RDl Nvit (FJ8510971)

275

Amino acid positions

Loop G Loop D Loop A Loop E Loop B Loop F Loop C

RDl Pxut (XM0133236021)

Fig 2 Alignment of amino acid sequences of loop structures containing putative agonist binding sites The residues are deduced from the alignments toC elegans GluCla the 3D structure of which has been determined (Hibbs and Gouaux 2011) Asterisks indicate conserved sites across all molecules Grayboxes indicate the sites that differ among the channels in a manner reflecting their specific agonists Accession numbers for each sequence are shown inparentheses Sequences between loops are omitted from the figure and represented by hyphens The number of hyphens does not indicate the length of thesequences omitted GluCla glutamate-gated chloride channel alpha GluCl glutamate-gated chloride channel HCLA histamine-gated chloride channel subunitA HCLB histamine-gated chloride channel subunit B RDl resistant-to-dieldrin and GABA-activated chloride-selective receptor Cele Caenorhabditis elegansAmel Apis mellifera Dmel Drosophila melanogaster Mdom Musca domestica Nvit Nasonia vitripennis Pxut Papilio xuthus

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of cells expressing PxHCLA homomer PxHCLB homomer acombination of the two ie PxHCLAB or neither of them Wecarried out more than 30 independent transfections throughoutthe study

Electrophysiological recordingsWe performed whole-cell patch-clamp recordings as describedpreviously (Takayama et al 2017) A coverslip with cultured cellson the top was placed in the chamber mounted on a Nikon EclipseTE2000-S inverted microscope Standard bath solution for thewhole-cell patch-clamp recording contained (in mmol lminus1) 140NaCl 5 KCl 2 CaCl2 2 MgCl2 10 glucose and 10 Hepes Thissolution was also used as a vehicle for chemicals (histamineGABA tyramine serotonin L-D-glutamate and glycine) tested inthe following experiments The bath solutions were maintained atabout 26degC and perfused through silicone tubes by gravity Thesolution in the pipette used for thewhole-cell patch-clamp recordingcontained (in mmol lminus1) 140 KCl 5 EGTA and 10 Hepes The pHof all solutions was adjusted to 74 by NaOH The whole-cellrecording datawere collected at a sampling frequency of 10 kHz andlow-pass filtered with a cut-off of 5 kHz for analysis (Axopatch200B Amplifier Molecular Devices) which was performed usingpCLAMP 104 software (Molecular Devices) For the experiments

analyzing the responses of PxHCLs to agonists voltage-ramppulses (300 ms duration) from minus100 to +100 mV were appliedevery 5 s to the patch-clamped cell which was held at 0 mV To testcurrent stability during the 300 ms of voltage-ramp pulses voltage-step pulses (300 ms) from minus100 mV to +100 mV with an incrementof 20 mVwere applied to the patch-clamped cell held at 0 mV every60 s to obtain a stable base line Whole-cell patch-clamp recordingswere performed 16ndash22 h after transfection of HEK293 cells All ofthe patch-clamp experiments were performed at room temperature

Statistical analysisWe obtained the current responses at minus60 mV in the voltage-ramppulses for analysis To analyze the histamine and GABA dosedependency of PxHCLs the current responses were normalized tothe maximal current response (IImax) We used these normalizedcurrents to draw dosendashresponse curves by fitting the data using thesimple Hill function

I

Imaxfrac14 1

1thorn ethethEC50THORN=ethfrac12agonistTHORNTHORNnH eth1THORN

where EC50 represents the concentration of histamine or GABArequired to elicit 50 of Imax lsquoagonistrsquo is either histamine orGABA and nH represents the Hill coefficient To analyze the effects

GABA10 mmol lndash1

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Fig 3 Representative current traces of PxHCLA expressed in HEK293 cells under histamine and GABA application (AB) Representative currenttraces showing activation of PxHCLA by histamine (HA 10 μmol lminus1 and 1 mmol lminus1 A) and GABA (10 and 50 mmol lminus1 B) in a dose-dependent mannerBars indicate the duration of chemical application (CD) Currentndashvoltage (IndashV ) relationship for PxHCLA at two HA (C) and GABA (D) concentrations A linearrelationship exists at high histamine and GABA concentrations but weak rectification is observed at low concentrations (EF) Representative traces ofcurrent through PxHCLA recorded under 20 micromol lminus1 HA (E) and 10 mmol lminus1 GABA (F) during 300 ms voltage steps between minus100 mV and +100 mV withan increment of 20 mV Currents were stable over 300 ms at all voltages

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of histamine and GABA on PxHCLs the current responses elicitedby the initial histamine application were used to normalize thesubsequent current responses We avoided using a saturatingconcentration of histamine to reduce the risks of cell damage forthe later chemical applications Statistical significance wasevaluated using Studentrsquos paired t-test at a value of Plt005 Datain figures are presented as meansplusmnsem

RESULTSMolecular phylogenyWe determined the full-length sequences of cDNAs encodingPxHCLA (378 amino acids) and PxHCLB (427 amino acids) Thephylogenetic analysis of Cys-loop chloride channels includingthese sequences showed that they were clustered according totheir specific ligands (Fig 1) Agonists are thought to bind atthe interfaces between adjacent receptor subunits (Hibbs andGouaux 2011 Karlin 2002 Sine 2002) As the 3D structure of theglutamate-gated chloride channel has been determined inC elegans(Hibbs and Gouaux 2011) we used this sequence as a reference todeduce the agonist binding sites or loop AndashG of Cys-loop chloridechannels The amino acid sequences at the putative agonist bindingsites are highly conserved particularly for the loop A structure(Fig 2) Of the seven amino acids that comprise loop A five areidentical across all analyzed channels In addition we detected five

sites (gray boxes in Fig 2) that are more or less specific to thereceptorsrsquo agonists For example the amino acid at position 115 inloop D is threonine for glutamate receptors phenylalanine forhistamine receptors and tyrosine for GABA receptors

PhysiologyDuring the 16ndash22 h period after transfection whole-cell currentscould be recorded upon application of histamine and GABA to thecells transfected with PxhclA PxhclB or both We could not detectany currents from cells with other tested chemicals (tyramineserotonin L-D-glutamate and glycine) In control cells noresponses were recorded to any chemicals including histamineand GABA

Fig 3AB shows representative current traces recorded fromPxhclA transfected cells under histamine and GABA applicationrespectively Fig 3CD shows the currentndashvoltage (IndashV )relationship of PxhclA transfected cells under the application ofhistamine and GABA respectively The IndashV relationship wasbasically linear at higher concentrations but weak outwardrectification was observed at lower concentrations As theintracellular and extracellular concentrations of chloride ions wereapproximately equal in our experiments this implies a reversalpotential for chloride ions of around 0 mV which was consistentwith our results (Fig 3CD) The voltage-step pulses produced

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HA 33 micromol lndash1HA 1 mmol lndash1

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Fig 4 Representative current traces of PxHCLB expressed in HEK293 cells under histamine and GABA application (AB) Representative currenttraces showing activation of PxHCLB by histamine (HA 33 μmol lminus1 and 1 mmol lminus1 A) and GABA (1 and 50 mmol lminus1 B) in a dose-dependent mannerBars indicate the duration of chemical application (CD) IndashV relationship of PxHCLB at two HA (C) and GABA (D) concentrations A linear relationship exists athigh histamine and GABA concentrations but weak rectification is observed at low concentrations (EF) Representative traces of current through PxHCLBrecorded under 66 micromol lminus1 HA (E) and 25 mmol lminus1 GABA (F) during 300 ms voltage steps between minus100 mV and +100 mV with an increment of 20 mVCurrents were stable over 300 ms at all voltages

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histamine and GABA-induced currents that were stable over a300 ms interval (Fig 3EF)Fig 4 shows the results obtained from analysis of PxhclB

transfected cells under histamine and GABA application in thesame format as Fig 3 for PxhclA transfected cells While weobserved dose-dependent responses in PxhclB transfected cells asfor PxhclA transfected cells the maximal current responseswere generally smaller in the former than the latter (see scales ofFigs 3 and 4) PxhclB transfected cells showed a linear IndashVrelationship at high agonist concentrations but weak outwardrectification at low concentrations (Fig 4CD) Histamine- andGABA-induced currents were stable over 300 ms (Fig 4EF)Fig 5 shows histamine and GABA dose dependency fitted

with the simple Hill function for cells transfected by PxhclA PxhclBor both The fitted parameters of EC50 and the slope nH aresummarized in Table 1 PxhclB transfected cells showed the highestsensitivity to both histamine and GABA For both histamine andGABA the slope of the dosendashresponse curves was shallowest forPxhclAPxhclB co-transfected cells while the slopes were similarbetween PxhclA and PxhclB transfected cells We used 1 mmol lminus1

histamine and 50 mmol lminus1 GABA across all three transfections toobtain their maximal current responses for each agonist Howeverthe currents recorded from those cells reached their maxima at loweragonist concentrations According to the dosendashresponse curves thesaturating concentrations of histamine were in fact approximately300 micromol lminus1 and 30 micromol lminus1 for PxhclA and PxhclB transfectedcells respectively and that of GABA was 10 mmol lminus1 for PxhclBtransfected cells (Fig 5)Simultaneous application of histamine and GABA elicited

synergistic effects Fig 6AndashC shows representative response

traces of PxhclA transfected PxhclB transfected and PxhclAPxhclB co-transfected cells respectively The sum of the currentresponses elicited from individual application of histamine andGABAwas significantly smaller than the current responses elicitedfrom histamine and GABA mixtures in all transfection cases(Fig 6DndashF)

Note that we used HEK293 cells to express the moleculesPhysiological responses of ectopically expressed HCLs ofD melanogaster and M domestica were analyzed using insect S2cells (Pantazis et al 2008) and Xenopus oocytes (Kita et al 2017)respectively These studies including our present one have revealedsimilar characteristics indicating that the effect of using differentexpression systems would be minimal

DISCUSSIONMolecular characteristicsPhylogenetic analysis suggests PxhclA and PxhclB genes arehomologs of other insectsrsquo hcl genes as they cluster according totheir specific ligands (Fig 1) The amino acid sequences in putativeagonist binding sites provide some candidate residues in loop B CD and F for determining agonist specificity among Cys-loopchloride channels (Fig 2) In D melanogaster and M domesticahistamine activates all HCL conformations but GABA onlyactivates HCLB homomers (Gisselmann et al 2004 Kita et al2017) Interestingly both histamine and GABA activate all HCLconformations in Papilio Although the GABA-mediated activationmechanism in HCLs remains unknown we found one amino acidresidue that distinguishes the subunits capable of binding GABAsuch as PxHCLA PxHCLB fly HCLB and RDls (resistant-to-dieldrin or GABA-activated chloride-selective receptors) from the

[Agonist] (mmol lndash1)100101010010001

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IIm

ax

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PxHCLA HA

PxHCLB HA

PxHCLAB HA

PxHCLA GABA

PxHCLB GABA

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Fig 5 Dosendashresponse curves of PxHCLA PxHCLB and PxHCLAB for histamine and GABA Current amplitudes were normalized by the current at1 mmol lminus1 (IImax) for histamine (HA) and 50 mmol lminus1 for GABA Each point is based on the measurement from at least three separate cells taken from sixindependent transfection sessions in total The curves were drawn by simple Hill fittings (Eqn 1)

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subunits capable of binding histamine only such as fly HCLA Theresidue is at position 275 which is outside of the putative agonistbinding sites (Fig 2 Fig S1) The HCLs and RDls in Fig 2 haveglycine at this position except for the fly HCLA which has valinethere This suggests that the mutation from glycine to valine atposition 275 (G275V) caused the loss of function in the fly HCLAlineage Further electrophysiological analysis on HCLs from beelineages and HCLs with site-directed mutations would provideclues to understand the significance of amino acid substitutions forGABA binding

Physiological properties and functional implicationsElectrophysiological studies on HCLs in D melanogaster andM domestica have shown that the EC50 values of native channels

on the LMCs for histamine (24 and 34 micromol lminus1 respectively)are similar to those of in vitro-expressed HCLA homomers forhistamine (25 and 33 micromol lminus1 respectively) (Kita et al 2017Pantazis et al 2008 Skingsley et al 1995) As PxHCLAhomomers have an EC50 for histamine (2187 micromol lminus1) similarto that of D melanogaster PxHCLA homomers most likelyhave a similar biological function ie synaptic transmission fromphotoreceptors to LMCs In fact PxHCLA is expressed in neuronspostsynaptic to photoreceptors in both the lamina and the medullaof P xuthus (Chen et al 2016)

PxHCLB homomers have a much lower EC50 for histamine thanPxHCLA homomers Histamine at the concentration of thePxHCLB homomer EC50 barely activates PxHCLA reaching onlyaround 5 of its maximum current (Fig 5) Although the lowerEC50 for histamine of the HCLB homomer than of HCLA isconsistent with previous reports in flies (Gisselmann et al 2004Kita et al 2017) its significance remains unclear RecentlySchnaitmann et al (2018) demonstrated inD melanogaster that twolvfs (R7 and R8) originating from the same ommatidia directlyinhibit each other at their axon terminals in the medulla via HCLBresulting in spectral opponency in these photoreceptors The higherhistamine sensitivity of HCLB would cause it to activate morequickly than HCLA which would allow photoreceptors to pre-process each otherrsquos signals in this way before the informationreached higher order neurons We have detected PxHCLB atinterphotoreceptor connections in the lamina and the medulla of

Table 1 Histamine and GABA dosendashresponse parameters of PxHCLs

Receptor type

Histamine GABA

EC50

(micromol lminus1) nH

EC50

(mmol lminus1) nH

PxHCLAhomomer

219plusmn16 24plusmn03 94plusmn08 25plusmn07

PxHCLBhomomer

55plusmn02 22plusmn01 27plusmn01 23plusmn02

PxHCLAB 248plusmn29 11plusmn01 93plusmn09 19plusmn03

All fitted parameters are meansplusmnsem

Time(s)0 100 200 300

10

ndash10

5

0

ndash5

Cur

rent

(nA

)

HA10

HA33

GABA33

Mix

2

ndash2

1

0

ndash1

3

ndash30 100 200 300

Time(s)

HA5

HA1

micromol lndash1

GABA500

micromol lndash110

ndash10

5

0

ndash5

0 100 200 300Time(s)

HA10

HA33

micromol lndash1

GABA33

mmol lndash1

MixSum(HA+GABA)

05

0

01

02

03

04

Nor

mal

ized

cur

rent

P=003Paired t-test 03

02

01

0

P=001Paired t-test 08

06

04

0

02

P=001Paired t-test

A B C

D E F

Mix Mix

MixSum(HA+GABA)

MixSum(HA+GABA)

micromol lndash1 micromol lndash1 mmol lndash1 micromol lndash1 micromol lndash1

Fig 6 Possible synergistic effects of histamine and GABA on PxHCLs (AndashC) Representative traces from HEK293 cell expressing PxHCLA (A) PxHCLB(B) and PxHCLAB (C) showing the synergistic effects observed under histamine (HA) and GABA application Bars indicate the duration of chemicalapplications lsquoMixrsquo indicates the simultaneous application of histamine and GABA at concentrations equal to their concentrations on the second and thirdapplications in the trial respectively (DndashF) Synergistic effects of histamine andGABA on PxHCLA (D) PxHCLB (E) and PxHCLAB (F) Lines connect responsesrecorded from the same cell lsquoSumrsquo (HA+GABA) indicates the sum of the currents recorded from the second and third chemical applications Pie chartsindicate the ratio of histamine (white)- and GABA (black)-induced currents lsquoMixrsquo indicates the currents recorded from the application of histamine and GABAmixtures (gray) Both sum and mix currents were normalized to the currents recorded from the first application of histamine

7

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

entalB

iology

of histamine and GABA on PxHCLs the current responses elicitedby the initial histamine application were used to normalize thesubsequent current responses We avoided using a saturatingconcentration of histamine to reduce the risks of cell damage forthe later chemical applications Statistical significance wasevaluated using Studentrsquos paired t-test at a value of Plt005 Datain figures are presented as meansplusmnsem

RESULTSMolecular phylogenyWe determined the full-length sequences of cDNAs encodingPxHCLA (378 amino acids) and PxHCLB (427 amino acids) Thephylogenetic analysis of Cys-loop chloride channels includingthese sequences showed that they were clustered according totheir specific ligands (Fig 1) Agonists are thought to bind atthe interfaces between adjacent receptor subunits (Hibbs andGouaux 2011 Karlin 2002 Sine 2002) As the 3D structure of theglutamate-gated chloride channel has been determined inC elegans(Hibbs and Gouaux 2011) we used this sequence as a reference todeduce the agonist binding sites or loop AndashG of Cys-loop chloridechannels The amino acid sequences at the putative agonist bindingsites are highly conserved particularly for the loop A structure(Fig 2) Of the seven amino acids that comprise loop A five areidentical across all analyzed channels In addition we detected five

sites (gray boxes in Fig 2) that are more or less specific to thereceptorsrsquo agonists For example the amino acid at position 115 inloop D is threonine for glutamate receptors phenylalanine forhistamine receptors and tyrosine for GABA receptors

PhysiologyDuring the 16ndash22 h period after transfection whole-cell currentscould be recorded upon application of histamine and GABA to thecells transfected with PxhclA PxhclB or both We could not detectany currents from cells with other tested chemicals (tyramineserotonin L-D-glutamate and glycine) In control cells noresponses were recorded to any chemicals including histamineand GABA

Fig 3AB shows representative current traces recorded fromPxhclA transfected cells under histamine and GABA applicationrespectively Fig 3CD shows the currentndashvoltage (IndashV )relationship of PxhclA transfected cells under the application ofhistamine and GABA respectively The IndashV relationship wasbasically linear at higher concentrations but weak outwardrectification was observed at lower concentrations As theintracellular and extracellular concentrations of chloride ions wereapproximately equal in our experiments this implies a reversalpotential for chloride ions of around 0 mV which was consistentwith our results (Fig 3CD) The voltage-step pulses produced

0 100 200 300Time (ms)

1

0

ndash1Cur

rent

(nA

)

E

2

ndash2

1

0

ndash1Cur

rent

(nA

)

F

2

ndash2

0 100 200 300Time (ms)

10

ndash10

05

0

ndash05

I (nA

)

ndash100 ndash50 0 50 100V (mV)

C

HA 33 micromol lndash1HA 1 mmol lndash1

4

ndash4

2

0

ndash2

ndash100 ndash50 0 50 100

I (nA

)

V (mV)

D

GABA 1 mmol lndash1GABA 50 mmol lndash1

10

ndash10

0

Cur

rent

(nA

)

0 50 100 150

A HA33 micromol lndash1

HA1 mmol lndash1

Time (s)200

05

ndash05

GABA1 mmol lndash1

GABA50 mmol lndash13

0

Cur

rent

(nA

)

B

0 50 100 150 200Time (s)

2

1

ndash1

ndash2

ndash3

Fig 4 Representative current traces of PxHCLB expressed in HEK293 cells under histamine and GABA application (AB) Representative currenttraces showing activation of PxHCLB by histamine (HA 33 μmol lminus1 and 1 mmol lminus1 A) and GABA (1 and 50 mmol lminus1 B) in a dose-dependent mannerBars indicate the duration of chemical application (CD) IndashV relationship of PxHCLB at two HA (C) and GABA (D) concentrations A linear relationship exists athigh histamine and GABA concentrations but weak rectification is observed at low concentrations (EF) Representative traces of current through PxHCLBrecorded under 66 micromol lminus1 HA (E) and 25 mmol lminus1 GABA (F) during 300 ms voltage steps between minus100 mV and +100 mV with an increment of 20 mVCurrents were stable over 300 ms at all voltages

5

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

entalB

iology

histamine and GABA-induced currents that were stable over a300 ms interval (Fig 3EF)Fig 4 shows the results obtained from analysis of PxhclB

transfected cells under histamine and GABA application in thesame format as Fig 3 for PxhclA transfected cells While weobserved dose-dependent responses in PxhclB transfected cells asfor PxhclA transfected cells the maximal current responseswere generally smaller in the former than the latter (see scales ofFigs 3 and 4) PxhclB transfected cells showed a linear IndashVrelationship at high agonist concentrations but weak outwardrectification at low concentrations (Fig 4CD) Histamine- andGABA-induced currents were stable over 300 ms (Fig 4EF)Fig 5 shows histamine and GABA dose dependency fitted

with the simple Hill function for cells transfected by PxhclA PxhclBor both The fitted parameters of EC50 and the slope nH aresummarized in Table 1 PxhclB transfected cells showed the highestsensitivity to both histamine and GABA For both histamine andGABA the slope of the dosendashresponse curves was shallowest forPxhclAPxhclB co-transfected cells while the slopes were similarbetween PxhclA and PxhclB transfected cells We used 1 mmol lminus1

histamine and 50 mmol lminus1 GABA across all three transfections toobtain their maximal current responses for each agonist Howeverthe currents recorded from those cells reached their maxima at loweragonist concentrations According to the dosendashresponse curves thesaturating concentrations of histamine were in fact approximately300 micromol lminus1 and 30 micromol lminus1 for PxhclA and PxhclB transfectedcells respectively and that of GABA was 10 mmol lminus1 for PxhclBtransfected cells (Fig 5)Simultaneous application of histamine and GABA elicited

synergistic effects Fig 6AndashC shows representative response

traces of PxhclA transfected PxhclB transfected and PxhclAPxhclB co-transfected cells respectively The sum of the currentresponses elicited from individual application of histamine andGABAwas significantly smaller than the current responses elicitedfrom histamine and GABA mixtures in all transfection cases(Fig 6DndashF)

Note that we used HEK293 cells to express the moleculesPhysiological responses of ectopically expressed HCLs ofD melanogaster and M domestica were analyzed using insect S2cells (Pantazis et al 2008) and Xenopus oocytes (Kita et al 2017)respectively These studies including our present one have revealedsimilar characteristics indicating that the effect of using differentexpression systems would be minimal

DISCUSSIONMolecular characteristicsPhylogenetic analysis suggests PxhclA and PxhclB genes arehomologs of other insectsrsquo hcl genes as they cluster according totheir specific ligands (Fig 1) The amino acid sequences in putativeagonist binding sites provide some candidate residues in loop B CD and F for determining agonist specificity among Cys-loopchloride channels (Fig 2) In D melanogaster and M domesticahistamine activates all HCL conformations but GABA onlyactivates HCLB homomers (Gisselmann et al 2004 Kita et al2017) Interestingly both histamine and GABA activate all HCLconformations in Papilio Although the GABA-mediated activationmechanism in HCLs remains unknown we found one amino acidresidue that distinguishes the subunits capable of binding GABAsuch as PxHCLA PxHCLB fly HCLB and RDls (resistant-to-dieldrin or GABA-activated chloride-selective receptors) from the

[Agonist] (mmol lndash1)100101010010001

0

02

04

06

08

10

IIm

ax

00001

PxHCLA HA

PxHCLB HA

PxHCLAB HA

PxHCLA GABA

PxHCLB GABA

PxHCLAB GABA

Fig 5 Dosendashresponse curves of PxHCLA PxHCLB and PxHCLAB for histamine and GABA Current amplitudes were normalized by the current at1 mmol lminus1 (IImax) for histamine (HA) and 50 mmol lminus1 for GABA Each point is based on the measurement from at least three separate cells taken from sixindependent transfection sessions in total The curves were drawn by simple Hill fittings (Eqn 1)

6

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

entalB

iology

subunits capable of binding histamine only such as fly HCLA Theresidue is at position 275 which is outside of the putative agonistbinding sites (Fig 2 Fig S1) The HCLs and RDls in Fig 2 haveglycine at this position except for the fly HCLA which has valinethere This suggests that the mutation from glycine to valine atposition 275 (G275V) caused the loss of function in the fly HCLAlineage Further electrophysiological analysis on HCLs from beelineages and HCLs with site-directed mutations would provideclues to understand the significance of amino acid substitutions forGABA binding

Physiological properties and functional implicationsElectrophysiological studies on HCLs in D melanogaster andM domestica have shown that the EC50 values of native channels

on the LMCs for histamine (24 and 34 micromol lminus1 respectively)are similar to those of in vitro-expressed HCLA homomers forhistamine (25 and 33 micromol lminus1 respectively) (Kita et al 2017Pantazis et al 2008 Skingsley et al 1995) As PxHCLAhomomers have an EC50 for histamine (2187 micromol lminus1) similarto that of D melanogaster PxHCLA homomers most likelyhave a similar biological function ie synaptic transmission fromphotoreceptors to LMCs In fact PxHCLA is expressed in neuronspostsynaptic to photoreceptors in both the lamina and the medullaof P xuthus (Chen et al 2016)

PxHCLB homomers have a much lower EC50 for histamine thanPxHCLA homomers Histamine at the concentration of thePxHCLB homomer EC50 barely activates PxHCLA reaching onlyaround 5 of its maximum current (Fig 5) Although the lowerEC50 for histamine of the HCLB homomer than of HCLA isconsistent with previous reports in flies (Gisselmann et al 2004Kita et al 2017) its significance remains unclear RecentlySchnaitmann et al (2018) demonstrated inD melanogaster that twolvfs (R7 and R8) originating from the same ommatidia directlyinhibit each other at their axon terminals in the medulla via HCLBresulting in spectral opponency in these photoreceptors The higherhistamine sensitivity of HCLB would cause it to activate morequickly than HCLA which would allow photoreceptors to pre-process each otherrsquos signals in this way before the informationreached higher order neurons We have detected PxHCLB atinterphotoreceptor connections in the lamina and the medulla of

Table 1 Histamine and GABA dosendashresponse parameters of PxHCLs

Receptor type

Histamine GABA

EC50

(micromol lminus1) nH

EC50

(mmol lminus1) nH

PxHCLAhomomer

219plusmn16 24plusmn03 94plusmn08 25plusmn07

PxHCLBhomomer

55plusmn02 22plusmn01 27plusmn01 23plusmn02

PxHCLAB 248plusmn29 11plusmn01 93plusmn09 19plusmn03

All fitted parameters are meansplusmnsem

Time(s)0 100 200 300

10

ndash10

5

0

ndash5

Cur

rent

(nA

)

HA10

HA33

GABA33

Mix

2

ndash2

1

0

ndash1

3

ndash30 100 200 300

Time(s)

HA5

HA1

micromol lndash1

GABA500

micromol lndash110

ndash10

5

0

ndash5

0 100 200 300Time(s)

HA10

HA33

micromol lndash1

GABA33

mmol lndash1

MixSum(HA+GABA)

05

0

01

02

03

04

Nor

mal

ized

cur

rent

P=003Paired t-test 03

02

01

0

P=001Paired t-test 08

06

04

0

02

P=001Paired t-test

A B C

D E F

Mix Mix

MixSum(HA+GABA)

MixSum(HA+GABA)

micromol lndash1 micromol lndash1 mmol lndash1 micromol lndash1 micromol lndash1

Fig 6 Possible synergistic effects of histamine and GABA on PxHCLs (AndashC) Representative traces from HEK293 cell expressing PxHCLA (A) PxHCLB(B) and PxHCLAB (C) showing the synergistic effects observed under histamine (HA) and GABA application Bars indicate the duration of chemicalapplications lsquoMixrsquo indicates the simultaneous application of histamine and GABA at concentrations equal to their concentrations on the second and thirdapplications in the trial respectively (DndashF) Synergistic effects of histamine andGABA on PxHCLA (D) PxHCLB (E) and PxHCLAB (F) Lines connect responsesrecorded from the same cell lsquoSumrsquo (HA+GABA) indicates the sum of the currents recorded from the second and third chemical applications Pie chartsindicate the ratio of histamine (white)- and GABA (black)-induced currents lsquoMixrsquo indicates the currents recorded from the application of histamine and GABAmixtures (gray) Both sum and mix currents were normalized to the currents recorded from the first application of histamine

7

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

entalB

iology

histamine and GABA-induced currents that were stable over a300 ms interval (Fig 3EF)Fig 4 shows the results obtained from analysis of PxhclB

transfected cells under histamine and GABA application in thesame format as Fig 3 for PxhclA transfected cells While weobserved dose-dependent responses in PxhclB transfected cells asfor PxhclA transfected cells the maximal current responseswere generally smaller in the former than the latter (see scales ofFigs 3 and 4) PxhclB transfected cells showed a linear IndashVrelationship at high agonist concentrations but weak outwardrectification at low concentrations (Fig 4CD) Histamine- andGABA-induced currents were stable over 300 ms (Fig 4EF)Fig 5 shows histamine and GABA dose dependency fitted

with the simple Hill function for cells transfected by PxhclA PxhclBor both The fitted parameters of EC50 and the slope nH aresummarized in Table 1 PxhclB transfected cells showed the highestsensitivity to both histamine and GABA For both histamine andGABA the slope of the dosendashresponse curves was shallowest forPxhclAPxhclB co-transfected cells while the slopes were similarbetween PxhclA and PxhclB transfected cells We used 1 mmol lminus1

histamine and 50 mmol lminus1 GABA across all three transfections toobtain their maximal current responses for each agonist Howeverthe currents recorded from those cells reached their maxima at loweragonist concentrations According to the dosendashresponse curves thesaturating concentrations of histamine were in fact approximately300 micromol lminus1 and 30 micromol lminus1 for PxhclA and PxhclB transfectedcells respectively and that of GABA was 10 mmol lminus1 for PxhclBtransfected cells (Fig 5)Simultaneous application of histamine and GABA elicited

synergistic effects Fig 6AndashC shows representative response

traces of PxhclA transfected PxhclB transfected and PxhclAPxhclB co-transfected cells respectively The sum of the currentresponses elicited from individual application of histamine andGABAwas significantly smaller than the current responses elicitedfrom histamine and GABA mixtures in all transfection cases(Fig 6DndashF)

Note that we used HEK293 cells to express the moleculesPhysiological responses of ectopically expressed HCLs ofD melanogaster and M domestica were analyzed using insect S2cells (Pantazis et al 2008) and Xenopus oocytes (Kita et al 2017)respectively These studies including our present one have revealedsimilar characteristics indicating that the effect of using differentexpression systems would be minimal

DISCUSSIONMolecular characteristicsPhylogenetic analysis suggests PxhclA and PxhclB genes arehomologs of other insectsrsquo hcl genes as they cluster according totheir specific ligands (Fig 1) The amino acid sequences in putativeagonist binding sites provide some candidate residues in loop B CD and F for determining agonist specificity among Cys-loopchloride channels (Fig 2) In D melanogaster and M domesticahistamine activates all HCL conformations but GABA onlyactivates HCLB homomers (Gisselmann et al 2004 Kita et al2017) Interestingly both histamine and GABA activate all HCLconformations in Papilio Although the GABA-mediated activationmechanism in HCLs remains unknown we found one amino acidresidue that distinguishes the subunits capable of binding GABAsuch as PxHCLA PxHCLB fly HCLB and RDls (resistant-to-dieldrin or GABA-activated chloride-selective receptors) from the

[Agonist] (mmol lndash1)100101010010001

0

02

04

06

08

10

IIm

ax

00001

PxHCLA HA

PxHCLB HA

PxHCLAB HA

PxHCLA GABA

PxHCLB GABA

PxHCLAB GABA

Fig 5 Dosendashresponse curves of PxHCLA PxHCLB and PxHCLAB for histamine and GABA Current amplitudes were normalized by the current at1 mmol lminus1 (IImax) for histamine (HA) and 50 mmol lminus1 for GABA Each point is based on the measurement from at least three separate cells taken from sixindependent transfection sessions in total The curves were drawn by simple Hill fittings (Eqn 1)

6

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

entalB

iology

subunits capable of binding histamine only such as fly HCLA Theresidue is at position 275 which is outside of the putative agonistbinding sites (Fig 2 Fig S1) The HCLs and RDls in Fig 2 haveglycine at this position except for the fly HCLA which has valinethere This suggests that the mutation from glycine to valine atposition 275 (G275V) caused the loss of function in the fly HCLAlineage Further electrophysiological analysis on HCLs from beelineages and HCLs with site-directed mutations would provideclues to understand the significance of amino acid substitutions forGABA binding

Physiological properties and functional implicationsElectrophysiological studies on HCLs in D melanogaster andM domestica have shown that the EC50 values of native channels

on the LMCs for histamine (24 and 34 micromol lminus1 respectively)are similar to those of in vitro-expressed HCLA homomers forhistamine (25 and 33 micromol lminus1 respectively) (Kita et al 2017Pantazis et al 2008 Skingsley et al 1995) As PxHCLAhomomers have an EC50 for histamine (2187 micromol lminus1) similarto that of D melanogaster PxHCLA homomers most likelyhave a similar biological function ie synaptic transmission fromphotoreceptors to LMCs In fact PxHCLA is expressed in neuronspostsynaptic to photoreceptors in both the lamina and the medullaof P xuthus (Chen et al 2016)

PxHCLB homomers have a much lower EC50 for histamine thanPxHCLA homomers Histamine at the concentration of thePxHCLB homomer EC50 barely activates PxHCLA reaching onlyaround 5 of its maximum current (Fig 5) Although the lowerEC50 for histamine of the HCLB homomer than of HCLA isconsistent with previous reports in flies (Gisselmann et al 2004Kita et al 2017) its significance remains unclear RecentlySchnaitmann et al (2018) demonstrated inD melanogaster that twolvfs (R7 and R8) originating from the same ommatidia directlyinhibit each other at their axon terminals in the medulla via HCLBresulting in spectral opponency in these photoreceptors The higherhistamine sensitivity of HCLB would cause it to activate morequickly than HCLA which would allow photoreceptors to pre-process each otherrsquos signals in this way before the informationreached higher order neurons We have detected PxHCLB atinterphotoreceptor connections in the lamina and the medulla of

Table 1 Histamine and GABA dosendashresponse parameters of PxHCLs

Receptor type

Histamine GABA

EC50

(micromol lminus1) nH

EC50

(mmol lminus1) nH

PxHCLAhomomer

219plusmn16 24plusmn03 94plusmn08 25plusmn07

PxHCLBhomomer

55plusmn02 22plusmn01 27plusmn01 23plusmn02

PxHCLAB 248plusmn29 11plusmn01 93plusmn09 19plusmn03

All fitted parameters are meansplusmnsem

Time(s)0 100 200 300

10

ndash10

5

0

ndash5

Cur

rent

(nA

)

HA10

HA33

GABA33

Mix

2

ndash2

1

0

ndash1

3

ndash30 100 200 300

Time(s)

HA5

HA1

micromol lndash1

GABA500

micromol lndash110

ndash10

5

0

ndash5

0 100 200 300Time(s)

HA10

HA33

micromol lndash1

GABA33

mmol lndash1

MixSum(HA+GABA)

05

0

01

02

03

04

Nor

mal

ized

cur

rent

P=003Paired t-test 03

02

01

0

P=001Paired t-test 08

06

04

0

02

P=001Paired t-test

A B C

D E F

Mix Mix

MixSum(HA+GABA)

MixSum(HA+GABA)

micromol lndash1 micromol lndash1 mmol lndash1 micromol lndash1 micromol lndash1

Fig 6 Possible synergistic effects of histamine and GABA on PxHCLs (AndashC) Representative traces from HEK293 cell expressing PxHCLA (A) PxHCLB(B) and PxHCLAB (C) showing the synergistic effects observed under histamine (HA) and GABA application Bars indicate the duration of chemicalapplications lsquoMixrsquo indicates the simultaneous application of histamine and GABA at concentrations equal to their concentrations on the second and thirdapplications in the trial respectively (DndashF) Synergistic effects of histamine andGABA on PxHCLA (D) PxHCLB (E) and PxHCLAB (F) Lines connect responsesrecorded from the same cell lsquoSumrsquo (HA+GABA) indicates the sum of the currents recorded from the second and third chemical applications Pie chartsindicate the ratio of histamine (white)- and GABA (black)-induced currents lsquoMixrsquo indicates the currents recorded from the application of histamine and GABAmixtures (gray) Both sum and mix currents were normalized to the currents recorded from the first application of histamine

7

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

entalB

iology

subunits capable of binding histamine only such as fly HCLA Theresidue is at position 275 which is outside of the putative agonistbinding sites (Fig 2 Fig S1) The HCLs and RDls in Fig 2 haveglycine at this position except for the fly HCLA which has valinethere This suggests that the mutation from glycine to valine atposition 275 (G275V) caused the loss of function in the fly HCLAlineage Further electrophysiological analysis on HCLs from beelineages and HCLs with site-directed mutations would provideclues to understand the significance of amino acid substitutions forGABA binding

Physiological properties and functional implicationsElectrophysiological studies on HCLs in D melanogaster andM domestica have shown that the EC50 values of native channels

on the LMCs for histamine (24 and 34 micromol lminus1 respectively)are similar to those of in vitro-expressed HCLA homomers forhistamine (25 and 33 micromol lminus1 respectively) (Kita et al 2017Pantazis et al 2008 Skingsley et al 1995) As PxHCLAhomomers have an EC50 for histamine (2187 micromol lminus1) similarto that of D melanogaster PxHCLA homomers most likelyhave a similar biological function ie synaptic transmission fromphotoreceptors to LMCs In fact PxHCLA is expressed in neuronspostsynaptic to photoreceptors in both the lamina and the medullaof P xuthus (Chen et al 2016)

PxHCLB homomers have a much lower EC50 for histamine thanPxHCLA homomers Histamine at the concentration of thePxHCLB homomer EC50 barely activates PxHCLA reaching onlyaround 5 of its maximum current (Fig 5) Although the lowerEC50 for histamine of the HCLB homomer than of HCLA isconsistent with previous reports in flies (Gisselmann et al 2004Kita et al 2017) its significance remains unclear RecentlySchnaitmann et al (2018) demonstrated inD melanogaster that twolvfs (R7 and R8) originating from the same ommatidia directlyinhibit each other at their axon terminals in the medulla via HCLBresulting in spectral opponency in these photoreceptors The higherhistamine sensitivity of HCLB would cause it to activate morequickly than HCLA which would allow photoreceptors to pre-process each otherrsquos signals in this way before the informationreached higher order neurons We have detected PxHCLB atinterphotoreceptor connections in the lamina and the medulla of

Table 1 Histamine and GABA dosendashresponse parameters of PxHCLs

Receptor type

Histamine GABA

EC50

(micromol lminus1) nH

EC50

(mmol lminus1) nH

PxHCLAhomomer

219plusmn16 24plusmn03 94plusmn08 25plusmn07

PxHCLBhomomer

55plusmn02 22plusmn01 27plusmn01 23plusmn02

PxHCLAB 248plusmn29 11plusmn01 93plusmn09 19plusmn03

All fitted parameters are meansplusmnsem

Time(s)0 100 200 300

10

ndash10

5

0

ndash5

Cur

rent

(nA

)

HA10

HA33

GABA33

Mix

2

ndash2

1

0

ndash1

3

ndash30 100 200 300

Time(s)

HA5

HA1

micromol lndash1

GABA500

micromol lndash110

ndash10

5

0

ndash5

0 100 200 300Time(s)

HA10

HA33

micromol lndash1

GABA33

mmol lndash1

MixSum(HA+GABA)

05

0

01

02

03

04

Nor

mal

ized

cur

rent

P=003Paired t-test 03

02

01

0

P=001Paired t-test 08

06

04

0

02

P=001Paired t-test

A B C

D E F

Mix Mix

MixSum(HA+GABA)

MixSum(HA+GABA)

micromol lndash1 micromol lndash1 mmol lndash1 micromol lndash1 micromol lndash1

Fig 6 Possible synergistic effects of histamine and GABA on PxHCLs (AndashC) Representative traces from HEK293 cell expressing PxHCLA (A) PxHCLB(B) and PxHCLAB (C) showing the synergistic effects observed under histamine (HA) and GABA application Bars indicate the duration of chemicalapplications lsquoMixrsquo indicates the simultaneous application of histamine and GABA at concentrations equal to their concentrations on the second and thirdapplications in the trial respectively (DndashF) Synergistic effects of histamine andGABA on PxHCLA (D) PxHCLB (E) and PxHCLAB (F) Lines connect responsesrecorded from the same cell lsquoSumrsquo (HA+GABA) indicates the sum of the currents recorded from the second and third chemical applications Pie chartsindicate the ratio of histamine (white)- and GABA (black)-induced currents lsquoMixrsquo indicates the currents recorded from the application of histamine and GABAmixtures (gray) Both sum and mix currents were normalized to the currents recorded from the first application of histamine

7

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P xuthus (Chen et al 2016) suggesting that they serve similarfunctions in butterfliesFor co-transfected cells we observed partly discrepant results

between P xuthus and D melanogaster In D melanogaster hclAhclB co-transfectants show the lowest EC50 for histamine among thethree cell types (Gisselmann et al 2004 Pantazis et al 2008)In contrast PxhclAPxhclB co-transfectants have an EC50 forhistamine similar to that of PxhclA transfected cells (Table 1) TheEC50 of GABA in PxhclAPxhclB co-transfectants is also similarto that of PxhclA transfectants However nH the slope of thehistamine dosendashresponse curve was smaller in PxhclAPxhclBco-transfectants than those of PxhclA and PxhclB transfectantsalthough the curve for GABA was pretty close to that of PxhclAtransfectants We need to carefully consider any possibilities ofheteromeric channels andor mixed expression of PxhclA andPxhclB homomers in the co-transfected cells in varying proportionsIn fact a shallower slope of the histamine dosendashresponse curve hasalso been observed in the fly hclAhclB co-transfectant (Pantaziset al 2008) however the underlying mechanism remains unclearWhile HCLA has been localized in LMCs and medulla neuronsHCLB has been localized in lamina glial cells and lvfs R7 and R8 inD melanogaster (Pantazis et al 2008 Schnaitmann et al 2018)Therefore HCLAB heteromers seem unlikely to exist in vivo atleast in the visual systemIn several experiments we applied histamine and GABA

simultaneously We always found an increase in current uponco-application in all three transfectants (Fig 6) This indicates thepossibility that histamine and GABA might have synergistic effectson PxHCLs (Fig 6) while simultaneous application of histamineand GABA affect HCLB of M domestica only in an additivemanner (Kita et al 2017) The high sequence similarity betweenPxHCLs and fly HCLB particularly in the putative agonist bindingsites implies that a small number of amino acid substitutions areresponsible for the functional divergence if any The activation ofPxHCLA or PxHCLB homomers by histamine and GABA mightbe functionally significant In P xuthus for instance a subset ofLMCs are GABAergic (Hamanaka et al 2012) They terminate inthe distal region of the medulla along with the lvfs which releasehistamine upon light stimulation Thus the possible synergisticeffects of histamine and GABA could modulate synaptictransmission in this regionThe variability in the response to GABA between HCLA and

PxHCLA may reflect differences in the underlying visual circuitsbetween flies and butterflies In flies LMCs are postsynaptic to svfsR1ndashR6 which all have identical spectral sensitivity (Rivera-Albaet al 2011) and they are assumed to be responsible mainly formotion vision In contrast the spectrally heterogeneous lvfs R7 andR8 which are thought to be essential for color vision have nosynaptic interactions with photoreceptors or LMCs in the laminaUnlike in flies the Papilio lvfs have a number of lateral processes inthe lamina making local contacts with both svfs and LMCs(Takemura and Arikawa 2006) As a result the medulla neuronsexpressing PxHCLA in Papilio receive not only chromatic inputsfrom histaminergic lvfs but also mixed chromatic and motionsignals from LMCs including GABAergic ones (Hamanaka et al2012) In contrast the medulla neurons expressing HCLA in fliesonly receive chromatic signals from histaminergic lvfs because theirHCLAs are insensitive to GABA Therefore we propose that flieshave evolved an optimal achromatic motion vision system primarilydependent on R1ndashR6 broadband photoreceptors (Hardie 1985)while in butterflies pathways for color and motion vision are lesssegregated at the early stages of visual processing (Stewart et al

2015) We note that the sensitivity of PxHCLs to GABA is about500-fold less than that of histamine Whether or not this isphysiologically relevant has to be confirmed for example by directmeasurement of transmitter concentrations at the synaptic sites

Skingsley et al (1995) conducted whole-cell patch-clamprecordings on isolated LMCs in five dipteran species with diversevisual ecologies in order to study the physiological characteristicsof HCLA channels The dosendashresponse functions of LMCs in fast-flying diurnal species exhibit lower sensitivity (higher EC50 value)and a narrower dynamic range (larger nH) than those in slow-flyingcrepuscular species Under the histamine concentration that elicits10 of Imax in each species single-channel analyses of HCLAshave revealed similar properties between species Therefore theobserved species differences should be attributed mainly to synapticgain and perhaps also to the absolute sensitivity of single channels(Skingsley et al 1995) To better understand the evolution of earlyvisual processing in insects it would be worthwhile to compare theLMC membrane response properties as well as single-channelproperties for example between butterflies with flight speeds thatdiffer greatly (Dudley and Srygley 1994)

AcknowledgementsWe thank Dr Finlay Stewart for critically reading the manuscript

Competing interestsThe authors declare no competing or financial interests

Author contributionsConceptualization P-JC KA Methodology Y Takayama MT Formal analysisHDA P-JC MW Y Takayama Investigation HDA P-JC TA Y TeraiMW Y Takayama KA Data curation HDA TA Y Terai Y Takayama KAWriting - original draft HDA KA Writing - review amp editing HDA P-JC TAY Terai MW Y Takayama MT KA Supervision Y Takayama MT KAProject administration KA Funding acquisition MT KA

FundingThis work was financially supported by Grants-in-Aid for Scientific Research(KAKENHI) from the Japan Society for the Promotion of Science (JSPS) to KA(26251036 18H05273) and to P-JC (16J07688) A major part of experiments wasconducted under the collaborative research program of National Institute forPhysiological Sciences

Data availabilityPxhclA and PxhclB sequences are deposited in DDBJ (accession numbersLC373508 and LC373509 respectively)

Supplementary informationSupplementary information available online athttpjebbiologistsorglookupdoi101242jeb183129supplemental

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Gisselmann G Pusch H Hovemann B T and Hatt H (2002) Two cDNAscoding for histamine-gated ion channels in D melanogaster Nat Neurosci5 11-12

8

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Journal

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perim

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iology

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Nishikawa H Iijima T Kajitani R Yamaguchi J Ando T Suzuki YSugano S Fujiyama A Kosugi S and Hirakawa H (2015) A geneticmechanism for female-limited Batesian mimicry in Papilio butterfly Nat Genet47 405-409

Pantazis A Segaran A Liu C-H Nikolaev A Rister J Thum A SRoeder T Semenov E Juusola M andHardie R C (2008) Distinct roles fortwo histamine receptors (hclA and hclB) at theDrosophila photoreceptor synapseJ Neurosci 28 7250-7259

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Ribi W A (1976) The first optic ganglion of the bee II Topographical relationshipsof the monopolar cells within and between cartridges Cell Tissue Res 171359-373

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Sine S M (2002) The nicotinic receptor ligand binding domain Dev Neurobiol53 431-446

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9

RESEARCH ARTICLE Journal of Experimental Biology (2018) 221 jeb183129 doi101242jeb183129

Journal

ofEx

perim

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iology