Intron Retention in mRNA Encoding Ancillary Subunit of Insect Voltage-Gated Sodium Channel

16 Pages·2013·3.28 MB·English

Intron Retention in mRNA Encoding Ancillary Subunit of
Insect Voltage-Gated Sodium Channel Modulates
Channel Expression, Gating Regulation and Drug
Sensitivity
Ce´line M. Bourdin1, Be´ne´dicte Moignot1, Lingxin Wang2, Laurence Murillo1,3, Marjorie Juchaux4,
Sophie Quinchard1, Bruno Lapied1, Nathalie C. Gue´rineau5, Ke Dong2, Christian Legros1,5*
1LaboratoireRe´cepteursetCanauxIoniquesMembranaires(RCIM)UPRESEA2647/USCINRA1330,SFRQUASAV4207,UFRSciences,Universite´d’Angers,Angers,France,
2DepartmentofEntomology,GeneticsandNeurosciencePrograms,MichiganStateUniversity,EastLansing,Michigan,UnitedStatesofAmerica,3LaboratoireLIttoral
ENvironnementetSocie´te´s(LIENSs),UMR7266CNRS,InstitutduLittoraletdel’Environnement,Universite´deLaRochelle,LaRochelle,France,4IMAC,SFRQUASAV4207,
Universite´d’Angers,Beaucouze´,France,5LaboratoiredeBiologieNeurovasculaireetMitochondrialeInte´gre´e,UMRCNRS6214,INSERM1083,UFRdeSciencesMe´dicales,
Universite´d’Angers,Angers,France
Abstract
Insectvoltage-gatedsodium(Na )channelsareformedbyawell-knownpore-forminga-subunitencodedbypara-likegene
v
andancillarysubunitsrelatedtoTipEfromthemutation‘‘temperature-induced-paralysislocusE.’’Theroleoftheseancillary
subunitsinthemodulationofbiophysicalandpharmacologicalpropertiesofNa+currentsarenotenoughdocumented.The
uniqueneuronalancillarysubunitTipE-homologousprotein1ofDrosophilamelanogaster(DmTEH1)stronglyenhancesthe
expression of insect Na channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and
v
functionalexpressionoftwoneuronalDmTEH1-homologsofthecockroach,Periplanetaamericana,PaTEH1AandPaTEH1B,
encodedbyasinglebicistronicgene.InPaTEH1B,thesecondexonencodingthelast11-amino-acidresiduesofPaTEH1Ais
shiftedto39UTRbytheretentionofa96-bpintron-containingcoding-message,thusgeneratinganewC-terminalend.We
investigated the gating and pharmacological properties of the Drosophila Na channel variant (DmNa 1-1) co-expressed
v v
withDmTEH1,PaTEH1A,PaTEH1BoratruncatedmutantPaTEH1D(270-280)inXenopusoocytes.PaTEH1Bcauseda2.2-fold
current density decrease, concomitant with an equivalent a-subunit incorporation decrease in the plasma membrane,
comparedtoPaTEH1AandPaTEH1D(270-280).PaTEH1Bpositivelyshiftedthevoltage-dependencesofactivationandslow
inactivationofDmNa 1-1channelstomorepositivepotentialscomparedtoPaTEH1A,suggestingthattheC-terminalendof
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both proteins may influence the function of the voltage-sensor and the pore of Na channel. Interestingly, our findings
v
showed that the sensitivity of DmNa 1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW
v
dependsonassociatedTEH1-likesubunits.Inconclusion,ourworkdemonstratesforthefirsttimethatdensity,gatingand
pharmacologicalpropertiesofNa channelsexpressedinXenopusoocytescanbemodulatedbyanintronretentionprocess
v
inthe transcription of theneuronalTEH1-likeancillary subunitsofP. americana.
Citation:BourdinCM,MoignotB,WangL,MurilloL,JuchauxM,etal.(2013)IntronRetentioninmRNAEncodingAncillarySubunitofInsectVoltage-Gated
SodiumChannelModulatesChannelExpression,GatingRegulationandDrugSensitivity.PLoSONE8(8):e67290.doi:10.1371/journal.pone.0067290
Editor:ZheZhang,XuzhouMedicalCollege,China
ReceivedFebruary15,2013;AcceptedMay16,2013;PublishedAugust15,2013
Copyright:(cid:2)2013Bourdinetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyINRAandtheRe´gionPaysdelaLoire(Ph.D.fellowshipsofBe´ne´dicteMoignotandCe´lineBourdin).Thefundershadnorole
instudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction InsectauxiliarysubunitsofNa channelsarestructurallyunrelated
v
towell-knownauxiliaryb-subunitsofvertebrateNa channels.While
v
Voltage-gated sodium (Nav) channels are critical for the mammalianNa auxiliaryb-subunitsaresinglemembrane-spanning
v
initiation of action potentials (APs) in excitable cells [1]. Because
proteins [7], Drosophila melanogaster auxiliary subunits (TipE and
of their crucial role in the regulation of membrane excitability in
TEH1-4) contain two membrane-spanning segments [8,9,10]. In
the nervous system, the Na channels are major targets of
v contrast with most mammalian Na a-subunit, expression of para
v
numerousnaturaltoxinsandtherapeuticcompounds,suchaslocal (DmNa1)alonegeneratesverysmallNa+currentsinXenopusoocytes
v
anesthetics(LAs),antiepilepticdrugsandantiarrythmics[2].They
[8,9,10].Thefirstinsectauxiliarysubunitdescribed,TipE,strongly
also constitute a main molecular target of insecticides like enhancesNa+currentswhenco-expressedwithDmNa1inXenopus
pyrethroids and pyrazoline-type insecticides (PTIs) used in pest v
oocytes, indicating that these proteins have strong chaperone or
control [3,4]. They consist in a pore-forming subunit (a-subunit)
stabilizing effects on channel expression [8,9,10]. More recently,
associated with ancillary subunits. In insects, two para-related
Derst et al. [8], show that TEH1 displays a stronger expression
genes encode a-subunits of Na channels [5,6], sharing high
v stimulating effect than TipE on DmNa1 expression. By contrast,
sequence identity withthose ofvertebrate. v
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IntronRetentionofNa+ChannelAncillarySubunit
TEH2 and TEH3 stimulate channel expression to a lower extent, RNAextraction,RT-PCRandcloningoffulllengthPaTEH1
while TEH4 does not at all. Co-expression of DmNav1 and TipE cDNAs
results in Na+ currents with same activation properties, but with
Total RNA extraction was isolated from various tissues using
accelerated current decay and also faster rate of recovery from
theTRIzolH Reagent (Ozyme/Biogentex, France). For polymer-
inactivation[8,10].Moreover,Na+currentselicitedbyco-expression
ase chain reaction (PCR) experiments, first strand cDNAs were
of DmNa1 channels and TipE or TEH1-4, display specific
v synthesized from 5 mg of total RNA using SuperScriptTM III
inactivation and repriming properties, reminding functional prop-
First-StrandSynthesisSystemSuperMix(Invitrogen,USA)inthe
ertiesofmammalianb-subunits[8,10].
presenceofoligo(dT) .Thesequencesoftheprimersusedinour
20
In D. melanogaster, TEH1 was found exclusively expressed in
experimentsarelistedinTable1.ForcloningPaTEH1cDNAin
neuronaltissues,whileTipE,TEH2toTEH4haveabroadtissue
the nerve cord, a degenerated primers pair (DPS1 and DPR1)
distribution, including non-excitable cells [8]. This suggests that
wasdesignedfromconservedregionsobservedinanalignmentof
TEH1andTEH1-likeproteinsofotherinsectspeciesarerelevant
available TEH1 subunit sequences. A PCR amplification of
elements in the modulation of neuronal excitability through the
cDNA fragment was carried out using EuroTaq polymerase
modulation of the gating and pharmacological properties of Na
v (Eurobio, France). PCR products were separated by agarose gel
channels. Indeed, TEH1 dramatically changes the inactivation
electrophoresis and purified using NucleospinH Gel and PCR
properties(voltage-dependenceandrecoveryfrominactivation)of
clean up (Macherey-Nagel, Germany). The cDNA fragments
Na+currentsgeneratedbyDmNa 1channelsinXenopusoocytesin
v were cloned into PCRH 4 TOPOH (Invitrogen). Each clone was
comparison to TipE [8]. Thus, we hypothesized that DmNa 1/
v sequenced in both strands by the company Millegen Biotechnol-
TEH1 channels display different pharmacological properties
ogy.ForRACE(rapidamplification ofcDNAends)of59and39
compared toDmNa 1/TipEchannels.
v endsofPaTEH1,theRNAligase-mediatedrapidamplificationof
PTIs belong to a relatively new class of insecticides, including cDNAendsmethod(GeneRacerTMkit,Invitrogen)wasused.For
indoxacarb and metaflumizone which kill a broad spectrum of amplification of 59end and 39end regions, the gene-specific
insects by inhibiting Na channels [11]. Indoxacarb acts as a
v primer used were P-R1, P-R2, P-S1 and P-S2. PCR products
proinsecticide, which is biotransformed into a toxic N-decarbo-
wereclonedandsequencedasdescribedabove.Finally,theORF
methoxyllated metabolite DCJW [12]. DCJW irreversibly blocks
ofPaTEH1cDNAswasamplifiedusingthegene-specificprimers
APs in preparations of lepidopteran (Manduca sexta) larval motor
P-S3andP-R3,containingtherestrictionenzymesitesXmaIand
nerveandinducesadose-dependentinhibitionofNa channelsin
v XbaI respectively, for directional cloning into pGEM-HEJUEL
cockroach Periplaneta americana neurons [13,14]. The similarity
plasmid suitable for expression in Xenopus oocytes (kindly
betweentheblockingmechanisminducedbyPTIsandLAs,such
provided by Pr. Olaf Pongs, Institute for Neural Signal
as lidocaine was early reported [15]. Indoxacarb/DCJW and
Transduction, Hamburg, Germany). Sequences analyses were
lidocaine preferably bind to inactivated channels resulting in
performedusingBioEditsequenceanalysisSoftware.Full-length
stabilization of this state [13,14,15,16,17,18]. Since PTIs potency
ORFs were identified using BLAST research in the GenBank
dependsontheinactivationpropertiesofNa channels[13,14,15],
v database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Amino
TEH1 could also differentially modulate the sensitivity of Na
v acidsequencesalignmentwascarriedoutusingClustalWmethod
channel toinsecticides.
as described [6].
Thecockroachmodelisa usual tooltoinvestigatewhetherand
Semi-quantitative RT-PCR experiments were performed to
howdoneurotoxicinsecticidesmodulateionchannels.P.americanais
studythetissuedistributionpatternofPaTEH1usingtheP-S3/R3
suitable neurobiological model, for which electrophysiological and
primer pair. Theexperiments were repeated five times.
pharmacologicalstudieshaveshown theexistenceofdifferent Na+
currentswithdistinctbiophysicalandpharmacologicalpropertiesin
Extraction of genomic DNA and PCR
isolated neurons [14,19,20]. To gain further insights into the
Genomic DNA was extracted from five cockroach individuals
molecularcharacterizationofNa channelsinP.americana,wecloned
v usingtheproceduremodifiedfromBlinandStafford[21].Tissues
and characterized two novel variants of teh1-like gene. We showed
werefrozeninliquidnitrogenandstoredat280uC.Frozentissues
that these TEH1-like variants are exclusively expressed in the
werehomogenizedinanextractionbuffer(1 mlper100mgtissue)
nervous system of P. americana and resulted from exclusion or
containing Tris-Cl 10mM (pH8.0), EDTA 0.1 M (pH 8.0),
retention of an intron that modulates expression and gating
pancreaticRNAase20mg/ml,0.5%SDSandincubatedfor1hat
properties of the DmNa1-1 variant expressed in Xenopus oocytes.
Furthermore, we examvined the biophysical properties of Na+ 37uC.ProteinaseKwasaddedtoafinalconcentrationof100mg/
ml and the suspension was incubated at 50uC for 3h. Three
currents resulting from the co-expression of DmNa1-1 with
v phenol extractions were carried out and the DNA in aqueous
TEH1andTEH1-likevariantsofP.americana.Next,wecharacter-
phase was precipitated with 0.2 volume of 10 M acetate
izedthepharmacologicalconsequencesoftheco-expressionofthese
ammonium and 2 volumes of ethanol. High molecular weight
auxiliary subunits and we observed marked differences in the
genomic DNA was recovered with a Pasteur pipette. DNA was
blockingeffectsoflidocaineandDCJWonDmNa1-1channels.
v dissolved in TE (pH 8.0) and stored at 4uC. Pateh1 gene was
amplifiedbynestedPCRusingtheP-S4/R4primerpair,followed
Materials and Methods
bytheP-S3/R3primerpairwithgenomicDNAastemplate.The
Insects amplifiedproductofthesecondPCRwasclonedandsequencedas
described above.
Adult male American cockroaches P. americana were obtained
from our laboratory stock colony maintained at 29uC on 12-h
light/darkcyclewithfoodandwateradlibitum.Intestine,ganglia, Co-expression of DmTEH1 or PaTEH1 variants with
reproductiveglands,muscles(extractedfromthecoxaofthelegs), DmNav1.1 in Xenopus laevis oocytes
headandtheventralnervecord(thoracicandabdominalganglia, A splice variant of the sodium channel from D. melanogaster,
together with the connectives) were dissected from adult male DmNa 1.1,wasusedforco-expressionexperiments[22].pGEM-
v
cockroaches. DmTEH1 construct was kindly provided by Dr Christian Derst
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IntronRetentionofNa+ChannelAncillarySubunit
Table1. Sequencesofthe oligonucleotidesused inPCR andtheircorresponding region.
Primersname Nucleotidesequence Domain
1-DegeneratedprimerusedtoamplifyPaTEH1
DPS1 59-GTGCCGCTSTACGTSGAYCCS-39 MS1–MS2
DPR1 59-ATGCATTGYGACTGCMGSTG-39 MS1–MS2
2-SpecificprimersusedinRACE
P-R1 59-CGATGGGGATGTTGATGTTGAC-39 59UTRpartialORFPaTEH1(PCR#1)
P-R2 59-CGTACATGGTGGTGAAGTTGTC-39 59UTRpartialORFPaTEH1(PCR#2)
P-S1 59-GTCAACATCAACATCCCCATCG-39 39UTRpartialORFPaTEH1(PCR#1)
P-S2 59-GACAACTTCACCACCATGTACG-39 39UTRpartialORFPaTEH1(PCR#2)
3-Primersusedtoamplifythefull-lengthORF
P-S3 59-CAGATCCCGGGATGAGGAGCAGCAGCTCGGAG-39 Full-lengthORFPaTEH1
P-S4 59-AAAAGTTGTCAGCTGTTCGGCG-39 CompletecDNAPaTEH1
P-R3 59-CTGATTCTAGACCGAATCATGTTCTATCTTCT-39 Full-lengthORFPaTEH1
P-R4 59-(T)23CAAAATATAGGCCATGTATTTCTACC-93 CompletecDNAPaTEH1
Designationofoligonucleotidemixtures:S=G+C;Y=C+T;M=A+C.
UTR,untranslatedregion;ORF,open-readingframe.
RestrictionenzymerecognitionsequencesareunderlinedandcorrespondtoXmaIsite(CCCGGG)andXbaIsite(TCTAGA).
doi:10.1371/journal.pone.0067290.t001
(Institute for Integrative Neuroanatomy, Charite´, Berlin, Ger- experiments are performed at room temperature (18–21uC) in
many). All recombinant plasmids were linearized with SpeI SOS.Capacitivetransientandleakcurrentswerecorrectedusing
(Promega, Madison, USA) or NotI (Invitrogen), and capped P/N (N=6) subtractions. For activation experiments, families of
RNA were transcribed in vitro using the T7 mMESSAGE Na+ current traces were generated by step depolarisations to test
mMACHINEkit (Ambion,Austin, USA). potentials from 270 to +40mV (10mV increment) from a
OvarianlobesweresurgicallyharvestedfromadultXenopuslaevis holdingpotentialof2100mV.Na+channelconductance(G)was
previously anesthetized in tricaine (0.15 M, Sigma-Aldrich) and calculatedusingthefollowingequationG=I/(V2Vrev)whereIis
washedinastandardoocytesaline(SOS)solutioncomposedof(in thecurrentamplitude,VisthetestpotentialandVrevisthereverse
mM): NaCl 100, KCl 2, CaCl 1.8, Hepes 5, pH 7.5. potential [23]. The reverse potential was determined from I/V
2
Defolliculated oocytes were obtained after 1 h-treatment with curve fitted using Stu¨hmer’s equation: I=G*(12(1/
2 mg/ml collagenase (type 1A, Sigma-Aldrich, Saint Quentin (1+exp((V2V1/2)/K))))*(V2Vrev) [23]. Conductance values (G)
Fallavier,France)and0.8mg/mltrypsininhibitor(Sigma-Aldrich) were normalized to the maximum conductance and plotted
incalcium-freeSOS.DependingonthecombinationofDmNa 1- against the test voltage. The resulting curves were fitted with the
v
1andauxiliarysubunits,variousamounts(2to35ng)ofDmNav1- following Boltzmann’s equation G=1/(1+exp((V1/22V)/k))
1 and auxiliary subunits RNA mixture were injected in oocytes where V1/2 is the potential at which half of the Nav channels
(1:1 ratio). Injected oocytes were incubated in sterile medium are activated and k is the slope factor [19]. To determine the
composed of SOS supplemented with gentamycin (50 mg/ml), voltagedependenceofsteadystatefastinactivation,familiesofNa+
penicillin (100U/ml), streptomycin (100 mg/ml) and sodium current traces were generated by a two-pulse protocol beginning
pyruvate (2.5mM)at 18uCfor 2to10days before recordings. withastepofpotentialrangingfrom280mVto+40mV(5 mV
increment) for 200ms. A second 12ms test pulse was applied to
25 mV,apotentialatwhichthecurrentamplitudewasmaximal.
Ethics Statement
Eachsetofdoublepulseswasseparatedby10s.Fordetermination
This study was carried out in strict accordance with the
ofthevoltagedependenceofsteady-stateslowinactivation,atwo-
recommendations in the Guide for the Care and Use of
pulse protocol was used, starting with a first step of potential
Laboratory Animals of the European Community. The protocol
ranging from 290mV to +20mV (10 mV increment) for 60s,
was approved by the «Direction De´partementale des services
followed by a 100ms interval at the holding potential. A second
Ve´te´rinaire du Maine et Loire» (NuB49071) and by the ‘‘Comite´
pulse to 25 mV for 10ms allowed measuring the available
d’e´thique en expe´rimentation des Pays de la Loire» (Nu
current. For both fast and slow inactivation, the peak current
CEEA.2012.68).
recordedduringthesecondpulsewasnormalizedtothemaximum
current peak and the data were fitted with Boltzmann’s equation
Electrophysiological recordings and analysis
I/Imax=1/(1+(exp((V2V )/k)) where V is the potential at
Na+ currents were recorded using the two electrode voltage- which half of the Na chan1/n2els are inactiva1t/e2d and k is the slope
v
clamp (TEVC)technique. Oocyteswere testedusing 1M-KCl/2 factor [23]. For recovery from inactivation experiments, Na+
M-Kacetate-filled borosilicate glass electrodes connected to a currentsweregeneratedbyatwo-pulseprotocolbeginningwitha
TEVC amplifier (TEV-200, Dagan Corporation, Minneapolis, first pulse to 25 mV for 50ms followed by a second pulse to
Minessota,USA).Digidata1440Ainterface(AxonCNSMolecular 25 mV for 14.5ms. The duration between the two pulses was
Devices,California,USA)andpCLAMP10software(AxonCNS increased by 1 ms every set of double pulses and each set was
MolecularDevices)wereusedforacquisition(samplingrate:10or separated by10s.Fractional recoverywascalculated bydividing
100kHz; band pass-filter: 5 kHz) and stimulation protocols. All the maximum current amplitude the second test pulse by the
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IntronRetentionofNa+ChannelAncillarySubunit
maximumcurrentamplitudeofthecorrespondingfirstpulse.The groups, or the unpaired Student’s t-test. p values are specified.
recovery data were fit with the following exponential equation: Statisticalprobabilitiesareexpressedas*p,0.05,**p,0.01and
I=I +(Imax2I )*(12exp(2k*(t2t )))wherekistherateconstant *** p,0.001.
0 0 0
and t is the time (GraphPad Software; equation name: plateau
followed by onephase associationequation). Results
Analysis of cDNA clones unmasks possible intron
DCJW and lidocaine sensitivity assays
Oocytes were perfused with 2mM DCJW (DPX-JT333, retention in PaTEH1 transcript
according to a Material Transfer Agreement between Angers Since the genome sequence of Periplaneta americana is not
University and DuPont Crop Protection, Newark, USA) for available in databases, a conventional RT-PCR strategy was
45min. Before DCJW was perfused, several stable recordings performed to clone the cDNAs encoding the homolog of
were performed. Oocytes were perfused with 2mM lidocaine DmTEH1 in the nerve chord of P. americana. Briefly, using
(Sigma-Aldrich) for 10min and Na+ channel blockade was degenerated primers P-S1 and P-R1 (Table 1) designed from
measured at 25 mV. Then the ratio of peak Na+ current after conserved amino acid regions deduced from an alignment of
lidocaine perfusion was measured and compared to the control TEH1 subunits of Drosophila melanogaster (Genbank accession
peak. Protocols described above for activation, slow and fast number NP_649959), Anopheles gambiae (accession number
steady-state inactivation and recovery from fast inactivation were XP_310354), Apis mellifera (accession number XP_001120804),
also performed afterdrug application. Nasoniavitripennis(accessionnumberXP_001606835)andTribolium
castaneum (number XP_969821), a 561bp cDNA fragment was
Proteinexpressionanalysisusingconfocallaserscanning amplified. Blast analysis indicated that the deduced amino acid
sequence of this cDNA shares 51% of sequence identity with the
microscopy
extracellular loop of DmTEH1 and it was thus identified as
For confocal microscopy experiments, albinos Xenopus oocytes
TEH1-likeproteinofP.americana(PaTEH1).ThispartialPaTEH1
were used. To detect auxiliary subunits, a histidine tag
cDNA allowed us to design specific primers for 59 and 39 RACE
(RGHHHHHH) was introduced by PCR at the 59 end of
experiments. By 59 RACE experiment, we obtained a cDNA
PaTEH1A and PaTEH1B constructs. Five days after injection
fragmentof986bpcontaining432bpoftheORFand554pbof
withwaterorwith3 ngRNAencodingDmNa 1.1andPaTEH1A
v the59untranslatedregion(59UTR).39RACEamplificationledto
or PaTEH1B, oocytes were fixed in phosphate buffered saline
the identification of two cDNAfragments of 618bp and 712bp.
(PBS)containing2%paraformaldehydefor30minat4uC.Fixed
Thesequenceofthelongfragmentdifferedfromthatoftheshort
oocytes were washed in PBS and blocked in PBS-Triton (PBS-T)
oneonlyby94bpinsertion37bpupstreamthestopcodonofthe
0.2% fish gelatine for 1h at RT. Then, oocytes were incubated
shortfragment.Thisindicatedthepresenceoftwotranscriptswith
10h at 4uC with mouse RGS-His antibody (dilution 1:500;
distinct ORF 39ends. The 618bp fragment contained the last
Qiagen) and rabbit Anti-Pan Na (SP19) antibody (1:100;
v 197bp of the first ORF; followed by a 421bp 39UTR, and the
Euromedex, France). Oocytes were washed three times in PBS-
712bp fragment contained the last 194bp of the second ORF,
T 0.2% for 5 min and were next incubated 90min with Goat
followed by 518bp 39UTR. Both 39UTR sequences exhibited
Anti-Mouse IgG Alexa FluorH 546 antibody and Goat Anti-
polyAtailwithouttheconsensus‘AATAAA’polyadenylationsite.
Rabbit IgG Alexa FluorH 488 (dilution 1:3000, Invitrogen, USA)
Finally,usingaprimerpairencompassingtheORF,weamplified
and washed three times in PBS. Oocytes were placed in dishes
two different cDNAs. Ten clones contained an ORF of 843bp
(ibidi, Martinsried, Germany) in PBS/glycerol 5:1 solution and
encodinga280aminoacidsprotein,namedPaTEH1A(accession
imaged through the 610/0.3 numerical aperture objective of a
number KC206367). Six other clones displayed a longer insert
Nikon (Nikon Instruments, Melville, NY) A1S1 confocal laser
(937 bp), containing an ORF of 840bp encoding a 279 amino
scanningmicroscopeequippedwithargon-ion(488nm)anddiode
acids protein, named PaTEH1B (accession number KC206368).
(561 nm)lasers.AlexaFluorH488wasexcitedat488nmandthe
The nucleotide sequence of PaTEH1B clones corresponded to
fluorescence emission was collected using a 500–550nm filter; PaTEH1A but with an insertion of 94bp after the position 807.
Alexa FluorH 546 was excited at 561nm and the fluorescence This 94bp insert in PaTEH1B sequence contained a novel stop
emissionwascollectedusinga570–600nmfilter.ImajeJsoftware codonandmodifiedthecodingsequenceofthe39end,leadingtoa
(version 1.4.3.67) was used to measure relative fluorescence. To deduced amino acid protein, PaTEH1B that diverged from
quantify relative fluorescence intensity for each tested oocyte, an PaTEH1A sequence only by the last 11 residues of their C-
identical areaof5002.2 mm2was delimitedandthetotalnumber termini (Figure 1A). These data indicated that PaTEH1A and
ofpixelscorrespondingtoAlexaFluorH488orAlexaFluorH546in PaTEH1Blikelyresultfromanalternativesplicingmechanismofa
this area was determined. No fluorescence was measured for singlegene.
controls in which primary antibodies were omitted. Control of Hydrophobicity analysis [24] of amino acid sequences of
immunostaining specificity was performed using the same PaTEH1A and PaTEH1B (Figure 1B) revealed two hydrophobic
procedure in separate experiments: first with rabbit Anti-Pan domains(frompositions40to67and225to243)thatalignedwith
Nav (SP19) and secondary Goat Anti-Mouse IgG Alexa FluorH the putative transmembrane domain of DmTEH1. Both hydro-
546 antibodies, and second with mouse RGS-His and secondary phobicprofileswereoverlappeduptotheaminoacidresidue269.
Goat Anti-Rabbit IgG AlexaFluorH488antibodies. The three PaTEH1 variants contained four putative N-glycosyl-
ation sites (Asn residues at positions 98, 135, 161 and 205)
Statistical analysis commonwithDmTEH1(Asnresiduesatpositions101,129,142
Statistical analysis of all data was performed using GraphPad and 191) (Figure 1A). Twenty putative O-glycosylation sites (Thr
Prism software (version 5). All data are presented as means 6 residuesatpositions137,139,141,145,147to155and163and
standard error of the mean (SEM). Significance tests between Ser residues at positions 138, 140, 142, 144, 146 and 157) were
groupsofdatawereperformedusingavarianceanalysis(one-way detected in the region between the two membrane-spanning
ANOVA)followedbyaTukeyposthoctestforcomparisonofall domainsof PaTEH1 variants(Figure 1A).
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IntronRetentionofNa+ChannelAncillarySubunit
Figure1.PrimarystructureanalysisofTEH1-likeauxiliarysubunitsofP.americana.A.ClustalWalignmentofPaTEH1A(GenBankaccession
numberKC206367),PaTEH1B(accessionnumberKC206368)andDmTEH1(accessionnumberNP_649959).Transmembranesegments(M1andM2)
areindicatedwithboldlineabovethesequences.ConservedN-glycosylationsitesareindicatedbyclosedinvertedtriangles(.)andO-glycosylation
sitesofPaTEH1ssubunitsareindicatedbyasterisk(*)(www.cbs.dtu.dk).Gapsareindicatedbydashes.Theundecapeptide(VALLDCEEDRT)andthe
decapeptide(YVPLSVHDTR)attheC-terminalendsofPaTEH1variantsareboxed.B.Hydrophobicityprofileanddeducedtopologicalorganizationof
PaTEH1AandPaTEH1B.HydrophobicityanalysiswasperformedusingthealgorithmofKyteandDoolittle(1982).Theaminoacidresiduepositionis
plotted along the x-axis and the calculated mean hydrophobicity is plotted along the y-axis. Regions above the line are hydrophobic. The two
putativemembrane-spanningsegmentsareindicatedasM1andM2.
doi:10.1371/journal.pone.0067290.g001
TodeterminewhetherPaTEH1AandPaTEH1Bweretheresult partialPateh1genesequencewasdelimitedbydonorandacceptor
of an alternative splicing event, a PCR was carried out using splice sites (Figure 2A). While the 59 splice site sequence
genomicDNAofP.americanaandprimersencompassingtheORF conformed to the consensus sequence that is known to promote
of PaTEH1A. A single 900bp PCR fragment was cloned and its splicing,the39splicesitesequence(tttttgtgctgtg)didnot[25].This
sequence totally matched with the sequence of PaTEH1B cDNA. 39 splice site was considered as weak since it contained three
A94bpinsert,whichwasnotpresentinPaTEH1A,foundinthis unusual ‘‘G’’ (at positions 28, 26 and 23) and it ended with
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IntronRetentionofNa+ChannelAncillarySubunit
‘‘TG’’ instead of the highly conserved dinucleotide ‘‘AG’’ DmTEH1,PaTEH1AandPaTEH1BinXenopusoocytes(Figure4).
(Figure 2A). The unusual TG splice acceptor was reported in No current was measurable in oocytes injected with DmTEH1,
both insect and mammalian genes [26,27]. The additional 94bp PaTEH1AandPaTEH1Baloneascontrol(datanotshown).We
sequencefoundinPateh1genealsoshowedaconsensusbranchsite observed that the required amounts of RNA (1.8 to 35ng) to
(TAGCGAC) close to the end of the putative intron. In recordNa+currentswithappropriateamplitudes(withmaximum
conclusion, assuming that only one copy of teh1 gene is present peak comprised between 0.25 and 2 mA) varied in each case.
inP.americanagenome,the94bpinsertmostlikelycorrespondsto Unfortunately, we were unable to compare these effects with the
an intron that is unspliced in PaTEH1B (Figure 2B). Altogether, co-expression of the Nav a-subunit of P. americana [6] and
these observations demonstrate that the variant PaTEH1B results DmTEH1, DmTipE, PaTEH1A or PaTEH1B, because Na+
froman intronretention mechanism. currentswerenotdetectableevenafterprolongedincubationtime
(FigureS1).AsillustratedinFigure4A,35ngofDmNa 1-1RNA
v
Tissue distribution of PaTEH1 transcripts wereinjectedinoocytestoobtainamaximumpeakof,0.25mA
aftertendayswithoutanyauxiliarysubunits.Ontheotherhand,
Semi-quantitative RT-PCRs using P-S3 and P-R3 primers
whenco-injectedwithDmTEH1,4.2-foldfewerDmNa 1-1RNAs
encompassing the ORF were performed to determine the v
were needed to yield 0.4mA maximum current after 3-days
expression pattern of PaTEH1 transcripts, in six different tissues.
incubation, indicating a chaperone-like effect that had been
First, amplification of PaTEH1 cDNAs were detected in head,
previously reported [8]. Interestingly, this effect was significantly
thoracic ganglia and nerve cord (Figure 3), indicating that
stronger with PaTEH1A (1.2 mA, 3-days incubation) and Pa-
PaTEH1 variants were exclusively expressed in neuronal tissues.
TEH1B (0.6 mA, 4-days incubation), even for lower amounts of
The agarose gel showed two bands with different intensity. The
injected RNAs (1.8 and 2.8 ng, respectively). For an accurate
lower band matched perfectly with PaTEH1A (expected size:
comparison of the chaperone-like effect of these different b-
843bp) and the upper band exactly corresponded to PaTEH1B
subunits,thecurrentdensitiesofNa+currentsweremeasuredasa
(expected size: 936bp). PaTEH1A transcripts appeared to be less
ratioofmaximumcurrentdensityandRNAamountafter3days
abundant in head than PaTEH1B. By contrast, PaTEH1A
of injection (Figure 4B). In comparison to DmTEH1, PaTEH1A
transcripts were expressed at higher levels than PaTEH1B both
had the strongest chaperone-like effect (6.3-fold, p,0.001),
in thoracicgangliaand innervecord.
followed by PaTEH1B (2.8-fold, p,0.001). We also observed a
decrease in the proportion of oocytes expressing detectable
Na+ current densities are modulated by PaTEH1 variants currents with PaTEH1B (,50%) compared with PaTEH1A
in Xenopus oocytes (,90%). PaTEH1A appeared to be more efficient (2.2-fold,
We first characterized the Na+ currents elicited by the p,0.001)thanPaTEH1BtoincreaseNa+currentdensities.Since
expression of DmNa 1-1 [22], alone or in combination with both differed in sequence only by their last 11 amino acids
v
Figure2.OrganizationofgenomicregionandmRNAandencodingTEH1-likesubunitofP.americana.A.ThegenomicregionofPateh1
consistsintwoexonsinterruptedbyashortintron.Theintronsequenceisinlowercaselettersandsplicedonorsite,branchsiteandspliceacceptor
siteareunderlined.Sizesofexonsandintronareindicatedinparentheses.*:stopcodon.B.ExclusionorretentionoftheintronledtoPaTEH1Aand
PaTEH1BvariantswithdifferentC-terminalends.The94bpintronsequencecontainsashortnewcodingsequencefollowedbyanin-framestop
codon.Thisgeneratesasecondprotein(PaTEH1B)withanovelC-terminalend.
doi:10.1371/journal.pone.0067290.g002
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IntronRetentionofNa+ChannelAncillarySubunit
Figure3.Semi-quantitativeRT-PCRanalysisofPaTEH1expressioninvariousP.americanatissues.RT-PCRwasperformedusing5mgof
mRNAextractedfromhead,thoracicganglia,nervecord,muscles,gutandmushroom-shapedaccessorygland.Actin(Genbankaccessionnumber
AY116670)wasusedasinternalquantitativecontrol.
doi:10.1371/journal.pone.0067290.g003
residues, the unique modification by PaTEH1B was the result of anism is likely involved in the regulation of Na+ channels
the novel C-terminal extremity generated by the intron retention expression inneurons of P.americana.
mechanism. A truncated form of PaTEH1s lacking the 11 last
amino-acidresidues(PaTEH1D(270-280))modulatedNa+current Biophysical properties of Na+ currents are modified by
densities similarly to PaTEH1A (Figure 4B). The high sequence TEH1 variants in Xenopus oocytes
identitysharedbyDmNav1withPaNav1(81%)andwiththeNav Activation and inactivation properties of the DmNav1-1
channeloftheGermancockroachBlatellagermanica,BgNav1(77%) channel co-expressed with DmTEH1, PaTEH1A, PaTEH1B or
[6] led us to postulate that the consequence of the interaction PaTEH1D(270-280)were reported in Table2. The expression of
between a-subunit and auxiliary subunit would be conserved in DmNa 1-1 alone led to very small current amplitude either with
v
insects. To confirm this assumption, we co-expressed the variant injection of large amount of RNA (up to 35ng) and long
BgNav1-1a, containinga similar exonorganization asDmNav1-1 incubation time (up to 10 days). Thus, we could not accurately
with PaTEH1A or PaTEH1B (Figures S2). The resulting Na+ assay Na+ currents elicited by DmNa 1-1 expression alone.
v
current densities were roughly twice-fold higher in oocytes Figure 6A shows the voltage-dependence of activation of
expressing BgNav1-1a/PaTEH1A than BgNav1-1a/PaTEH1B, DmNav1-1 with DmTEH1, PaTEH1A, PaTEH1B or Pa-
arguing that the C-terminal end of PaTEH1B modulated TEH1D(270-280). We observed a significant negative shift in the
Drosophila and cockroach Nav channel expression to a similar voltage-dependence of activation by PaTEH1A compared with
extent. Altogether, our data indicate that the last 11 amino-acid DmTEH1 (8.1mV, p,0.001) and PaTEH1B (5.1 mV, p,0.05)
residues of PaTEH1A are not involved in the chaperone-like (Table 2). In contrast, the voltage-dependence of activation
effects and the presence of the unique C-terminal sequence in regulated by PaTEH1B was not significantly different from that
PaTEH1B encoded by the retained intron interferes with the of DmTEH1. The voltage-dependences of activation of Na+
chaperone-like properties. currents were positively shifted to the same extent by PaTEH1B
In order to assess whether the enhancement of Na+ current (3.6mV,p,0.01)whenco-expressedwithBgNa 1-1aincompar-
v
densitiesreflectanincreaseofDmNav1-1accumulationinXenopus ison with PaTEH1A (Figure S3A, Table S1). Interestingly, the
oocytes membrane, we designed two Histag-PaTEH1A and voltage-dependence of activation of DmNa 1-1 with Pa-
v
PaTEH1B constructs for double immunostaining. We ensured TEH1D(270-280) was similar to that of DmNa 1-1/PaTEH1A.
v
that the addition of the N-terminal His-tag to PaTEH1A and This indicated that the 11-amino-acid C-terminal end of
PaTEH1B did not modify the electrophysiological properties of PaTEH1B modulated activation properties of Na+ currents, but
Na+currentselicitedbyco-expressionwithDmNav1-1,eventheir not that of PaTEH1A.
chaperone-like effects (data not shown). Figure 5A and 5B show Figure6Band6Cshowthevoltage-dependencesofsteady-state
immunofluorescent confocal images recorded for oocyte injected fast and slow inactivation. The voltage-dependences of fast
with water (negative control) and with DmNav1.1 together with inactivationofNa+currentsarenotsignificantlydifferentbetween
PaTEH1A or PaTEH1B RNAs. The data show a significant DmNa 1-1/DmTEH1 and DmNa 1-1/PaTEH1B, and between
v v
increase (1.8 fold, p,0.01, Figure 5C) in the membrane relative DmNa 1-1/PaTEH1A and DmNa 1-1/PaTEH1D(270-280)
v v
fluorescence intensity (AlexaH488 for DmNav1-1) in oocytes (Table 2). Compared with PaTEH1B, both PaTEH1A and
expressingDmNa 1-1/PaTEH1Acomparedwithoocytesexpress- PaTEH1D(270-280) caused a small but significant negative shifts
v
ing DmNa 1-1/PaTEH1B. This indicated that PaTEH1A in- in the voltage-dependences of fast inactivation (2.8mV and
v
ducedastrongerexpressionoftheDmNa 1-1channelsinoocytes 2.9 mV for PaTEH1A and PaTEH1D(270-280), respectively,
v
membrane than PaTEH1B. Unexpectedly, we also observed a p,0.01). By contrast, no significant difference in the voltage-
higher level of membrane relative fluorescence intensity dependences of fast inactivation was found between BgNa 1-1a/
v
(AlexaH546forPaTEH1AorPaTEH1B)inoocytesinjectedwith PaTEH1A and BgNa 1-1a/PaTEH1B (Figure S3B, Table S1).
v
DmNa 1-1/PaTEH1B than with DmNa 1-1/PaTEH1A RNAs Also, the voltage-dependence of slow inactivation between Na+
v v
(2.2fold,p,0.001,Figure5C).Thesefindingsdemonstratedthat currentsobtainedwithDmTEH1,PaTEH1B,orPaTEH1D(270-
the C-terminal extremity encoded by the intronic sequence of 280) was not modified. Interestingly, PaTEH1A shifted the
PaTEH1B was responsible for the decrease in the density of voltage-dependence of slow inactivation to a more negative
DmNa 1-1 channels, suggesting that the intron retention mech- potential,comparedtoDmTEH1(26.8 mV,p,0.05),PaTEH1B
v
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IntronRetentionofNa+ChannelAncillarySubunit
Figure4.ModulationofNa+currentdensitiesbydifferentTEH1auxiliarysubunitsubtypes.A.FamilyofNa+currentsmeasuredattest
potentialsof270mVto40mVfromaholdingpotentialof2100mVforDmNa1-1alone(a)orco-expressedwithDmTEH1(a+DmTEH1),PaTEH1A
v
(a+PaTEH1A),PaTEH1B(a+PaTEH1B)orPaTEH1D(270-280)(a+PaTEH1D(270-280))injectedwithabout35ng,8ng,2ng,3ngand2ngofRNA(a:b,
1:1),respectively.Theprotocolofdepolarizingvoltage-clampstepsisshown.B.Na+currentdensityperngofinjectedRNAafter3-daysincubation,
exceptforDmNav1-1(10daysafterinjection).ResultsareexpressedinmApernFperngofinjectedRNA(One-wayANOVA:F(4,55)=15.11,p,0.0001
posthocTukeytest).Thenumberoftestedoocytesisindicatedinthebarhistogram.
doi:10.1371/journal.pone.0067290.g004
(26.1mV, no significant) and PaTEH1D(270-280) (25.0mV, (p=0.0340), 24.3 mV for PaTEH1B (p=0.0009), 23.0mV for
p,0.05) (Table 2). For all combinations, the rate of recovery as DmTEH1 (p=0.0280) and 23.3mV for PaTEH1D(270-280)
illustrated in Figure 6D, showed no significant difference (One- (p=0.0396)comparedtothecontrol.Similarly,lidocaineinduces
way ANOVA: F =2.104, p=0.1098, post hoc Tukey test) a shift in the voltage-dependence of slow inactivation towards
(3,60)
between thedifferent TEH1s. hyperpolarizing potentials: 215mV for PaTEH1A (p=0.0121),
29.7mV for PaTEH1B (p=0.0426), 214.2 mV for DmTEH1
Blocking effects by Na channel inhibitors depend on (p=0.0069) and 216.5 mV for PaTEH1D(270-280) (p=0.0101).
v
TEH1 subtypes Lidocaine significantly increased the time constant of recovery
We next examined the involvement of the C-terminal end of from inactivation by 1.2ms (PaTEH1A, p=0.0006), 1.4ms
TEH1onthepharmacologicalpropertiesofDmNa 1-1channels. (PaTEH1B, p,0.0001), 0.8 ms (DmTEH1, p=0.0338) and
v
First, we measured the inhibition of Na+ currents by the LA, 1.1 ms (PaTEH1D(270-280), p=0.0115) compared to control.
lidocaine (2 mM) (Figure 7A and 7B). Interestingly, lidocaine Lidocaine slowed down the recovery of all tested Na+ channels
caused a significant (p,0.01) greater inhibition of Na+ currents combinations to the same extent (One-way ANOVA:
with PaTEH1B (44.863.62%) than with PaTEH1A F(3,20)=1.178, p=0.35, post hoc Tukey test). In addition, in the
(28.462.97%). No significant difference was observed between presenceoflidocaine,Na+currentsrecoveredpartially,meaninga
DmTEH1 (34.862.58%) and PaTEH1A. We also found no partialblockofNa+channelsintheinactivationstate(Figure9A).
significantdifferencebetweenPaTEH1BandPaTEH1D(270-280) The recovery of lidocaine-blocked Na+ currents was significantly
(36.662.9%).ThesedataindicatedthattheparticularC-terminal lower (p,0.01) with PaTEH1A (87.0862.65%), than with
end of PaTEH1A (VALLDWEEDRT) played a role in the DmTEH1 (76.2061.85%), PaTEH1B (79.2161.54%) and (Pa-
decreaseoftheNa+currentsensitivitytolidocaine.Tounderstand TEH1D(270-280)(75.7161.29%) (Figure 9B).
these differences, we analysed the effect of lidocaine on Na+ WealsoassessedthesensitivityofDmNav1-1co-expressedwith
channel gating properties (Figure 8A–D). Lidocaine significantly the four TEH1 subtypes to DCJW, the active metabolite of
shiftedthevoltage-dependenceofactivationtowardsdepolarizing indoxacarb which is a Na+ channel-targeting insecticide [12,13].
potentials in each case (Figure 8A and Table 2) by 9.3 mV The Na+ currents obtained with DmTEH1 were found more
(PaTEH1A, p=0.0036), 10.5 mV (PaTEH1B, p=0.0005), sensitive (39.463.6% inhibition) to DCJW (2 mM), than those
5.7 mV (DmTEH1, p=0.0466) and 14.7 mV (PaTEH1D(270- obtained with PaTEH1A (25.162.1%, p,0.05) and with the
280),p=0.008)comparedtothecontrol(Table2).Lidocainealso truncated form, PaTEH1D(270-280) (8.361.9%, p,0.001)
induced a shift of the voltage-dependence of fast inactivation (Figure 7C and 7D). By contrast, no significant difference of
towards hyperpolarizing potentials: 23.4mV for PaTEH1A sensitivities to DCJW was observed between DmTEH1 and
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IntronRetentionofNa+ChannelAncillarySubunit
Figure5.ModulationofNa channelsexpressionbyintronretentionmechanismofteh1-likegeneofP.americana.Immunofluorescent
v
confocalimagesofrepresentativeoocytesinjectedwithwater(control)orwith3ngofDmNa1.1/PaTEH1A(a+PaTEH1A)orPaTEH1B(a+PaTEH1B)
v
RNAs.A. Immunostaining representative snapshot with an anti Pan-Na (AlexaH488)antibodyand associatedwireframeplots drawnwithImajeJ
v
software(version1.4.3.67).B.ImmunostainingrepresentativesnapshotwithanRGS-His(AlexaH546)antibodyandassociatedwireframeplotsdrawn
withImajeJsoftware.C.RelativefluorescencewasmeasuredusingtheImajeJsoftware(version1.4.3.67).Foreachmeasurement,anidenticalregion
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IntronRetentionofNa+ChannelAncillarySubunit
ofinterestof5002.2mm2(292636pixels)wasdefined.MeanrelativefluorescenceintensityvaluesmeasuredwithAlexaH488(white)andAlexaH546
(grey)aremean6SEMfor6oocytes.PaTEH1Ashowsastrongerexpressionofthea-subunitthanPaTEH1Binoocytes(One-wayANOVA:AlexaH488:
F =24.86,p,0.0001andAlexaH546:F =39.81,p,0.0001,posthocTukeytest).a.u:arbitraryunit.
(2,16) (2,15)
doi:10.1371/journal.pone.0067290.g005
PaTEH1B (31.467.1%). Thus, our findings indicate that the We also analyzed the effects of DCJW (2mM) on the gating
truncation of 11 amino acid residues at the C-terminal end of properties of DmNa 1-1 channels. DCJW shifted the voltage-
v
TEH1 b-subunits strongly decreased the sensitivity of Na+ dependence of activation towards depolarized potentials by
channelstoDCJW. 8.7 mV (p=0.0036; Figure 8A, Table 2) only for DmNa 1-1 co-
v
expressedwithPaTEH1B.ItappearedthatDCJWslightlyshifted
Figure6.BiophysicalpropertiesofNa+currentselicitedbyco-expressionofDmNa 1-1withDmTEH1orPaTEH1-likesubunits.A.
v
Voltage-dependence of activation (One-way-ANOVA: F =6.67, p,0.0005, post-hoc Tukey test). G represents the conductance. B. Voltage
(3,65)
dependenceoffaststeady-stateinactivation(One-way-ANOVA:F =6.84,p=0.0009,post-hocTukeytest).C.Voltage-dependenceofslowsteady-
(3,75)
stateinactivation(One-way-ANOVA:F =4.49,p=0.014,post-hocTukeytest).D.Recoveryfromfastinactivation(One-way-ANOVA:F =2.104,
(3,23) (3,62)
p=0.1098,post-hocTukeytest).Na+currentamplitudes(I)weremeasuredusingthepulseprotocolsdescribedintheMaterialsandMethodssection
andwerenormalizedtothelargestcurrentamplitude(Imax).Valuesaremean6SEM.Thenumberofindividualexperiments,eachperformedwitha
differentoocyte,isindicatedinparentheses.Standardprotocolsareshownininsets.
doi:10.1371/journal.pone.0067290.g006
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