The role of an ancestral hyperpolarization activated cyclic nucleotide-gated K+-channel in ...

33 Pages·2016·1.3 MB·English

©2016.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
RESEARCHARTICLE
The role of an ancestral hyperpolarization-activated cyclic
+
nucleotide-gated K channel in branchial acid-base regulation in
the green crab, Carcinus maenas
SandraFehsenfeld1,2,*andDirkWeihrauch2
ABSTRACT uptake of Na+ and Cl− in crustaceans (Riestenpatt et al., 1996;
Numerous electrophysiological studies on branchial K+ transport in Onkenetal.,2003).BecauseofthepresenceofapicalNKCCand
apical K+ channels, as well as the accompanying basolateral Cl−
brachyurancrabshaveestablishedanimportantroleforpotassium
channel, the moderately leaky gill epithelium of C. maenas has
channelsinosmoregulatoryionuptakeandammoniaexcretioninthe
been compared with the thick ascending limb (TAL) of the
gillepitheliumofdecapodcrustaceans.However,hardlyanythingis
mammalian kidney (Riestenpatt et al., 1996). The apical K+
knownoftheactualnatureofthesechannelsincrustaceans.Inthe
channelpresentintheTALisamemberoftheinwardlyrectifying
presentstudy,theidentificationofahyperpolarization-activatedcyclic
K+ channel family K (Kir1.1) and promotes apical efflux of K+
nucleotide-gated potassium channel (HCN) in the transcriptome of IR
to energize the apical NKCC. Additionally, Kir4.1/5.1 has been
the green crab Carcinus maenas and subsequent performance of
identifiedtobeco-localizedbasolaterallywiththeNa+/K+-ATPase
quantitativereal-timePCRrevealedtheubiquitousexpressionofthis
in the kidney’s distal tubule, providing a basolateral back flow of
channelinthisspecies.EventhoughmRNAexpressionlevelsinthe
K+ out of the cell into the body fluids to maintain the
cerebral ganglion were found to be approximately 10 times higher
electrochemical gradient (Hibino et al., 2010).
compared with all other tissues, posterior gills still expressed
Asageneral,relativelyunspecificinhibitorofK+currents,apical
significant levels of HCN, indicating an important role for this
transporter in branchialion regulation. The relativelyunspecific K+- 15mmoll−1Ba2+reducedtheshort-circuitcurrent (ISC)andtrans-
channelinhibitorBa2+,aswellastheHCN-specificblockerZD7288, epithelial conductance (GTE) of split gill lamellae of C. maenas
(Riestenpatt, 1995; Riestenpatt et al., 1996). In gills of Neohelice
as applied in gill perfusion experiments and electrophysiological
(Chasmagnathus)granulata,basolateralapplicationof10mmoll−1
studiesemployingthesplitgilllamellaerevealedthepresenceofat
least two different K+/NH+-transporting structures in the branchial BaCl2 altered ISC without affecting the conductance, but had no
4 effect when applied apically even though apical Cs+, another
epitheliumofC.maenas.Furthermore,HCNmRNAlevelsinposterior
unspecificK+channelblocker,resultedinasignificantdecreaseof
gill 7 decreased significantly in response to the respiratory or
I (Onken et al., 2003). At these relatively high concentrations,
metabolic acidosisthat was induced byacclimation of green crabs SC
Ba2+ is believed to target voltage-gated/delayed-rectifier K+
to high environmental P and ammonia, respectively.
CO2 channels (Rudy, 1988; Wollmuth, 1994), while specifically
Consequently, the present study provides first evidence that HCN-
promotedNH+epithelialtransportisinvolvedinbothbranchialacid- inhibitingCa2+-activatedK+channelsandK+channelsoftheKIR
4 family only in lower concentrations of up to 100µmol l−1
baseandammoniaregulationinaninvertebrate.
(Wollmuth, 1994; Franchini et al., 2004). The fact that the K+-
KEYWORDS:Ammonia,Gillperfusion,HCN,Hypercapnia,HEA dependent current in the C. maenas gill could be more potently
inhibited by 10mmoll−1 apical Cs+, however, indicates the
INTRODUCTION presence of at least two, a Ba2+-sensitive and a Ba2+-insensitive
Potassium channels form a diverse family of ubiquitously K+channelpopulation(Riestenpatt,1995;Riestenpattetal.,1996).
expressed transmembrane proteins that are crucial for a number Furthermore,applicationofBa2+insplitgillsoftheChinesemitten
of important physiological functions (Tian et al., 2014). In crab,Eriocheirsinensis,resultedinacomplexdi-phasicresponseof
y
particular,K+fluxesareessentialforthemembraneexcitabilityof theshort-circuitcurrentI (Onkenetal.,1991),alsoindicatingthe g
neurons and muscles (Choe, 2002). In the crustacean gill participationofmorethaSnConekindofK+channel. o
l
o
epithelium, K+ channels have been identified to participate in In addition to their role in osmoregulation, basolateral K+ i
B
the establishment of an electrochemical gradient over the channels also participate in branchial ammonia excretion in
l
basolateral membrane mainly generated by a basolateral Na+/K+- C. maenas, with NH+ substituting for K+ because of its similar a
4 t
ATPase (Onken et al., 1991; Riestenpatt et al., 1996). sizeandcharge(Lignon,1987;Skou,1960;Weihrauchetal.,1998). n
e
Furthermore, apical K+ channels seem to co-localize with apical Apical application of Cs+, however, did not affect branchial m
Na+/K+/2Cl− (NKCC) to allow K+ circulation and thereby an ammonia excretion in this species (Weihrauch et al., 1998). i
r
Furthermore, branchial ammonia regulation in C. maenas has e
p
been observed to be closely linked to acid-base regulation x
1DepartmentofZoology,UniversityofBritishColumbia,6720UniversityBlvd, (Fehsenfeld and Weihrauch, 2013), but the specific role of K+ E
Vancouver,BritishColumbia,CanadaV6T1Z4.2DepartmentofBiological f
Sciences,UniversityofManitoba,190DysartRoad,Winnipeg,Manitoba,Canada channelsinthiscontextisnotclear. o
R3T2N2. Interestingly, a K+ channel of the hyperpolarization-activated al
cyclic nucleotide-gated channel family (HCN2) has recently been n
*Authorforcorrespondence([email protected]) r
shown to be involved in acid-base and ammonia regulation in the u
o
Received6November2015;Accepted11January2016 collectingductofthemammaliankidney(Carrisoza-Gaytánetal., J
887
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
2011). Although to date, HCN2 has been mainly associated with 0.03, P =96±7Pa), until gill perfusions were performed (see
CO2
pacemaker activities (DiFrancesco, 1993), Carrisoza-Gaytán and below). Green crabs were fed ad libitum with blue mussels,
colleagues (2011) showed that this gene is also expressed in the Mytilus edulis, twice per week. Animals were starved for a
basolateral epithelium of rat kidney cells and is capable of minimum of 2days before experiments.
promotingNH+transportoverthemembrane,thusparticipatingin Forquantitativereal-timePCRandUssingchamberexperiments,
4
mammalianrenalpHhomeostasis. malegreencrabswerecaughtinBarkleySoundattheopeningofthe
The present study aims to investigate the role of K+ channels Pipestem Inlet (Vancouver Island, BC, Canada) in summer 2013
inbranchialacid-baseregulationoftheosmoregulatinggreencrab under Department of Fisheries and Oceans collection permit XR-
C.maenas,andparticularlyofanHCN-likeproteinasidentifiedin 235-2013, and transported back to Winnipeg, MB, Canada. They
the transcriptome. By application of two different K+ channel were kept at the animal holding facility of the University of
inhibitors, Ba2+ and the HCN-specific ZD7288, acid-base and Manitobainaerated1200litretankswithartificialseawateradjusted
ammonia regulatory properties of the gill epithelium will be to a salinity of 32ppt at 14°C (Seachem Marine Salt®) until
characterizedwithrespecttoK+andNH+transport.Apotentialrole experimentation. Foracclimationtodilutesalinityof11ppt,eight
4
forK+channels,andspecificallyforHCN,willbeevaluated. animals were transferred into a 120 litre tank for a minimum of
7days and fed ad libitum with squid once a week. Water was
MATERIALSANDMETHODS exchanged at least every second day and always on the day after
IdentificationandgenetreeanalysisofCmHCN feeding.Greencrabswerestarvedforaminimumof2dayspriorto
To identify potential candidates for the hyperpolarization-activated experiments.
cyclic nucleotide-gated potassium channel family in C. maenas Forsubsequentacclimationofgreencrabstohighenvironmental
(CmHCN),thepartialaminoacidsequenceforthehyperpolarization- ammonia(HEA)for7days,thebrackishwaterofthe120litretanks
activated ion channel of Cancer borealis (GenBank accession no. was enriched with1mmoll−1NH Cl. Waterwas exchanged ona
4
AAZ80094.3)wasusedasassemblyscaffoldforarecentlygenerated dailybasisandanimalswerenotfedduringHEAacclimation.
transcriptomeforC.maenas(Verbruggenetal.,2015)usingthebasic
local alignment search tool (BLAST)feature in Geneious© version Quantitativereal-timePCR
7.1 (Biomatters Limited, Auckland, New Zealand). Accordingly, Geneexpressionanalysiswasperformedontissuesobtainedfrom
potential candidates for the basic voltage-gated (K ), inward- animalsheldatUniversityofManitoba.Posteriorgill7andallother
V
rectifying (K ), two-pore domain (K ) and Ca2+-activated (K ) tissuesamplesofosmoregulatingC.maenas(11ppt)acclimatedto
IR 2P Ca
K+channelswereidentifiedintheC.maenastranscriptomeusingthe control conditions and HEA (1mmoll−1 NH Cl, 7days) were
4
sequences for the potassium voltage-gated channel subfamily H harvested from animals acclimated as described above, while
member 6 isoform 1 (accession no. NP_110406.1) and the ATP- samplesforhypercapnia-acclimatedgreencrabs(400Pa)wereused
dependent inward-rectifying potassium channel KIR4.1 and 5.1 of as described in the recent study by Fehsenfeld and Weihrauch
Homosapiens (accession nosAAB07046.1and AAF73240.1), the (2013). Tissue samples representing anterior and posterior gills
acid-sensitivetwo-poredomainK+channeldTASK-7ofDrosophila were generated by pooling equal tissue amounts of the respective
melanogaster (accession no. CAI72673.1), as well as the calcium- gillspriortoisolatingtheRNA(gills4and5foranteriorgills,and
activated potassium channel of C. borealis (accession no. gills7,8and 9forposteriorgills).
AAZ80093.4). Briefly, total RNA was isolated under RNase-free conditions
The best protein model for the gene analysis on all HCN usingTRIZOL(Invitrogen,Carlsbad,CA,USA).FollowingDNase
candidates as listed in Table 1 was chosen according to the test I (Ambion™, Thermo Fisher Scientific, Pittsburgh, PA, USA)
function provided in MEGA 6.06 (Tamura et al., 2013). treatment, regular PCR was performed to ensure the absence of
Subsequently, a gene tree was constructed in MEGA 6.06, DNAtraces(RbS-3primers;forward:5′GTCCCTTTTCACCAA-
applying the maximum likelihood method using the gamma- GGACA; reverse: 3′ CAAGGCCAAACTCAACA GGTT).
distributed GL model with invariant sites (LG+I+G; Le and Subsequently, verified DNA-free RNA was transcribed with the
Gascuel, 2008). A matrix of pairwise distances estimated using a iScript cDNA synthesis kit (Bio-Rad, Mississauga, ON, Canada).
JTTmodel(Jonesetal.,1992)wasusedtoautomaticallygenerate Quantitativereal-timePCR(qPCR)wasperformedon20ngcDNA
an initial tree(s) for the heuristic search by applying neighbour- andafinalprimerconcentrationof0.4µmoll−1in15µlreactions
joining and BioNJ algorithms and subsequently selecting the with the SSO FastEvaGreen Supermix (Bio-Rad). The gene-
y
topologywiththesuperiorlog-likelihood value.Evolutionary rate specific primers for CmHCN were designed based on the g
differences among sites were modelled by discrete gamma identifiedtranscriptomecontig(forward:5′GTCTTCATGCGCA- lo
o
distribution (six categories). All positions with lessthan 95% site TCTTCAA; reverse: 5′-GTTGATTGCCACCCAAGAGT) and i
B
coveragewereeliminated(i.e.lessthan5%alignmentgaps,missing resulted in the expected specific 123bp fragment. The primers
l
a
dataandambiguousbaseswereallowedatanyposition).Bootstrap were ensured to result in a single signal in the qPCR byadding a
t
analyseswereperformedwith1000replicates. meltingcurveanalysisfollowingtheregularqPCR.IneveryqPCR n
e
run,anegativecontrol(notemplate)wasincludedtoverifythatthe m
Animals assaywasnotcontaminated,aswellasoneofthestandardstreated ri
e
For gill perfusion experiments, male green crabs, Carcinus as an unknown sample to ensure homogeneous performance. A
p
maenas (Linnaeus 1758), were caught in traps in the Kiel Fjord standardcurvewasincludedineachqPCRrunbasedonadilution x
E
(Baltic Sea) in front of the GEOMAR pier (west shore building, series of known quantities of the respective gel-extracted gene
f
54°19.8′N, 10°9.0′E) at 3–4m depth in May 2012 (collection fragment (0.01–100fg; QIAquick Gel Extraction Kit, Qiagen). o
permit issued September 2009, file no. V313-72241.121-19). Additionally,thehousekeepinggeneribosomalRbS-3wasusedfor al
n
Animalswerekeptinca.250litreflow-throughtanksinaclimate internalnormalizationofhypercapnia-relatedtissuesinaccordance
r
chamberat GEOMAR, directly fed by water from the Kiel Fjord withFehsenfeldandWeihrauch(2013).Nohousekeepinggene(i.e. u
o
(salinity=14.3±0.0ppt, temperature=17.2±0.4°C, pH =7.96± RbS-3, arginine kinase) with constant mRNA expression levels J
NBS
888
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
Table1.InformationonthepotassiumchannelsasincludedinthegenetreeanalysisasdepictedinFig.2
Species (Infra)Class Accessionno. Description
Homosapiens Mammalia NP_001185.3 HCN(2)
NP_110406.1 K (H6isoform1)
V
Q9H427.2 K (KT3.3)
2P
NP_002238.2 K (subunitalpha-1b)
Ca
AAB07046.1 K 4.1
IR
AAF73240.1 K 5.1
IR
Musmusculus Mammalia NP_032252.1 HCN(2)
NP_001032801.1 K (H6)
V
AAC53367.1 K (TWIK-related)
2P
NP_001240307.1 K (subunitalpha-1.22)
Ca
BAA92432.1 K 4.1
IR
BAA34723.1 K 5.1
IR
Daniorerio Teleostei XP_686078.4 HCN
NP_998002.1 K (H6)
V
NP_001122021.1 K (K9)
2P
NP_001139072.1 K (subunitalpha-1)
Ca
NP_001092204.1 K 4.1
IR
NP_001265731.1 K (J2a)
IR
Strongylocentrotuspurpuratus Echinoidea NP_001028182.1 HCN
XP_011669414.1 K (H7isoformX1)
V
XP_782284.2 K (sup-9-like)
2P
XP_011666091.1 K (subunitalpha-1)
Ca
XP_789112.1 K (12)
IR
Aedesaegypti Insecta XP_001658860.1 HCN(AAEL008056-PA)
XP_001653553.1 K (AAEL001542-PA)
V
XP_001652733.1 K (AAEL007386-PA)
2P
AAL40812.1 K (AAEL003765-PA)
Ca
AGA61793.1 K (1)
IR
Drosophilamelanogaster Insecta AAX78393.1 HCN(DMIH-A1B1C1)
NP_476713.1 K (seizure,isoformA)
V
CAI72673.1 K (dTASK-7)
2P
AAA28651.1 K
Ca
CAC87638.1 K
IR
Daphniapulex Branchiopoda EFX81385.1 HCN(DAPPUDRAFT_2754)
EFX90436.1 K (DAPPUDRAFT_39704)
V
EFX84367.1 K (DAPPUDRAFT_25421)
2P
EFX85873.1 K (DAPPUDRAFT_313756)
Ca
EFX88146.1 K (DAPPUDRAFT_311738)
IR
Carcinusmaenas Malacostraca GBXE01087917.1 HCN(Transcriptomecontig)
GBXE01103936.1 K (Transcriptomecontig)
V
GBXE01029129.1 K (Transcriptomecontig)
2P
GBXE01102019.1 K (Transcriptomecontig)
Ca
GBXE01116654.1 K (Transcriptomecontig)
IR
Cancerborealis Malacostraca AAZ80094.3 HCN(HAIC)
AAZ80093.4 K
Ca
AccessionnumbersareaccordingtoGenBankandspecificdescriptionsarebasedontheNCBIproteindatabase.HCN,potassium/sodiumhyperpolarization-
activatedcyclicnucleotide-gatedchannel;K ,voltage-gatedpotassiumchannel;K ,two-poredomainK+-channel;K ,calcium-activatedpotassiumchannel;
V 2P Ca
K ,inwardlyrectifyingpotassiumchannel.
IR
y
g
o
could be identified for HEA-acclimated green crabs. Hence, Seaindividuals.Accordingly,theperfusionsolutionconsistedof(in l
absolute quantification was solely assessed using the included mmoll−1):340NaCl,9CaCl2,7MgCl2,9KCl,14NaHCO3,0.13 Bio
standard curve. In this case, a standard curve based on diluted NH Cl, 0.5 glutamine, 0.5 glucose and 0.1 glutathione. The pH
4 l
a
cDNAsampleswasperformedtoensurecomparableamplification (NBS scale) was adjusted to 7.9. Posterior gills (no. 7) were
t
efficiency [PCR fragments: 99.4±0.2% (N=3); cDNA templates: perfused according to the procedure described in Fehsenfeld and n
e
100.5±0.7%(N=3)]. Weihrauch(2013),followingthetechniqueofSiebersetal.(1985). m
Each perfusion consisted of a control step, followed by the ri
e
Perfusionexperimentsonisolatedposteriorgill7 applicationoftherespectiveinhibitorandathird wash-outstepto p
For gill perfusion experiments in Kiel, hemolymph ammonia, ensurethesurvivalofthegilltissue.Eachperfusionstepallowedfor x
E
hemolymph carbonate system parameters, as well as hemolymph 10min adjustment of the gill, after which the perfusate was
f
cationconcentrations(Na+,K+andCa2+;flamephotometeranalysis collectedfor30min.ToblockbasolateralK+channels,BaCl was o
2
performed by T. Stegmann, Institute of Physiology, Christian- appliedbasolaterallyinthesecondperfusionstepataconcentration al
Albrechts-UniversityKiel)ofcontrolgreencrabswereassessedfor of 12mmoll−1 (Schirmanns and Zeiske,1994; Riestenpatt,1995; n
r
theadjustmentoftheperfusionsolutionasdescribedinFehsenfeld Riestenpattetal.,1996;Onkenetal.,2003).TheinhibitorZD7288 u
o
andWeihrauch(2013)toaccountforinvivoconditionsoftheBaltic (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium J
889
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
chloride)wasappliedbasolaterallyataconcentrationof10µmoll−1 thattheagarbridgesdidnotproduceexcessiveresistancebypassing
to specifically block the K+ current generated by the identified current in this open setup (resulting in ca. 60µA). Using the fluid
HCN, according to Carrisoza-Gaytán et al. (2011). Gill ammonia resistance compensation function of the clamping device, the
excretion rates were calculated based on the difference in the potentiometer was adjusted so that the voltage was again nullified
perfusate compared with the perfusion solution. Perfusate proton whilepassingcurrent.Theresistanceofthecrabsaline(R )couldbe
S
concentration was calculated based on the pH, whereas perfusate read from the potentiometer knob and was determined as 8.2
P andHCO−werecalculatedusingtheequationsandconstants ±0.1Ω cm−2. After mounting the six split gill lamellae, the (area-)
CO2 3
followingTruchot(1976).Anincreaseinperfusate[H+],CO and/ corrected short-circuit current I (as a measure of ion/charge
2 SC
or [HCO−] in comparison to the initial perfusion solution was movements over the gill epithelium; Riestenpatt et al., 1996) was
3
consideredequivalenttoadecreaseinexcretion. directly recorded simultaneously for every channel using Acquire
Analyzesoftware(version2.2).Voltagepulses(5mVevery60sfor
Ussingchamberexperimentsonsplitgilllamellae(posterior 1s) were applied to assess the epithelial conductance {G =1/R ,
TE TE
gill7) R =[(ΔV/ΔI)−R ]=[(5mV/ΔI )−8.2Ω cm−2], where R is the
TE S SC TE
Ussingchamberexperimentswereperformedatroomtemperature transepithelialresistanceandΔVistheamplitudeofthevoltagepulse}.
in Winnipeg, Canada.Single gill lamellae of posterior gill 7 were The dose–response curve for ZD7288 was obtained by
splitaccordingtoSchwarzandGraszynski (1989)andmountedin increasing the concentration basolaterally in a step-wise fashion.
theEM-CSYS-6Ussingchambersystem(PhysiologicInstruments, As soon as I levelled off forone concentration (ca. 10–15min)
SC
San Diego, CA, USA) with specific sliders (P2308), providing a andremainedstablefor>1min,thenexthigherconcentrationwas
circularapertureof1mmthatresultedinanopenepithelialsurface applied. In case of the dose–response curve for BaCl , preceding
2
areaof8×10−3cm2.Silicongreasewasusedtosealtheedgesofthe thebasolateralapplicationofeachconcentrationofBaCl wasthe
2
preparation to prevent potential edge damage. Preparations were application of an equivalent concentration of choline chloride
aerated throughout the experiment. Providing six channels and apicallytoaccountforpotentialCl−-dependentchangesinI .To
SC
chambers, the EM-CSYS-6 Ussing chamber system together with account for time effects on the I , one split gill lamella per
SC
theautomaticclampingdevice(VCC600,PhysiologicInstruments) experimentalrunwaskeptundercontrolconditionsduringthefull
allowedforsixsplitgilllamellaetobeinvestigatedinparallelinone duration of the experiment.
experimentalrun.
Symmetrical conditions were used to account foronly active ion Statistics
transport,withthefollowingidenticalcrabsalinebeingappliedtoboth AllstatisticalanalyseswereperformedwithPAST3(Hammeretal.,
theapicalandbasolateralsides(inmmoll−1):248NaCl,5CaCl ,4 2001).Datasetsweretestedfornormaldistribution(Shapiro–Wilk
2
MgCl ,5KCl,2NaHCO ,5HEPESand2glucose(afterRiestenpatt test) and homogeneity of variances (F-test) to qualify for
2 3
etal.,1996).ThepH wasadjustedto7.9.TwopairsofAg/AgCl parametrical testing. If one or both requirements were not met,
NBS
electrodes were connected to the preparation via agar bridges (3% datawerelogtransformed.Iflogtransformationstilldidnotresultin
agaroseincrabsalinetoavoiddiffusiveeffectsofKCl):onepairserved adatasetappropriateforparametrictesting,non-parametricaltests
todeterminethetrans-epithelialpotentialdifference(PD ),andthe wereappliedinstead.Forcomparisonofsinglemeans,apairedt-test
TE
secondpairservedascurrentelectrodestoshort-circuitthePD with was applied (gill perfusion, mRNA expression changes stressors),
TE
theVCC600.Thereferenceelectrodewaspositionedatthebasolateral whereasKruskal–WalliswithpairwiseMann–Whitneycomparisons
side.Beforethetissuewasintroducedintotheexperimentalsetup,the was used to compare multiple means (tissue expression). Dose–
voltageoffsetbetweenpairedelectrodeswassetto0.0.Itwasensured responsecurveswereevaluatedbyrepeatedmeasuresANOVAwith
post hoc Tukey’s pairwise comparisons after verifying a normal
distribution (Shapiro–Wilk test) and homogeneity of variances
(Levene’s test). All results were considered significant at P<0.05.
Graphs were generated using the software Inkscape, version 0.48
(https://inkscape.org/). The dose–response curve for Ba2+ was
generated by GraphPad Prism version 6.00 for Windows
(GraphPadSoftware,SanDiego,CA,USA,www.graphpad.com).
y
RESULTS g
o
IdentificationofCmHCNandgenetreeanalysis l
o
The two different identified contigs (resulting in an open reading i
B
frame of >100 amino acids) in the green crab transcriptome
l
a
resembling HCN, GBXE01087917.1 (326 amino acids) and
t
GBXE01153810.1 (148amino acids), alignedto differentregions n
e
oftheHCNsequenceofC.borealis(positions291–712versus66– m
213, respectively) and HCN2 of H. sapiens (positions 360–659 ri
versus 180–299, respectively). Only GBXE01087917.1 contained e
p
Fig.1.ClustalWalignmentofCarcinusmaenasHCNandHomosapiens the specific motifs for voltage-gated potassium channels x
E
HCN2proteins.Indicatedinthesequencearethevoltage-dependent (PLN03192) and the cyclic nucleotide-monophosphate binding
f
potassiumchannelmotifPLNO3192(underlined),theCrpmotifforthe domain (cAMP/cNMP/Crp) as determined by the NCBI protein o
predictedcNMP/cAMPbindingsite(greybackground),andthebindingsites BLAST tool and was therefore chosen for further analysis. The al
Ala425andLeu432fortheinhibitorZD7288(blackboxes)afterChengetal. n
partial326aminoacidsequenceforCmHCNwashighlyconserved
(2007).‘*’,single,fullyconservedresidue;‘:’,conservationofstronggroups;‘.’, r
andresembledthehumanHCN2with69%identityand83%overall u
conservationofweakgroupsasdesignatedbyBiologyWorkBench(version o
3.2,http://seqtool.sdsc.edu/CGI/BW.cgi). conserved domains (Fig. 1). The major binding sites for ZD7288 J
890
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
Drosophila melanogaster HCN Fig.2.Moleculargenetreeanalysisof
Aedes aegypti HCN potassiumchannelsinvertebratesand
Daphnia pulex HCN Invertebrates invertebrates.Themaximumlikelihood
analysiswasconductedusingtheLGmodel(Le
Carcinus maenas HCN
Cancer borealis HCN HCN andGascuel,2008)basedonpairwise
Danio rerio HCN distancescalculatedbytheJTTmodel(Jones
Homo sapiens HCN2 etal.,1992)asimplementedinMEGA6.06
Vertebrates
Mus musculus HCN (Tamuraetal.,2013).Thetreewiththehighest
Strongylocentrotus purpuratus HCN log-likelihoodof−6915.8448isshownand
drawntoscale,sothatthebranchlengths
D. pulex KV
representthenumberofsubstitutionspersite
C. maenas KV
Invertebrates (scalebar).Numbersbesidethebranches
D. melanogaster KV
indicatethepercentageoftreesthatclustered
A. aegypti KV
S. purpuratus KV Kv togetherinthedisplayedway.HCN,
hyperpolarization-activatedcyclicnucleotide-
H. sapiens KV
gatedpotassiumchannel;K ,voltage-gated
M. musculus KV Vertebrates V
potassiumchannel;K ,two-pore
D. rerio KV 2P
domainpotassiumchannel;K ,calcium-
H. sapiens K2P Ca
activatedpotassiumchannel;K ,inward-
D. rerio K2P IR
rectifyingpotassiumchannel.
M. musculus K2P Vertebrates
S. purpuratus K2P
K
D. pulex K2P 2P
C. maenas K2P
D. melanogaster K2P Invertebrates
A. aegypti K2P
D. melanogaster KCa
A. aegypti KCa Invertebrates
D. pulex KCa
C. maenas KCa
K
C. borealis KCa Ca
D. rerio KCa
Vertebrates
M. musculus KCa
H. sapiens KCa
S. purpuratus KCa
H. sapiens KIR4.1
M. musculus KIR4.1
D. rerio KIR4.1
Vertebrates
H. sapiens KIR5.1
M. musculus KIR5.1
D. rerio KIR5.1
K
S. purpuratus KIR IR
C. maenas KIR
A. aegypti KIR
Invertebrates
D. melanogaster KIR
D. pulex KIR
0.5
were identified as Ala-425 and Leu-432, as recently described by was made for the K -related proteins of the arthropods, which
IR
Chengetal.(2007). seemedtoresembleKIR5.1channelsasfoundinmammalsandfish.
For each of the four classes of K+ channels (K , K , K and CmHCN was observed to be ubiquitously expressed in all
V IR 2P y
K ),arespectivecontigcouldbeidentifiedinthetranscriptomeof analyzed tissues. Although the cerebral ganglion exhibited the g
Ca o
C. maenas as generated by Verbruggen et al. (2015). A discrete highest mRNA expression levels (ca. 10-fold compared with l
o
gammadistributionwasusedtomodelevolutionaryratedifferences theothertissues),posteriorgillsstillexhibitedsignificantlevelsof i
B
amongsitesbetweenallsequencesincludedinthegenetreeanalysis thetranscript (Fig.3).
l
a
(six categories; +G, parameter=3.3). The rate variation model
t
allowed for some sitesto be evolutionarily invariable ([+I], 1.1% EffectsofK+-channelinhibitorsonbranchialacid-base n
e
sites). All positions with less than 95% site coverage were regulation,ammoniaexcretionandtheshort-circuitcurrent m
eliminated, resulting in a total of 141 positions in the final The excretion of ammonia, CO2 and HCO3− as well as the ri
dataset.Forallincludedinvertebrateandvertebratesequences,the concentration of H+ in the perfusate were significantly affected by e
p
respective K+-channel families formed distinct monophyletic both Ba2+ and the HCN-specific ZD7288 in posterior gill 7 of x
E
groups (Fig. 2). As expected, the HCNs represented a distinct osmoregulatinggreencrabswhenappliedbasolaterally(Fig.4).The
f
(sub)familyofthevoltage-gatedK+channels.Furthermore,thegene degreeofinhibition,however,variedwithbothinhibitors.Whereas o
tree analysis revealed that C. maenas grouped with the other branchialammoniaexcretiondecreasedtoagreaterextentwiththe al
arthropodchannelsinallcases.AlthoughfourdifferentHCNscould application of Ba2+ (ca. 70% versus 40% with ZD7288; Fig. 4A), n
r
befoundinmammals(onlyHCN2included,otherdatanotshown), excretion of acid equivalents (as equivalent to accumulation of u
o
arthropods seem to express onlyone HCN. A similarobservation protons in the hemolymph) decreased more drastically under the J
891
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
7 a conductance GTE accounted for 35.0±2.5mScm−2 (n=17).
AlthoughG exhibitedsimilarvaluesinallpreparations,I was
on 6 more variabTlEe, with values between −196 and −634µAScCm−2.
essi 0.5 b,c,d Withoutinfluenceofanyinhibitorandonlyaccountingforchanges
e exprbS-3)0.4 b b,d toivmeertpimereio,dtheanIdSCdreecmreaainseeddroenlataivveerlaygsetabbyle1o.v7e±r0t.h4e%exppeerri1m0emntianl
nR
geN/0.3 (n=5).Themaximaloveralldecreasewasobservedinoneoftheruns
alized (HC0.2 b,c,d c durWinhgetnhaepipnliiteiadlb2a0somlainteraanldlyaocncospulnittegdilflolarm5%ellaoef(thgeillin7i)tiianlUISsCs.ing
m c
or 0.1 chamberexperiments,bothinhibitors(Ba2+andZD7288)reduced
N theinitialshort-circuitcurrentinadose-dependentmanner,ascan
0Cergeabnrgallion Heart MuscleHypodermis Antennglaland Anteriorgill Posteriorgill brseteasbpseloeennseianncdthuervoeenxsleym(Fpislgea.erym7te)i.mdTehtcoeurvcdeoesncd(rFeuiacgstsean5c,se6li)graehsmtlwyaeinllewadisthtrheelahdtiiogvsheele–yr
concentrationsofBaCl .
2
Fig.3.NormalizedtissueexpressionofCmHCNinCarcinusmaenas For the dose–response curves, all measurements for I have
SC
acclimatedtobrackishwater(11ppt).mRNAexpressionwasnormalizedto beencorrectedfortheobservedmaximumtimeeffectof−5%per
theribosomalgeneRbS-3.Valuesaregivenasmeans±s.e.m.Significant 20min. In the case of Ba2+, the basolateral inhibition of I
differencesareindicatedbydifferentlowercaseletters(Kruskal–Wallistest SC
withpairwiseMann–Whitney,P<0.05,N=4–5). followedtypicalMichaelis–Mentenkineticsandcouldbefittedby
theHillequationwithΔI =−346.9µAcm−2(73%inhibition)
SCmax
influence of ZD7288 (1.3-fold; Fig. 4B). The effect of Ba2+ on andIC =0.76mmoll−1.TheapplicationofZD7288resultedinan
50
perfusateprotonconcentrationwassmall,yetsignificant.Inregardto almost linear decrease of the I and hence was not fitted by a
SC
CO excretion, the application of both inhibitors resulted in a typical dose–response curve. The maximum inhibition of I at
2 SC
significant,ca.1.1-foldincreaseofP intheperfusate(equivalent 10µmoll−1(plateauingafter1µmoll−1)accountedfor30%ofthe
toa1.1-folddecreaseinexcretion;FCigO.24C).Themostpronounced initial I (ΔI =−120.2µA cm−2) and the basolateral
SC SCmax
difference between Ba2+ and ZD7288 was observed in relation to applicationofZD7288resultedinanIC of6×10−7mmoll−1.
50
HCO−:whereasZD7288resultedinasmall,butsignificantincrease
3
of5%intheperfusate,Ba2+resultedintheoppositeresponse,namely, CmHCNinresponsetoenvironmentalstressors
a5%increaseinperfusate[HCO−](reducedexcretion;Fig.4D). mRNA levels of CmHCN in posterior gill 7 were significantly
3
Splitgilllamellaeofosmoregulatinggreencrabsemployedinthe downregulated in green crabs exposed to 7days of either high
present study generated an outside positive PD of 4.5±0.4mV environmental P (hypercapnia, 400Pa; ca. 50% reduction) or
(n=17) and an inward negative I of −445±28TEµA cm−2 (n=17) elevated environCmOe2ntal ammonia (1mmoll−1 NH Cl HEA, ca.
SC 4
in the Ussing chamber setup, indicating a net flux of negative 80%reduction;Fig.8).
charge from the apical to the basolateral side. The corresponding
DISCUSSION
1.5 A 1.4 B PotentialK+-permeablestructuresinthegillepitheliumof
* Carcinusmaenas
Fold change inmmonia excretion10..05 * * Fold change in+perfusate [H]1111....3210 * IocBnifarcds2tu+euicdtaanicpeduosrdtCroecsnrd+tuas(stOtetau,cndtekhiaeeensnpsorehentaseasspnlo.lc,niet1lygo9if9bl1lKee,l+an2mc0vhe0ealr3nlia;fnieReedlaisepbsipnytleyitnohipnneaggfttltiuhlelxeteaaipnnli.htd,his1behl9iitou9orm6rts-;
a Weihrauch et al., 1998, 1999). Because of its similar radius in
0 0 comparisontoK+,Ba2+isabletoenterthechannelporeandbindto
Control Inhibitor Wash Control Inhibitor Wash
the selection filter motif (SF4) domain of the respective K+
1.2 C 1.10 D channels,therebycompetingwithK+andblockingitsbindingsite
Fold change inPperfusate CO211..10 * * Fold change in–perfusate [HCO]3110...009505 * * cm2Httroh0aCoa1nornN3seunc)r.recpiklaAposnnttsooeinbnmwFettillehgeyfoe.daun2gbcnaeerslud,chyastsotthuhiawesscr,eosesa,uapolrngseefohcpgoiiriiftunelislcsttehdhtnnhaueatseatagulntarironevpeetieomonsbsfiaefctoleKirvanrk+ebiK-innpCcCgvrh.adoe,amomsrKtmgaiog2ee,tPaina,nt(aenRgKsddo.Im,RtsHob,seoiaKmdwseaVebettdrevaa.aenonlr.dne,, talBiology
The identified CmHCN, as described in the present study, thus n
0 0 e
Control Inhibitor Wash Control Inhibitor Wash provides the first (partial) sequence-based characterization of a m
Fig.4.Changesinammoniaandacid-baseexcretorypatternsbyisolated potentialK+/NH4+channelinadecapodcrustaceananddocuments ri
e
perfusedposteriorgill7ofbrackish-water(15ppt)acclimatedCarcinus itsinvolvementinbranchialacid-baseandammoniaregulation. p
maenasapplyinginhibitorsforpotassiumchannels.(A)Ammonia Importantly, Fig. 1 shows clearly that CmHCN exhibits the x
E
e[HxCcrOet3−io]nduraritnegs,c(oBn)trpoelrpfuesrfautseio[Hn,+]d,u(rCin)gpearpfpulsicaatetioPnCOof2baansdo(lDat)epraelrfiunshaibteitors, rdeesspcercibtievdebbyiCndhiennggseitteasl.(f2o0r07Z)D.E72v8en8,thAoulag-h42th5ealantdterLdeiuff-e4r3s2f,roams of
aawnpadpsldiaeupdrpianlitge1dt0haeµtwmaaocslohln−s1ct,eewnpth.raTetrhieoeanHsoCtfhN1e2-msmpoemrceoifuilcnl−iinv1e.hrVibsaaitloluirenZshDiab7riet2o8rg8iBv(eabnClala2csk(gmbraeeryasn)bwsa±rass) tshuebstmitaumtiomnaloiafnIleIlew-4i3th2,Ltehuedaiudthnoorts sohfiftthtahtesItuCd5y0 ssihgonwifeicdantthlyat. rnal
s.e.m.Asterisksdenoteasignificantdifferencebetweenthecontrolandthe Accordingly, this provided the basis for the functional studies ou
inhibitorstepoftherespectiveinhibitor(pairedt-testwithP<0.05,N=6). applyingZD7288ingillperfusionandUssingchamberexperiments. J
892
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
Time (min)
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
0
–100
–2) –200
m
c
A
µ
( C–300
S
I
–400
Control Control
1 2 3 4 5 6 7 8 9 10 11 12
saline saline
Fig.5.Representativetimecourseforthechangesofshort-circuitcurrent(I )acrosssplitlamellaeofposteriorgill7ofCarcinusmaenas
SC
applyingapicalcholinechlorideandbasolateralBaCl .I iscorrectedforfluidresistance,butnotforthegeneraldecreaseovertime(seeMaterialsand
2 SC
methodsfordetails).Theverticalcurrentdeflectionsreflecttheconductanceasassessedbytheapplicationof5mVvoltagepulses.(1)10µmoll−1apicalcholine
chloride;(2)10µmoll−1apicalcholinechlorideand10µmoll−1basolateralBaCl ;(3)100µmoll−1apicalcholinechlorideand10µmoll−1basolateralBaCl ;
2 2
(4)100µmoll−1apicalcholinechlorideand100µmoll−1basolateralBaCl ;(5)1mmoll−1apicalcholinechlorideand100µmoll−1basolateralBaCl ;(6)
2 2
1mmoll−1apicalcholinechlorideand1mmoll−1basolateralBaCl ;(7)5mmoll−1apicalcholinechlorideand1mmoll−1basolateralBaCl ;(8)5mmoll−1apical
2 2
cholinechlorideand5mmoll−1basolateralBaCl ;(9)10mmoll−1apicalcholinechlorideand5mmoll−1basolateralBaCl ;(10)10mmoll−1apicalcholine
2 2
chlorideand10mmoll−1basolateralBaCl ;(11)20mmoll−1apicalcholinechlorideand10mmoll−1basolateralBaCl ;(12)20mmoll−1apicalcholinechloride
2 2
and20mmoll−1basolateralBaCl .
2
MolecularcharacterizationofCmHCN thetransportprocessesthatareobservedinthemammaliankidney
As seen by the sequence and gene tree analysis, the level of (Riestenpatt et al., 1996) and it is therefore not surprising that, in
conservation of the HCN sequence between arthropods and additiontointhecerebralganglion,significantmRNAexpression
vertebratesingeneral,andC.maenasandH.sapiensspecifically, levelsofHCNcanbefoundintheosmoregulatoryposteriorgillsof
is significant. A similar level of conservation between C.maenas.Interestingly,thedifferenceofCmHCNmRNAlevels
invertebrate and vertebrate proteins is found in other important between anterior and posterior gills (ca. fivefold) mirrors what is
membrane transporters such as the α-subunit of the Na+/K+- seen for Na+/K+-ATPase (NKA) activity in these tissues (ca. 2
ATPase, one of the mostimportantepithelial proteins(C. maenas versus 11µmol P mg protein−1 h−1), emphasizing the postulated
i
versus Homo sapiens, 79% identity and 89% overall conserved close relationship between basolateral NKA and K+ channels in
domains, NCBI). Consequently, HCN can be hypothesized to mammalian (Hibino et al., 2010) as well as invertebrate epithelia
inherit a significant role in the course of evolution regarding (Riestenpattetal.,1996).
epithelialiontransport. Furthermore, the detected downregulation of the mammalian
Incollectingductsofratrenalcortexandmedulla–structuresthat renalHCN2mRNAinresponsetochronicmetabolicacidosisinrats
are responsible for both ammonia and acid-base regulation in (Carrisoza-Gaytán et al., 2011) was also observed in the present
mammals(WeinerandVerlander,2013)–HCN2hasrecentlybeen studyinthegillsofC.maenasinresponsetothemetabolicacidosis
observedtotransportnotonlyK+,butalsoNH+(Carrisoza-Gaytán resulting from acclimation to high environmental ammonia
4
etal.,2011).Asthemajorion,ammoniaandacid-baseregulatory (unpublished data). In addition, a role for CmHCN in general
organ (Larsen etal., 2014), the crustacean gill resembles manyof acid-baseregulationisindicatedbyitssignificantdownregulationas
Time (min) Fig.6.Representativetimecourseforthe
0 10 20 30 40 50 60 70 80 90 100 110 120 changesofISCacrosssplitlamellaeof gy
0 posteriorgill7ofCarcinusmaenasapplying o
basolateralZD7288.I iscorrectedforthefluid l
SC o
–250 resistance,butnotforthegeneraldecreaseover Bi
time(seeMaterialsandmethodsfordetails).The
l
verticalcurrentdeflectionsreflectthe a
–300 t
conductanceasassessedbytheapplicationof n
–2m) –350 5mVvoltagepulses. me
c i
A er
µ( SC–400 xp
I E
–450
f
o
–500 al
n
Control Control r
–550 saline 10–8 10–7 10–6 10–5 10–4 10–3 10–2 saline ou
ZD7288 (mmol l–1) J
893
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
A B
[Ba2+ ] (mmol l−1) [ZD7288 ] (mmol l−1)
basolateral basolateral
0 10–2 10–1 100 101 102 0 10–8 10–710–6 10–510–4 10–310–2
0 0
c cc
–100
–100
µ–2I(A cm) SC –––234000000 a a a b µ–2I(A cm) SC–––234000000 a a a,b b,c c,d d,e e,f f
–500
–600 –500
Fig.7.Dose–responsecurvesofpotassiumchannelinhibitorswithrespecttoI inUssingchamberexperimentsonsplitgilllamellaeof
SC
Carcinusmaenas.(A)Ba2+;(B)ZD7288.Allexperimentswereperformedonsplitgilllamellaeofposteriorgill7ofbrackish-water-acclimatedgreencrabs
(11ppt)undersymmetricalconditions.IC forBaCl=0.76mmoll−1;IC forZD7288=6×10−7mmoll−1.DifferentlettersindicatesignificantdifferencesinI
50 50 SC
(repeated-measuresANOVAwithTukey’sposthoccomparisons,P<0.05;N =5–8fromfourdifferentanimals,N =11fromfivedifferentanimals).
Ba2+ ZD7288
observed in the present study in response to a respiratoryacidosis nucleotide-gatedK+-channelfamily,CmHCN.Thefindingsofthe
caused by exposure to hypercapnia (Fehsenfeld and Weihrauch, presentstudythereforestrengthenresultsofearlierstudiesonsplit
2013).EventhoughHCNhomologshavebeenclonedfromseveral gill lamellae of C. maenas (Riestenpatt, 1995) and E. sinensis
invertebrate species and their hyperpolarization-activated current (Onkenetal.,1991)thatindicatedthatdifferentpopulationsofK+
characteristicshavebeenobservedtocloselyresemblemammalian channels promote transcellular K+ excretion because of the
HCNs (Biel et al., 2009), their potential function has so far only observed incomplete and complex di-phasic response of the I
SC
been associated with chemo-sensitivity in the fruit fly Drosophila when Ba2+ was applied to the apical and basolateral membranes,
melanogaster (Chen and Wang, 2012) and signal transduction in respectively.
olfactoryneuronsofthespinylobsterPanulirusargus(Gisselmann Moreover, the participation of the respective K+ channels in
etal.,2005). branchialacid-baseandammoniaexcretionbecameobviousbythe
Therefore,thepresentstudyisthefirsttodeliverevidencefora observed decrease of ammonia and CO excretion as well as the
2
functionofaconserved(ancestral)HCNasakeyplayerinacid-base accumulation of protons in the perfusate with the basolaterally
andammoniaregulationinaninvertebrate. appliedinhibitorsBa2+andZD7288inposteriorgill7.Therefore,
these results also support findings of arecent study thatclaim the
CharacterizationofK+-transportoverthegillepithelium ultimate linkage between acid-base and ammonia regulatory
Isolatedgills processes in the gill epithelium of this decapod crustacean by
The results of the gill perfusion experiments clearly indicated at using the same transporter inventory (Fehsenfeld and Weihrauch,
least two different K+/NH+-promoting structures in the branchial, 2013).Interestingly,HCO−excretionbytheposteriorgillisinduced
4 3
basolateral epithelial membranes of C. maenas. These include at by ZD7288 but decreased with Ba2+, thus verifying that the
least one Ba2+-sensitive K+ channel, as well as the identified observeddifferencesinexcretionratesofammonia,acidequivalents
ZD7288-sensitivememberofthehyperpolarization-activatedcyclic and CO are not solely based on differences in the effective
2
concentration of each inhibitor, but by the inhibitors targeting
A B differentK+/NH+-promotingstructures.
150 150 4
Both inhibitors in the present study were applied basolaterally
based on the fact that: (1) apical CsCl (10mmoll−1) had been
el
v shown to have no substantial effect on ammonia excretion in
e
e inon l100 100 C. maenas (Weihrauch et al., 1998) even though it significantly
ngssi reduced ISC (Riestenpatt et al., 1996), and (2) the novel candidate
chapre * gene CmHCN seemed to resemble the HCN2 as identified in y
Fold NA ex 50 50 * betasaol.l,a2te0r1a1l)maesmdibsrcaunsessedofabthoevev.eIrntetbhreatceaskeidonfeZyD(C72a8rr8is,ohzoaw-Gevaeyrt,áint olog
mR cannotbeexcludedthatittargetedtherespectivestructurealsointhe Bi
apical membrane because of its strong lipophilic character and
0 0 al
Control High PCO2 Control amHmigohnia s1u9b9s7e;qHueanrrtisdiafnfudsiCoonnsintatnoti,th1e99c5el)l. A(GpasrepdaorimniinaanndtlyDiinFtrraanceclelsuclaor, nt
e
presenceandactionofZD7288wouldalsoexplainwhytheeffectof m
Fig.8.mRNAexpressionchangesofCmHCNinposteriorgill7of
brackish-water-acclimatedCarcinusmaenas(11ppt)exposedto this inhibitor as observed in gill perfusion experiments was not ri
e
environmentalstressors.(A)Thelightgreybarrepresentsacclimationto reversedinthewashstep. p
highenvironmentalP (hypercapnia,400Pa,7days,N=6).mRNA x
CO2 E
expressionforhighPCO2acclimatedanimalswasnormalizedtotheribosomal Splitgilllamellae f
geneRbS-3priortocalculationofrelativegeneexpressionchanges.(B)The Further characterization of the effect of both inhibitors on overall o
d1amrkmgorlely−1bNarHr4eCplr,e7sednatyssa;cNc=li4m).aCtiohnantogehsigihnemnRviNroAnmleevenltsalinamHEmAonaicac(lHimEaAte,d ewpaisthaeclhiaielvioednimnoUvsesminegntcshaamndbethreexrpeseurilmtinegnttsraonns-seppliitthgeillliallamcuerlrleanet. nal
animalswereevaluatedapplyingastandardcurve(seeMaterialsandmethods r
Theobservedvaluesforgeneralcharacteristicsofsplitgill7were u
fordetails).Valuesaregivenasmeans±s.e.m.Asterisksdenotesignificant o
differencescomparedwithcontrolgeneexpression(Student’st-test,P<0.05). comparabletovaluesintheliterature.Eventhoughslightlyhigher J
894
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
outsidepositivepotentialdifferencesweremeasuredinposteriorgill In contrast to the high effective concentration of Ba2+, the
7ingreencrabsacclimatedto10pptingillperfusionexperiments unusuallylowIC ofZD7288isastonishing.Toverifytheeffectof
50
(7.6±0.2mV;Siebersetal.,1985),splitgillsseemedtogeneratea ZD7288 and exclude a simply time-dependent shift of the I , the
SC
slightly lower potential difference of 6.6±1.3mV (Onken and dose–response curve has been corrected for the maximal time-
Siebers, 1992), which is even more comparable to the value dependent shift observed in split gill preparations without the
observedinthepresentpreparation(4.4±0.4mV).Posteriorgillsof application of inhibitors. Hence, the analysis represents a
10ppt-acclimated C. maenas were also observed to generate an conservativeapproachandtheobservedeffectof30%inhibitionon
inwardnegativeI between−240and−375mAcm−2,aswellasa the I might slightly underestimate the actual inhibition (ca.5%).
SC SC
conductance of 40–45mScm−2 (Onken and Siebers, 1992; TheIC forZD7288of6×10−7mmoll−1inthepresentstudyis,to
50
Riestenpatt et al., 1996), values that were also reached in the our knowledge, 500-fold smaller than the next smallest IC for
50
presentstudy,validatingtheusedtechnique. ZD7288determinedinsinoatrialnodecellsisolatedfromtheguinea
The application of the inhibitors revealed the very different pig heart (3×10−4 mmol l−1; BoSmith et al., 1993) and can be
sensitivity of potential K+ channels to Ba2+ and ZD7288. Under interpretedasanextremelyhighaffinityofthisreagentforCmHCN
symmetrical conditions, the inward negative I generated by the and/oracomparablylowabundancyoftheproteininthebranchial
SC
split gill epithelium of C. maenas is mainly carried by an epithelium. The fact that CmHCN mRNA expression levels in
electrogenic inward-directed Cl− current dependent on the posteriorgillsofC.maenasaresimilarlyhighcomparedwiththose
negative membrane potential over the basolateral membrane that observedfortheNa+/K+-ATPase(FehsenfeldandWeihrauch,2013)
isgeneratedbythebasolateralNa+/K+-ATPaseandbasolateraland andapronouncedeffectof10µmoll−1wasobservedinisolatedgills
apical K+ channels (Riestenpatt et al., 1996). Increasing perfusion experiments, however, rather supports the high binding
extracellular Cl− concentrations by the addition of BaCl would capacity of this inhibitor. Generally, similar to Ba2+, the mode of
2
leadtotheweakeningofthedrivingforceforCl−uptakeandhence action of ZD7288 is voltage-dependent and related to the ambient
resultinapassivedecreaseofI .Hence,whennotcorrectedforby K+ concentration (Cheng et al., 2007), and might therefore differ
SC
additionofanequivalentconcentrationofCl−intheapicalbath,no substantially from other tissues and organisms, explaining the
saturation of the effect of BaCl on the I was observed, and observeddifferencesinIC valuesforZD7288,butalsoforBa2+.
2 SC 50
eventually, I switched to inward positive values (>20mmoll−1 Furthermore,thepotencyofZD7288wasshowntodependonthe
SC
BaCl , data not shown). Consequently, in the present study, respective HCN isoform in mammalian tissues and blocked the
2
preceding every increase in basolateral BaCl concentration, I (‘funny’) current in the sinoatrial node more effectively
2 F
choline chloridewas added to the external bath to correct for this (IC =3×10−4 mmol l−1; BoSmith et al., 1993) than the I
50 h
effect. As could be expected by an increased apical [Cl−], I (‘hyperpolarization’-activated) current in neurons (IC =2-
SC 50
becametransitionallymorenegative.Asaresult,however,theeffect 41×10−3 mmol l−1; Shin et al., 2001). Hence, even though highly
ofBa2+ontheI followeddistinctMichaelis–Mentenkineticsand conserved on the protein level, the potentially single HCN in
SC
theunbiasedIC couldbedetermined. C.maenasmightexhibitdifferentfeaturesregardingZD7288binding
50
The IC for Ba2+ (0.76mmoll−1) in the split gill lamellae of thatmakeitmoresensitivefortheblock[i.e.shiftsinhydrophobicity
50
greencrabasdeterminedinthepresentstudywasverysimilartowhat (Chengetal.,2007)andepigeneticfactorsalteringmethyl-dependent
was observed in the basolateral midgut epithelium of the tobacco polarization,asobservedforBa2+(Rossietal.,2013)].
hornworm,Manducasexta(0.50mmoll−1;Zeiskeetal.,1986),as Even though the present study clearly showed that the
wellastheapicalepitheliumoffrogskin(0.11mmoll−1;DeWolf involvement of HCN in branchial K+/NH+ transport accounted
4
and Van Driessche, 1986) and the basolateral epithelium of toad forca.30%oftheK+-generatedcurrent,thefullextentoftheBa2+-
urinary bladder (1mmoll−1; Van Driessche and Erlij, 1988). At relatedblockcannotbeexplainedatthispoint.Theanalysisofthe
these relatively high concentrations, however, Ba2+ is believed to C.maenastranscriptomeandsubsequentgenetreeanalysisrevealed
targetdelayedrectifierK+channelsandK channelsratherthanK apotentialroleforallmajorclassesofK+channelsinthegeneration
V IR
andK channels(Rudy,1988;Franchinietal.,2004).Hence,the of (branchial) K+ currents in the green crab. After verifying the
Ca
obtained results are not conclusive in regard to the nature of mRNA and/or protein expression pattern for each of the newly
theaffectedK+channel(s),takingintoaccountthatpotentiallyallof identified candidate genes in the green crab, further research also
theidentifiedK+channels(K ,K ,K ,K andHCN)mightbe needstobedonetofullycharacterizetheK+currentinthebranchial
v 2P Ca IR
present in the branchial epithelium and targeted by Ba2+. At the epithelium.
y
concentrationof100µmoll−1thatcanstillbeconsideredspecificfor g
o
KIRandKCachannels(Franchinietal.,2004;Hassinenetal.,2015), Conclusions ol
theobservedinhibitionofISCintheposteriorgill7ofosmoregulating Inconclusion,thepresentstudystronglyindicatestheinvolvementof Bi
C.maenasaccountedforca.10%oftheinitialI ,indicatingthata abasolateralHCN-likepotassiumchannelinbranchialacid-baseand
SC l
a
potentialinvolvementofbranchialK and/orK channelscannot ammoniaregulationinthedecapodcrustaceanC.maenas.Besides
IR Ca t
beexcluded,butifpresent,onlyaccountsforaminorfractionofthe potential other basolateral Ba2+-sensitive K+ channel(s), gill n
e
K+current.Furthermore,itcannotbeexcludedthatBa2+alsotargets perfusion experiments applying the HCN-specific inhibitor m
theHCN,ashasbeenobservedforhighmillimolarconcentrationsin ZD7288 show that this novel candidate gene is involved in NH4+ ri
e
the rat kidney (Carrisoza-Gaytán et al., 2011). According to the transportoverthebasolateralgillepithelialmembrane.Interestingly,
p
resultsofthepresentstudy,however,onlyca.30%ofthemeasured similarfeatures(i.e.thepotentialdualtransportcapabilitiesofK+and x
E
K+-relatedcurrentcanbeassignedtotheidentifiedCmHCNinthe NH+ and decreased mRNA expression changes in response to
4 f
branchial gill epithelium as demonstrated by the application of metabolicacidosis)havebeenobservedfortheHCN2expressedin o
ZD7288.Consequently,themaximumobservedeffectofBa2+(73% themammaliankidney,indicatingaconservedfunctionofthisgene al
n
inhibition of I ) remains partly unexplained for and additional forbasichomeostaticregulationthroughouttheanimalkingdom.The
SC r
experimentsarenecessarytofurthercharacterizetheBa2+-sensitivity high level of conservation of the amino acid sequence between u
o
oftheK+currentinthebranchialepitheliumofC.maenas. invertebrateandvertebrateHCNsstrengthenstheimportanceofthis J
895
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,887-896doi:10.1242/jeb.134502
geneasapotentialkeyplayerinacid-baseandammoniaregulationin Hibino,H.,Inanobe,A.,Furutani,K.,Murakami,S.,Findlay,I.andKurachi,Y.
vertebratesaswellasinvertebrates.However,asthisonlyaccounts (2010). Inwardly rectifying potassium channels: their structure, function, and
physiologicalroles.Physiol.Rev.90,291-366.
for ca. 30% of the K+-related trans-epithelial short-circuit current,
Jones,D.T.,Taylor,W.R.andThornton,J.M.(1992).Therapidgenerationof
furtherresearchshouldbecarriedouttoelucidatethenatureofthe mutationdatamatricesfromproteinsequences.Comput.Appl.Biosci.8,275-282.
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Acknowledgements Le,S.Q.andGascuel,O.(2008).Animprovedgeneralaminoacidreplacement
TheauthorsthankDrsGregG.Goss,ChrisM.WoodandTamzinBlewett,aswellas matrix.Mol.Biol.Evol.25,1307-1320.
thestaffoftheBamfieldMarineSciencesCenter,forthehelpincollectingthegreen Lignon,J.M.(1987).Ionicpermeabilitiesoftheisolatedgillcuticleoftheshorecrab
crabs.AdditionalthanksgototheAnimalHoldingFacilityoftheUniversityof Carcinusmaenas.J.Exp.Biol.131,159-174.
Manitobafortakingcareofthecrabs.ThanksgoalsotoDrFrankMelznerfor Onken,H.andSiebers,D.(1992).Voltage-clampmeasurementsonsinglesplit
providingsupervision,spaceandlaboratoryequipmentforgillperfusion lamellaeofposteriorgillsoftheshorecrabCarcinusmaenas.Mar.Biol.114,
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withthetranscriptomeanalysis. Onken,H.,Graszynski,K.andZeiske,W.(1991).Na+-independent,electrogenic
Cl−uptakeacrosstheposterior gillsoftheChinesecrab(Eriocheirsinensis):
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Competinginterests
Onken,H.,Tresguerres,M.andLuquet,C.M.(2003).ActiveNaClabsorption
Theauthorsdeclarenocompetingorfinancialinterests.
acrossposteriorgillsofhyperosmoregulatingChasmagnathusgranulatus.J.Exp.
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Authorcontributions Riestenpatt,S.(1995).DieosmoregulatorischeNaCl-AufnahmeüberdieKiemen
S.F.performedallexperimentsandalldataanalysesaspartofherPhDthesis.D.W. decapoder Crustaceen (Crustacea, Decapoda). Berlin: VWF Verlag für
helpedwiththedesignofthestudy,editedthemanuscriptandprovidedfinancial WissenschaftundForschung.
support,aswellaslaboratoryequipmentinWinnipeg,Canada. Riestenpatt,S.,Onken,H.andSiebers,D.(1996).ActiveabsorptionofNa+and
Cl−acrossthegillepitheliumoftheshorecrabCarcinusmaenas:voltage-clamp
Funding andion-fluxstudies.J.Exp.Biol.199,1545-1554.
Rossi,M.,Tkatchenko,A.,Rempe,S.B.andVarma,S.(2013).Roleofmethyl-
ThisworkwasfundedbyaNSERCDiscoveryGrant(D.W.)andaUniversityof
inducedpolarizationinionbinding.Proc.Natl.Acad.Sci.USA110,12978-12983.
ManitobaGraduateFellowship(S.F.),aswellasastudenttravelandresearchgrant
Rudy,B.(1988).DiversityandubiquityofKchannels.Neuroscience25,729-749.
oftheFacultyofGraduateStudies/FacultyofScience/DepartmentofBiologyofthe Schirmanns,K.andZeiske,W.(1994).AninvestigationofthemidgutK+pumpof
UniversityofManitoba(S.F.).
thetobaccohornworm(Manducasexta)usingspecificinhibitorsandamphotericin
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