and Keith Nehrke Erik Allman, Korrie Waters, Sarah Ackroyd elegans Rhythmic Behavior in ...

12 Pages·2013·2.14 MB·English

Signal Transduction:
Analysis of Ca2+ Signaling Motifs That
Regulate Proton Signaling through the
Na+/H+ Exchanger NHX-7 during a
Rhythmic Behavior in Caenorhabditis
elegans

Erik Allman, Korrie Waters, Sarah Ackroyd
and Keith Nehrke
J. Biol. Chem. 2013, 288:5886-5895.
doi: 10.1074/jbc.M112.434852 originally published online January 14, 2013
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THEJOURNALOFBIOLOGICALCHEMISTRYVOL.288,NO.8,pp.5886–5895,February22,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Analysis of Ca2(cid:1) Signaling Motifs That Regulate Proton
(cid:1) (cid:1)
Signaling through the Na /H Exchanger NHX-7 during a
Rhythmic Behavior in Caenorhabditis elegans*□S
Receivedforpublication,November8,2012,andinrevisedform,December20,2012Published,JBCPapersinPress,January14,2013,DOI10.1074/jbc.M112.434852
ErikAllman‡,KorrieWaters§,SarahAckroyd¶,andKeithNehrke‡¶1
FromtheDepartmentsof‡PharmacologyandPhysiologyand¶Medicine,UniversityofRochesterSchoolofMedicineandDentistry,
Rochester,NewYork14642andthe§DepartmentofFamilyMedicine,OhioUniversity,Athens,Ohio45701
Background:Ca2(cid:1)oscillationsstimulaterhythmicprotonsignalingbyNa(cid:1)/H(cid:1)exchangerNHX-7inC.elegans.
Results:ThecontributionofindividualregulatorymotifstoNHX-7activitywasdefinedbyinvivostructure-functionanalysis.
Conclusion:NHX-7activityisregulatedbybothCa2(cid:1)andpH,leadingtorobustbuttransientsignaling.
Significance:UnderstandingthemechanismsthatdistinguishprotonsignalingfrompHregulationiscriticalfordual-function
membranetransporters.
MembraneprotontransporterscontributetopHhomeostasis pHhomeostasisisvitalformaintainingproteinfolding/func-
buthavealsobeenshowntotransmitinformationbetweencells tion and cell volume and for facilitating the flow of ions and
in close proximity through regulated proton secretion. For nutrientsthroughvariousphysiologicallycoupledmechanisms
example, the nematode intestinal Na(cid:1)/H(cid:1) exchanger NHX-7 (forreview,seeRef.1).pHimbalancehasbeenimplicatedina
causesadjacentmusclecellstocontractbytransientlyacidifying number of human diseases, including renal tubular acidosis,
the extracellular space between the intestine and muscle. osteoporosis,andmentalretardation,aswellasinthetransfor-
NHX-7 operates during a Ca2(cid:1)-dependent rhythmic behavior mationandmetastasisofcancer(2–4).BecausepHregulation
and contains several conserved motifs for regulation by Ca2(cid:1) plays a ubiquitous role in cell physiology and is crucial to an
input,includingmotifsforcalmodulinandphosphatidylinositol organism’s health, it is not surprising that a wide variety of
4,5-bisphosphate binding, protein kinase C- and calmodulin- acid-basetransportersexist(1).
dependentproteinkinasetypeIIphosphorylation,andabind-
However, recent evidence suggests that protons them-
ingsiteforcalcineurinhomologousprotein.Here,wetestedthe
selvesmayhaveanadditionalroleinsignalingbetweencells.
idea that Ca2(cid:1) input differentiates proton signaling from pH
Several types of mammalian proton receptors have been
housekeepingactivity.Eachofthesemotifswasmutated,andtheir
identified,includingaclassofG-protein-coupledreceptors
contribution to NHX-7 function was assessed. These functions
(GPR4,OGR1,G2A,andTDAG8),adhesionthekinasePyk2,
includedpHrecoveryfromacidificationincellsincultureexpress- and cation channels of the Na(cid:1) channel/degenerin super-
ingrecombinantNHX-7,extracellularacidificationmeasureddur-
family(ASIC1/DRASIC)(5–9).Thesereceptorsarefoundin
ing behavior in live moving worms, and muscle contraction
thekidney,brain,andcentralnervoussystemandhavelong
strengthasaresultofthisacidification.Ourdatasuggestthatmul-
tiplelevelsofCa2(cid:1)inputregulateNHX-7,whosetransportcapac- been implicated in the perception of pain associated with
tissueacidosis(10,11).ReductionsinextracellularpHarise
itynormallyexceedstheminimumnecessarytocausemusclecon-
duringthecourseofischemia,epilepticseizures,andelectri-
traction. Furthermore, extracellular acidification limits NHX-7
cal stimulation in the brain, suggesting a potential role for
proton transport through feedback inhibition, likely to prevent
theseprotonreceptorsinhumandisease(12–16).Therefore,
metabolic acidosis from occurring. Our findings are consistent
withanintegratednetworkwherebybothCa2(cid:1)andpHcontribute local regulation of proton availability may have important
toprotonsignaling.Finally,ourresultsobtainedbyexpressingrat physiologic and pathophysiologic consequences through
NHE1inCaenorhabditiseleganssuggestthataconservedmecha- signal transmission processes. Membrane transporters that
nismofregulationmaycontributetocell-cellcommunicationor regulate pH homeostasis may also have a role in directing
protonsignalingbyNa(cid:1)/H(cid:1)exchangersinmammals. localized transient proton signals as part of an inter-
cellularcommunicationcascade.Ifso,itwillbeimportantto
determine what particular features of such proteins distin-
guishtheirbasalactivity(whichcontributestomaintaining
*ThisworkwassupportedbyRuthL.KirschsteinNationalResearchService
InstitutionalAwardGM068411fromtheNationalInstitutesofHealth(to pH) from their stimulated acute activity during cell
E.A.).ThisworkwasalsosupportedbyNationalScienceFoundationGrant signaling.
IOS0919848(toK.N.).
□S Thisarticlecontainssupplemental“ExperimentalProcedures,”Fig.S1,and Caenorhabditiselegansisageneticmodelorganisminwhich
additionalreferences. protonshavebeenrecentlyshowntobeactivelysecretedasan
1Towhomcorrespondenceshouldbeaddressed:Dept.ofMedicine,Uni- intercellular signaling molecule during a rhythmic behavior
versityofRochesterMedicalCenter,601ElmwoodAve.,Rochester,NY.
(17,18).Defecationisanultradianbehaviorthatoccurswitha
Tel.: [phone]; Fax: [phone]; E-mail: keith_nehrke@urmc.
rochester.edu. frequencyof(cid:2)45sinwellfedworms(19,20).Thedefecation
5886 JOURNALOFBIOLOGICALCDHoEwMnIlSoTaRdYed from http://www.jbc.org/ at University of Rochester on JuVnOe L1U7M, 2E012388•NUMBER8•FEBRUARY22,2013
Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
motorprogram(DMP)2ischaracterizedbythreeconsecutive of second messenger Ca2(cid:1) signaling. NHE1 and NHE3 both
setsofmusclecontractions(21).Thefirstsetisacontractionof respondtochangesinCa2(cid:1)throughanumberofmechanisms,
theposteriorbodywallmuscles(pBoc).Thisiscloselyfollowed including phosphorylation by Ca2(cid:1)/calmodulin-dependent
by a contraction of the anterior body wall muscles and then protein kinase type II (CaMKII) and interactions with Ca2(cid:1)-
expulsionoftheluminalcontents.Geneticanalyseshaveshown binding proteins such as calmodulin (CaM), calcineurin
that intestinal cell-autonomous oscillatory Ca2(cid:1) signaling is homologousprotein(CHP),andtescalin(reviewedinRefs.26)
centraltothedefecationpacemaker(22).Thefirstmotorstep and45).Thus,Ca2(cid:1)hastheabilitytobothpositivelyandneg-
of the DMP, pBoc, depends on Ca2(cid:1) signals initiating in the ativelyregulateNa(cid:1)/H(cid:1)exchangeandcandosoinanisoform-
posterior cells of the intestine (17, 18), whereas a subsequent andcell-specificmanner.
waveofCa2(cid:1)thatpropagatesposterior-to-anteriorisnecessary Here,wepresenttheresultsofastructure-functionanalysis
for the coupling and timing of the remaining motor steps focused on deciphering if and how Ca2(cid:1) signaling regulates
(23–25). NHX-7.WeuseddynamicfluorescentpHimagingtomeasure
AroleforprotonsignalingduringtheDMPemergedfrom NHX-7 activity both in cells in culture and in live moving
reportsindicatingthatincreasedCa2(cid:1)causedprotonstomove worms, where we could assess behavioral output. We hereby
fromtheintestinallumenintothecytoplasm,resultinginintra- describearoleforCaM-andphosphatidylinositol4,5-bisphos-
cellular acidification, and that this in turn triggered proton phate (PIP )-binding motifs in activation of the exchanger.
2
extrusion across the basolateral membrane via the Na(cid:1)/H(cid:1) AlthoughwewerefurtherabletodemonstratethatCaMinter-
exchanger NHX-7 (17, 18). Proton secretion was found to be actsdirectlywithNHX-7,wealsofoundthatitimpactsoscilla-
bothnecessaryandsufficienttotriggerpBoc.Mechanistically, tory Ca2(cid:1) signaling upstream of NHX-7. Finally, a potential
extracellular acidification resulting from NHX-7 activity was roleforCaMKIIphosphorylationinlimitingtheextentofpro-
foundtoprovideprotonsubstratesthatbounddirectlytoand ton transport may relate to the observation that extracellular
activatedaligand-gatedcationchannel,PBO-5/6(pH (cid:2)6.8), acidification provides feedback inhibition and likely prevents
50
expressed on the muscle membrane. Activation of PBO-5/6 metabolicacidosisfromoccurring.
resultedinmuscledepolarizationandpBoc(17).Thiswasthe
firstexampleofprotonsandaNa(cid:1)/H(cid:1)exchangercontributing EXPERIMENTALPROCEDURES
toacuteintercellularsignaling. Strains—Standard nematode culture techniques were used
Na(cid:1)/H(cid:1) exchangers are a 12-transmembrane-spanning (46). Strains containing pbo-4(ok583)X, pbo-5(n2303)V, and
family of phosphoglycoproteins that contain a hydrophobic pha-1(e2123ts)IIIalleleswereobtainedfromtheCaenorhabdi-
N-terminal region and a large cytoplasmic C-terminal tail. tis Genetics Center (University of Minnesota) and the
TheyexchangeoneextracellularNa(cid:1)foroneintracellularH(cid:1) C.elegansGeneKnockoutConsortium.Otherstrainsusedin
toregulateintracellularpH(pH)andtomaintainNa(cid:1)homeo- this work are described in detail under supplemental Experi-
i
stasis (26). They facilitate adhesion, migration, proliferation, mentalProcedures.”
andvolumeregulation(27–32)andhavebeenimplicatedina QuantitativeRT-PCR—Geneexpressionlevelswereassayed
numberofhumandiseases,includingepilepsy,mentalretarda- infirst-strandcDNAsamplesusingiQTMSYBR(cid:2)GreenSuper-
tion,andcancermetastasis,inadditiontobeingacomponentof mix (Bio-Rad) and standard protocols for real-time PCR and
ischemia/reperfusion injury (2–4). Mammals have nine iso- normalizedtobothpmp-3andcdc-42usingthefollowingprimer
formsofNa(cid:1)/H(cid:1)exchangerstermedNHEs(33–40),whereas pairs: nhx-7, 5(cid:3)-CAGTACACTTTGCGAGTCGCTTAT-3(cid:3)
the nematode C.elegans likewise codes for nine isoforms and 5(cid:3)-AGTTTGATCGTAGAACCCTGGATG-3(cid:3); pmp-3,
termedNHXs(41).Althoughsomeoftheseisoformsareubiq- 5(cid:3)-GTTCCCGTGTTCATCACTCAT-3(cid:3) and 5(cid:3)-ACACCGT-
uitouslyexpressed(NHE1(42)andNHX-4(41))ina“house- CGAGAAGCTGTAGA-3(cid:3);andcdc-42,5(cid:3)-CTGCTGGACAG-
keeping” manner, others demonstrate a high degree of cell- GAAGATTACG-3(cid:3) and 5(cid:3)-CTCGGACATTCTCGAAT-
specific expression, suggesting potentially unique modes of GAAG-3(cid:3).
regulationandfunction. MolecularBiology—Plasmidconstructswerecreatedbycon-
The basal activity of these NHEs can be modulated via a ventional restriction site-mediated cloning methods. Site-di-
cytoplasmicallostericproton-bindingsitethatenhancesactiv- rectedmutagenesiswasusedtointroducepointmutants,and
ityuponintracellularacidification(lowpH)(43)aswellasby inversePCRwasusedtocreatedeletions.Specificdetailsasto
i
various protein interactions with regulatory motifs located in the construction of the plasmids can be found under supple-
the C-terminal tail (26). This intracellular tail has also been mental “Experimental Procedures.” A plasmid containing the
showntobethesiteofregulationbyproteinkinases,andthese extracellularpHsensorwasakindgiftofDr.L.PabloCid(Cen-
regulatoryeventscanbeamorepowerfuldeterminantofactiv- tro de Estudios Científicos, Valdivia, Chile) and has been
itythantheoverallexpressionlevelofagiventransporter(44). describedpreviously(47).AllconstructswereverifiedviaDNA
Oneubiquitousandwellstudiedregulatorymechanismisthat sequencing.
Contraction Measurements—pBoc strength and duration
2Theabbreviationsusedare:DMP,defecationmotorprogram;pBoc,poste- were determined post hoc from transmitted light videos by
riorbodywallmusclecontraction;NHE,mammalianNa(cid:1)/H(cid:1)exchanger; measuringtheperimeteroflateL4wormsintheirmaximally
NHX,C.elegansNa(cid:1)/H(cid:1)exchanger;CaMKII,Ca2(cid:1)/calmodulin-dependent relaxed and contracted states. Worms in which the reference
proteinkinasetypeII;CaM,calmodulin;CHP,calcineurinhomologouspro-
points were obscured by movement, in either assay, were
tein;PIP ,phosphatidylinositol4,5-bisphosphate;rNHE1,ratNHE1;CBD,
2
CaM-bindingdomain;DIC,differentialinterferencecontrast. excludedfromanalysis.
FEBRUARY22,2013•VOLUME288•DNoUwMnlBoaEdRe8d from http://www.jbc.org/ at University of Rochester on JunJeO 1U7R, N20A1L3OFBIOLOGICALCHEMISTRY 5887
Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
FIGURE1.PhysiologiccharacterizationofrecombinantNHX-7activity.AP-1cells,aCHOcellderivativethatlacksNHEactivity,wereusedfortransient
expressionandfluorescence-basedpHmeasurementsofrecombinantNa(cid:1)/H(cid:1)activity.A,representativetracesofNa(cid:1)-dependentpHrecoveryfollowing
acidificationincellsexpressingC.elegansNHX-7orratNHE1asindicated.ThenegativecontrolwasthepcDNA3.1vectoralone.B,pH dependenceofNHX-7.
Tocalculateinstantaneousrecoveryratedata,abestlinefitequationwascalculatedfora1-minwindowfollowingthere-additionofeNa(cid:1)ofaplotofdpH/dt
versuspHandextrapolatedtopH6.1.Eachdatapointrepresentsthreetofourreplicateexperiments((cid:5)10cellsperexperiment).Themeanandmedianiare
designatedbythesmallinteriorboxandhorizontalline,respectively.Error(largebox)isS.E.,whiskersrepresentthemaximumandminimumvalues,andasterisks
indicatep(cid:6)0.05versuspH7.6viaanalysisofvariance.C,schematicofNHX-7protein:domainorganizationandCa2(cid:1)regulatorymotifs.Transmembrane
domainsarelabeledwithRomannumerals.Relevantnhx-7mutantallelesarelistedinitalics,withtheirpositionsdenotedbyarrows.ok583isadeletionallele
thatactsasanull,whereasn2568andox10resultintruncatedproteins.MotifstargetedformutagenesiswerebaseduponhomologywithratNHE1(supple-
mentalFig.S1)andaredenotedaswell.TheE271QporemutationisindicatedbyanasteriskintransmembranedomainVII.Othertargetsaremarked
schematicallyandbyaminoacidlocationwithinthenhx-7C-terminalcodingsequence,includingCHP,PIP ,andCBDmotifsandthesinglepotentialCaMKII
phosphorylationsiteatThr-618.FusionsbetweentheNterminusofNHX-7andtheCterminusofotherNa(cid:1)2/H(cid:1)exchangersoccurinaconservedsequencein
transmembranedomainXII.
Imaging Fluorescent Biosensors in C.elegans—Transgenic fusionproteinswith[35S]methionine.Proteinsassociatingwith
nematodeswereimagedliveandunrestrainedasdescribedpre- biotinylatedbovineCaM(5(cid:1)M;Calbiochem)wereprecipitated
viously (18). Imaging was performed with a 10(cid:4) Nikon Plan withstreptavidin-agarose.Equivalentfractionsofthestarting
Apoobjective.IlluminationwasprovidedbyaPolychromeIV andboundmaterialwereresolvedbySDS-PAGEandvisualized
monochromator (TILL Photonics) rigged to a Nikon Eclipse usingautoradiography.
TE2000-S inverted microscope equipped with a high-speed RNAiTreatmentandDMPAssay—Nematodesweregrown
charge-coupleddevicecamera(Cooke).TILLvisIONsoftware onbacteriaexpressingdouble-strandedRNAasdescribed(50).
wasusedforanalysis,datawerecompiledinMicrosoftExcel, ExecutionoftheDMPwasvisualizedunderadissectingmicro-
andgraphsweregeneratedusingOrigin. scope, and timing data were collected using Etho software, a
MeasurementsofNa(cid:1)/H(cid:1)ExchangeActivityinAP-1 Cells— keystrokerecorder(51).
AP-1cellswereculturedasdescribed(48)andtransfectedusing
RESULTS
LipofectamineLTXreagent(Invitrogen).After24h,cellswere
madequiescentbyplacingtheminlow-serummediumforat Recombinant NHX-7 Is an Authentic Na(cid:1)/H(cid:1) Exchanger—
least1h.ThecellswerethenloadedwiththepH-sensitivefluo- ToanalyzeNHX-7exchangeactivity,acDNAwasexpressedin
rescent dye 2(cid:3),7(cid:3)-bi-(2-carboxyethyl)-5(6)-carboxyfluorescein AP-1 cells, a mammalian cell line that lacks endogenous
acetoxymethyl ester (Molecular Probes) and superfused with Na(cid:1)/H(cid:1) exchanger activity (31). Antibody staining of a V5
physiologic saline on a Nikon/TILL Photonics fluorescent epitopetagbuiltintotheCterminusofNHX-7confirmedits
imagingrig(describedabove),andincubationwithhighK(cid:1)/ni- expression at the plasma membrane (see Fig. 3F). As is com-
gericin at varying pH values was used to calibrate the sensor monlyobservedwithoverexpression,someoftheproteinwas
(49). To measure Na(cid:1)/H(cid:1) exchange activity, an ammonium retainedintheendoplasmicreticulumaswell.Totestwhether
prepulsewasemployedasdescribed(48). ornottheexchangerwasactive,anammoniumprepulsewas
ConfocalMicroscopy—Confocalmicrographswereobtained usedtoacidifythecells(52),andtherateofNa(cid:1)-dependentpH
onanOlympusIX81invertedlaserscanningconfocalmicro- recovery was then measured. Eliciting NHX-7 activity from
scope.Thesameparameterswereusedforallofthemutants transfectedcellsrequiredthattheybestarvedofserumandthat
whenimagingacrosswormstrainsoracrosstransfections. the measurements be performed within 24 h of transfection.
Immunocytochemistry—Recombinant NHX-7 was detected Robustactivitywasobserved,althoughthecalculatedexchange
using a mouse anti-V5 monoclonal antibody (1:2000; Invit- rateofNHX-7wasslowerthanthatoftheubiquitousmamma-
rogen)andanAlexaFluor488-labeledgoatanti-mousesec- lianexchangerNHE1(Fig.1A).TheNHX-7exchangeratewas
ondaryantibody(1:5000;MolecularProbes)usingstandard sensitivetoextracellularpH(pH )(Fig.1B)andNa(cid:1)(K (cid:2)35
e m
techniques. mM).Surprisingly,agoniststhatraisedintracellularCa2(cid:1)levels
CaM Binding Assay—In vitro transcription/translation hadnosignificanteffectonrecombinantNHX-7activity(data
(TNT;Promega)oflinearizedtemplateDNAwasusedtolabel notshown).
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Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
FIGURE2.Invivostructure-functionanalysisofNHX-7.Atransgenicconstructcodingforannhx-7minigenefusedtoacDNAfortheredfluorescentprotein
mCherrywasmutatedatpotentialCa2(cid:1)regulatorymotifsorreplacedwithahomologousregionfromrNHE1orNHX-6.Theabilityoftheseconstructstorestore
pBocstrengthinannhx-7(ok585)nullmutantwasassessed.A,confocalmicrographofpnhx-7::NHX-7::mCherryrescueconstructexpressioninintestinalrings
7–9(lower)anddifferentialinterferencecontrast(DIC)(upper).Thewhitearrowpointsfromposteriortoanteriorandisscaledto50(cid:1)m.B,representative
contractionassayusingperimeteranalysis.ValueswerecalculatedposthocfromaDICvideobymeasuringtheperimeter(dashedlines)ofthewormatboth
maximalrelaxation(lower)andcontraction(upper).Arrowsindicatethevulvaandanus,whichwereusedasanteriorandposteriorreferencepoints,respec-
tively.Anexamplecalculationisshownintheinset.C,mutantpBocstrength.Transgenicconstructsaredenotedasfollows:NHX-7(cid:1)istheWTtransgene;ox10
recapitulatesaloss-of-functionmutationidentifiedthroughaforwardgeneticscreen(deletion/frameshiftstartingapproximatelyA702);E271Qisapore
mutation;NHX-7::rNHE1andNHX-7::NHX-6arefusionsbetweenthetransmembranedomainofNHX-7andthecytoplasmicdomainoftheindicatedNHEs;
(cid:7)PIP isamutationofthePIP -bindingsite(K569A/K570A);T618AisamutationofaCaMKIIrecognitionsite;(cid:7)CBDisadeletionoftheCBDfromaminoacids
695t2o723;(cid:7)CHPisamutation2ofthePBO-1-bindingsite(aminoacids541–545,MVQHLtoRRQHR).ErrorisS.D.forbetween7and27wormsperstrain.Asterisks
denotep(cid:6)0.05versusthewild-typecontrolviaanalysisofvariance.
TABLE1
CharacteristicsofinvivoNHX-7mutagenesis
ShownbelowaretherelativeacidificationratesandpBocstrengthanddurationintransgenicwormsexpressingmutantversionsofNHX-7.pBocdurationvalueswere
derivedposthocfromDICvideos.pbo-5mutantslackpBoc,butNHX-7activityisidenticaltowild-typecontrols.Thenhx-7(ok583)mutantstrainsexpressingrescue
constructsalsocontainedthepha-1andhim-5mutantalleles.ErrorisS.D.forcontractionstrengthanddurationandS.E.foracidificationrate.Thenvalueforeachtrialis
showninparentheses.ND,notdetermined.
Rescuing Contraction Contraction
Geneticbackground construct Activity strength duration
% % s
pbo-5(n2303) 100(cid:8)10(3) ND ND
pha-1(2123ts)/him-5(e1490) ND 14.1(cid:8)2.8(13) 4.1(cid:8)0.8(13)
nhx-7(ok583) 20(cid:8)1(3)a 3.9(cid:8)2.0(17)a 3.1(cid:8)0.8(16)a
nhx-7(ok583) NHX-7(cid:1) 120(cid:8)10(3) 13.3(cid:8)3.2(7) 5.0(cid:8)1.0(7)
nhx-7(ok583) ox10 26(cid:8)4(3)a 5.7(cid:8)4.2(27)a 3.3(cid:8)1.1(24)
nhx-7(ok583) E271Q 14(cid:8)2(3)a 4.4(cid:8)2.9(11)a 2.6(cid:8)0.3(9)a
nhx-7(ok583) NHX-7::rNHE1 67(cid:8)9(3) 13.2(cid:8)2.8(12) 3.8(cid:8)0.7(12)
nhx-7(ok583) NHX-7::NHX-6 26(cid:8)4(3)a 1.6(cid:8)0.4(8)a 1.1(cid:8)0.2(5)a
nhx-7(ok583) (cid:7)PIP 31(cid:8)6(3)a 13.2(cid:8)1.5(10) 3.4(cid:8)0.4(10)
nhx-7(ok583) T6182A 110(cid:8)20(3) 13.3(cid:8)1.8(11) 5.7(cid:8)0.5(11)a
nhx-7(ok583) (cid:7)CBD 50(cid:8)4(3)a 10.8(cid:8)1.7(8)a 3.4(cid:8)0.3(9)
nhx-7(ok583) (cid:7)CHP ND 3.0(cid:8)2.1(10)a 3.0(cid:8)0.3(10)a
nhx-7(ok583) rNHE1 30(cid:8)5(3)a 4.3(cid:8)1.7(9)a 3.9(cid:8)0.7(4)
a p(cid:6)0.05versustheNHX-7(cid:1)controlviaanalysisofvariance.
NHX-7IsRegulatedSimilarlytoMammalianNHEs—Asan “regulatory domain” (supplemental Fig. S1). These shared
alternative approach to AP-1 cells, we turned to an in vivo motifs are likely to be functionally sufficient for coordinating
modeltotestwhetherCa2(cid:1)regulatesNHX-7.First,theability upstream signaling, as the C-terminal coding sequence of rat
of recombinant NHX-7 to complement a C.elegans nhx- NHE1(rNHE1)effectivelysubstitutedforthatofNHX-7inthe
7(ok585) loss-of-function mutant was tested. An mCherry rescueconstruct(Fig.2C).Conversely,asimilardomainswap
fusion to NHX-7 (Fig. 2A) rescued the pBoc deficit in the betweenNHX-7andNHX-6,awormexchangerthatiscoex-
mutant(Fig.2CandTable1),consistentwithpreviousobser- pressed with NHX-7 in the intestine (41) but lacks notable
vations regarding an NHX-7::GFP fusion protein (18). The homologousmotifsforinputbyCa2(cid:1)signaling(supplemental
specificityofrescuewassuggestedbytwoobservations:reca- Fig.S1),didnotrescuethemutant(Fig.2C).However,NHE1
pitulatingannhx-7(ox10)loss-of-functionmutationthatarises could not fully substitute for NHX-7, suggesting that there is
fromadeletion/frameshiftstartingataboutA702suppressed someinnatepropertyofNHX-7outsideoftheCterminusthat
theabilitytorestorepBoc,asdidmutationofacriticalamino strongly influences its ability to effectively function, through
acid residue in the pore region (E271Q) that is essential for promoting proper localization, association with another pro-
Na(cid:1)/H(cid:1)exchangeactivity(Fig.2C). tein(s),ortransportactivityitself.
NHX-7hasseveralmotifsforinputbyCa2(cid:1)signaling(Fig. MutationofPutativeMotifsforCa2(cid:1)RegulationofNHX-7—
1C),whicharesharedwithmammalianNHE1initsC-terminal ConservedpotentialCa2(cid:1)regulatorymotifsinNHX-7(Fig.1C)
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Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
FIGURE3.Analysisofinvivo/invitroexchangeractivity.AgeneticallyencodedfluorescentpHsensorwastargetedtotheextracellularsideoftheposterior
intestinalbasolateralmembranetomeasureregionalchangesinpseudocoelomicpHduringdefecationintransgenicstrainsexpressingWTandmutantnhx-7
minigenes.Tomeasurethebasalactivityofrecombinantproteins,pH wasmonitoredintransfectedAP-1cellsinculture,andtherateofNa(cid:1)-dependentpH
i
recoveryfollowingacidificationwascalculated.A,confocalmicrographoftransgenicwormsexpressingtheextracellularpHsensoratthebasolateralmem-
braneofintestinalrings7–9.Thewhitearrowdenotesposterior-to-anteriororientationofthewormandisscaledto50(cid:1)m.B,schematicoftheextracellularpH
sensorwithprotonsmovingfromtheintestineintothespacebetweencells.C,representativetraceofextracellular(pseudocoelomic)pHfluctuationsthat
occurbetweentheintestineandbodywallmusclerelativetopBoc.DynamicfluorescentmeasurementsofpH infreelymovingwormswereusedtocalculate
e
theinitialrateofacidification(dpH/dt),whichisafunctionofNHX-7activity.D,extracellularacidificationratesresultingfromexpressionofthemutant
constructsarerepresentedasnormalizedtothecontrolstrain.TherestingpHofthepseudocoelomwasnotsignificantlydifferentbetweenstrains,andthe
overallextentofacidificationwasproportionaltotheinitialrateofacidification.ErrorisS.E.forthreeindependenttrials,withmorethanfivewormspertrial.
Asterisksindicatep(cid:6)0.05comparedwiththewild-typecontrolviaanalysisofvariance.E,instantaneousNa(cid:1)/H(cid:1)exchangeratedataformutantNHX-7
constructsexpressedinAP-1cellsinvitro.Tocalculateinstantaneousrecoveryratedata,abestlinefitequationwascalculatedfora1-minwindowfollowing
there-additionofNa(cid:1)ofaplotofdpH/dtversuspHandextrapolatedtopH6.1.Valueswereobtainedfrombetweenthreeandnineindependentexperiments,
with(cid:5)10cellsimagedperexperimenit.Themeanandmedianaredesignatedbythesmallinteriorboxandhorizontalline,respectively.Error(largebox)isS.E.,
whiskersrepresentthemaximumandminimumvalues,andasterisksindicatep(cid:6)0.05comparedwithcellsexpressingthewild-typeNHX-7viaanalysisof
variance.F,confocalmicrographofanAP-1cellexpressingrecombinantV5epitope-taggedNHX-7thathasbeenvisualizedusinganti-V5primaryand
fluorescentAlexaFluor488-conjugatedanti-mousesecondaryantibodies.Scalebar(cid:9)10(cid:1)m.G,schematicofpH measurementsincellculturewithprotons
movingoutofthecellintothemedium.H,representativetraceofpH duringNa(cid:1)-dependentpHrecoveryfolliowingacidification.Therecoveryratewas
i
calculatedbaseduponaplotofdpH/dtversuspH,withabestfitlineextrapolatedtopH6.1.
i i
were individually mutated, and the mutant constructs were CaMKII (T618A) mutants still rescued pBoc completely, but
tested for their ability to complement the loss-of-function deletion of the CBD ((cid:7)CBD) or CHP-binding sites ((cid:7)CHP)
mutant. These motifs included 1) a CaM-binding site of the reducedpBocstrength(Fig.2C).The(cid:7)CBDmutationresulted
1-5-8-14class,2)asiteforPIP binding,3)aCaMKIIphosphor- inasmallbutsignificantdecreaseinpBocstrength,whereasthe
2
ylation site, and 4) a binding site for the C.elegans CHP (cid:7)CHPmutationsuppressedtheabilityofthetransgenetores-
orthologPBO-1(54).Althoughthereareotherpotentialsites cuepBocentirely(Table1).Confocalmicroscopywasusedto
forCa2(cid:1)regulation,thesefoursitesrepresentedthemostlikely establishtheabundanceandmembranelocalizationofthefluo-
candidates for the following reasons. First, the nonfunctional rescentmCherry-taggedNHX-7mutantproteins,andwhereas
proteincodedforbythenhx-7(ox10)allelehasbeenshownto most of the mutants were indistinguishable from wild-type
be truncated immediately prior to the start of the potential NHX-7::mCherry (data not shown), the (cid:7)CHP protein accu-
CaM-binding domain (CBD), suggesting that CaM binding mulatedatintracellularlocationsratherthanatthebasolateral
might be import for NHX-7 function (17). Second, PIP is a membrane.3
2
necessarycofactorforavarietyofplasmamembranetransport- WenextconsideredtheideathatthestrengthofpBocmay
ers,includingNHEs(55),andduringdefecation,reductionsin notdirectlyreflectNHX-7activity.pBocistriggeredbyactiva-
PIP havebeenshowntoactivateTRPMchannels,hencecoor- tionofprotonreceptors,whosepK is(cid:2)6.8(17),onthebody
2 a
dinatingtheiractivitywithphospholipaseC-mediatedinositol wall muscle cells, but proton extrusion, receptor activation,
trisphosphate production (56). Third, CHP is a cofactor for muscle response, and proton clearance mechanisms all likely
NHEs that has been suggested to mediate Ca2(cid:1) regulation of impacttheoverallsignalingoutput.Therefore,itseemedpos-
the exchanger (57, 58), and a pbo-1 loss-of-function mutant siblethatreductionofNHX-7activitycouldresultincompen-
lacks pBoc (54). Finally, phosphoregulation by CaMKII of a sation from these other mechanisms or perhaps that NHX-7
conserved site in NHE1 increases its activity in response to normallyextrudesanabundanceofprotonsaboveandbeyond
Ca2(cid:1)signaling(59,60). thatnecessaryforanormalbehavioralresponse.Ifthatwerethe
Wewereinitiallyquitesurprisedtofindthatallofthemutant case, then reductions in NHX-7 activity might be tolerated
constructs supported robust pBoc (data not shown), at least withoutacorrespondingreductionincontractionstrength.
until we considered that the mutant transgenes were being WesurmisedthatmeasuringchangesinpH directlymight
e
overexpressed and that overexpression could conceivably be more informative regarding the mutations’ effects on
circumvent endogenous regulatory mechanisms. Hence, we NHX-7 activity. To accomplish this, an extracellular pH bio-
establishedstrainsthatapproximatedthewild-typeexpression
levels of nhx-7 as judged by quantitative RT-PCR (data not
shown).Withinthiscontext,thePIP -bindingsite((cid:7)PIP )and 3E.AllmanandK.Nehrke,unpublisheddata.
2 2
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Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
sensor(47)wasexpressedintheposteriorintestinalcellsand
directedtothebasolateralmembrane(Fig.3,AandB).Inagree-
mentwithpreviousresults(18),wewereabletodetectatran-
sientrobustacidificationoftheextracellularspacebetweenthe
posteriorintestineandtheoverlyingbodywallmuscleduring
pBocinlivemovingworms(Fig.3C).Theobservedacidifica-
tion was largely absent in nhx-7(ok583) mutant worms, and
measurements made in a pbo-5(n2303) background, which
lackstheprotonreceptorandpBoc,suggestedthatthesignal
wasnotinfluencedbymusclecontractility(Table1).Wethen
assessed the effect of each mutation on pH fluctuations and
e
extendedthisanalysistoincludestrainsexpressingthechime-
ric fusions with NHX-6 and rNHE1 described above (Fig. 3D
andTable1).
Ourresultssuggestthatthreedistinctcategoriesofmutant
phenotypesexist.TherearemutantsthatsupportnormalpH
oscillations and rescue pBoc, mutants that have reduced
exchangeactivitybutstillrescuepBoc,andmutantswithlittle
ornoexchangeactivityanddonotrescuepBoc.Thislastclass
mimics the nhx-7(ok583) null mutant. These results are con-
sistentwithourhypothesisthatareductioninNHX-7activity
canbetoleratedwithoutcausingavisiblephenotype.Forexam-
ple,the(cid:7)CBD,(cid:7)PIP ,andchimericrNHE1proteinsallrescued
2 FIGURE4.AnalysisofinvitrocalmodulinbindingbyNHX-7.Thecytoplas-
pBocatleasttosomeextent,butpH measurementssuggested micCterminiofbothNHX-7andrNHE1wereexpressedinvitroasradiola-
e
that they are less active than wild-type NHX-7 (Fig. 3D). In beledproteins,andtheirabilitytobindtobiotinylatedbovinecalmodulin
(bio-CaM;98%identicaltowormCMD-1)wasassessedbystreptavidinfrac-
contrast,theNHX-6chimera,(cid:7)CHPmutant,ox10mutant,and
tionation,gelelectrophoresisofthestartingandboundfractions,andauto-
E271Qporemutantresembledthenullmutantandexhibited radiographic detection. A, autoradiograms of gel-fractionated 35S-labeled
proteins.Thestartingproductsfrominvitrotranscription/translationreac-
low-amplitudepHoscillationsthatwereunabletoinvokecon-
tions(left)andboundfractions(right)areshown.Anequivalentfractionof
tractions.Theresidualoscillationsobservedinthenullmutant eachwasanalyzed.Luciferasewasusedasanegativecontrol.Multipleprod-
arelikelyduetotheactivitiesofotherpHhomeostaticmecha- uctsarelikelyduetotheuseofPCR-amplifiedtemplatesforinvitrotranscrip-
tionanddonotinterferewiththeconclusion.Predictedmolecularmassesare
nisms responding to intracellular acidification and causing asfollows:rNHE1,(cid:2)36kDa;NHX-7,(cid:2)31kDa;andluciferase,61kDa.B,the
somelowlevelofprotoneffluxintothepseudocoelom.Finally, CBD(identicalto(cid:7)CBD)wasdeleted,andtheabilityofthemutant(cid:7)CBD
wenotethattheT618AmutationoftheputativeCaMKIIphos- proteintobindCaMwasassessedasdescribedabove.Arrowsdenoteslight
residualCaMbinding.Thepredictedmolecularmassof(cid:7)CBDis(cid:2)28kDa.
phorylation site appears to have increased the activity of
NHX-7,resultinginastrongcontractionthatwasextendedin
duration(Table1). inAP-1cells(Fig.3E).Thiscouldconceivablybeduetohigher
Todirectlyassesswhetherthereducedactivityobservedwith levelsofPIP inwormsversuscellsinculturecompensatingfor
2
several of the mutants might be due to a reduction in basal reducedPIP bindingaffinityinthemutatedexchanger,butitis
2
levels of transport rather than disruption of Ca2(cid:1) signaling also possible that Ca2(cid:1) activation in worms overcomes the
input, these mutants were expressed in AP-1 cells, and their observedreducedbasalactivity.Regardless,thisresultisincon-
transportratesweremeasuredunderquiescentconditions(Fig. sistent with the idea that reductions in PIP might activate
2
3,E–H).ApproximatelyequallevelsofNHX-7expressionand NHX-7suchastheydoTRPMchannelsduringdefecation(56).
residenceattheplasmamembraneforeachofthemutantpro- NHX-7/Calmodulin Binding and Knockdown of cmd-1, a
teinsweresuggestedbyimmunolabelingandconfocalmicros- NematodeCalmodulinGene—Theresultsofourmutagenesis
copy (data not shown). Surprisingly, unlike our single-copy suggestedthatCaMbindingcontributestoNHX-7protonsig-
genewormmodel,wefoundthatenoughofthe(cid:7)CHPmutant nalingduringdefecation.Totestthismoredirectly,anNHX-7
was trafficked to the cell surface in AP-1 cells to permit C-terminalproteinfragmentwaslabeledwith[35S]Metviain
Na(cid:1)/H(cid:1)exchangeactivityatsimilarlevelsaswild-typeNHX-7 vitro transcription/translation, and the ability of the labeled
(Fig. 3E). Thus, the inability of the (cid:7)CHP mutant to trigger protein to associate with biotinylated CaM was assessed by
pBocwasnotduetoitlackingtransportactivity.Similarly,the coprecipitation.NHE1,whichisknowntobindCaM(61),was
reducedactivityofthe(cid:7)CBDmutantobservedinwormswas usedasapositivecontroltovalidatetheassay(Fig.4A).CaM
notasecondaryconsequenceofreducedbasaltransportcapac- associatedwiththesolublecytoplasmicCterminusofNHX-7
ity (Fig. 3E). On the other hand, the enhanced activity of the butdidnotassociatewithluciferase,whichwasusedasaneg-
T618AmutantinwormswasrecapitulatedinAP-1cells(Fig. ativecontrol(Fig.4A).WealsofoundthattheK forCaMwas
d
3E), suggesting that phosphorylation at Thr-618 by CaMKII similartothatforNHE1((cid:2)8nM)(datanotshown).Toconfirm
may normally suppress basal activity. We also found that the the specificity of this interaction, we demonstrated that the
(cid:7)PIP mutant,whichhadgreatlyreducedactivityinwormsbut (cid:7)CBDmutationsignificantlyreducedCaMbindingtoNHX-7
2
wasabletosupportanearlynormalpBoc,didnotfunctionatall (Fig.4B).
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Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
FIGURE5.Thecalmodulingenecmd-1isrequiredfornormaldefecationsignaling.RNAiwasusedtoreducecmd-1expressionspecificallyintheintestine.
Wormswereplacedontocmd-1orcontrolRNAiplatesasL3larva,andmeasurementsweremadefollowing0,18,36,and54hofRNAi.Thefluorescent
biosensorsD3cpvandpHluorinwereusedtofollowintestinalCa2(cid:1)andpHoscillations,respectively,ascmd-1expressionwasreducedovertime.A,composite
i
imageofatransgenicwormexpressingatranscriptionalfusionofthecmd-1promoterandthefluorescentproteinmCherry(left)andthecorrespondingDIC
image(right).Thearrowindicatesposterior-to-anteriororientationandisscaledto50(cid:1)m.B,dotplotsrepresentingpBoctiminginindividualwormsfollowing
exposuretocmd-1RNAiforthetimeperiodsindicated(0,18,36,and54h).Strongcontractionsareplottedassymbols,whereasshadowsorweakreiterative
contractionsareplottedassmallersymbolsofliketypeplacedlowerontheyaxis.C,representativeCa2(cid:1)oscillationsinacmd-1RNAiwormat54h.The
oscillatoryperiodshownhereissimilartothatofthebehavioralreiterationsshowninB.D,representativeintestinalpH oscillationsinacmd-1RNAiwormat
54h.NotethatthistracerepresentsfluctuationsinpH ratherthanpH andthattheperiodissimilartothatofbothCa2i(cid:1)oscillationsandpBoc.
i e
Having confirmed CaM binding and its physiologic rele- principalDMP.Atthispoint,thewormswereessentiallydefe-
vance to NHX-7 function, we next asked whether reducing catingevery(cid:2)18s(Fig.5).Continuedexposuretocmd-1RNAi
CaM levels in vivo would phenocopy the effect of the (cid:7)CBD ledtothecessationofdefecationentirely,followedbydeathof
mutant on pBoc. The C.elegans genome contains five genes theanimal(datanotshown).
thatencodeCa2(cid:1)-bindingCaM-likeproteins.Thecmd-1gene GiventhattheDMPisinitiatedbyoscillatoryCa2(cid:1)signaling
codes for a nematode CaM ortholog that is 100% conserved and cyclic acidification of the intestine, we used fluorescent
with human CALM3. A transcriptional fusion between the biosensors to measure Ca2(cid:1) and pH in the intestines of live
cmd-1promoterandtheredfluorescentproteinmCherrycod- moving worms subjected to cmd-1 RNAi. Following 54 h, we
ing sequence was broadly expressed in multiple cells and cell observedoscillationsinbothofthesesecondmessengersoccur-
types,includingbodywallmuscle,hypodermalcells,neurons, ringevery(cid:2)18s,mirroringtheobservedchangestothebehav-
and,mostrelevanttoourwork,posteriorintestinalcells(Fig. ioralperiod(Fig.5,CandD).WeconcludethatCaMdirectly
5A).Therefore,thefunctionalroleofcmd-1wasqueriedusing influences intestinal oscillatory Ca2(cid:1) signaling upstream of
RNAi-mediated knockdown. Unfortunately, global RNAi acidification,andhence,itslosshaseffectsthatprecludeanal-
screens have shown that cmd-1 is necessary for viability, and ysisofitsroleinregulatingNHX-7directly.Nevertheless,these
wormstreatedwithcmd-1RNAiforseveraldaysdied(datanot resultsareinterestinginthecontextofsignalingpathwaysthat
shown).Tocircumventthislimitation,weturnedtocell-spe- regulatetheperiodicityofCa2(cid:1)oscillationsandemphasizethe
cificRNAi.TheRDE-1proteinisrequiredforeffectiveRNAito integrationbetweenCa2(cid:1)andprotonsignalingduringdefeca-
occur,andanrde-1mutantstraincanbecomplementedwitha tioninworms.
transgene expressing recombinant RDE-1 in a limited set of
DISCUSSION
cellssuchastheintestine(25).
Here,wefoundthatintestinalRNAiofcmd-1resultedina Na(cid:1)/H(cid:1) exchangers are recognized to contribute to pH
i
progressivephenotypeinwhichthecumulativelossofcmd-1 homeostasisandelectrolyteandwaterbalance(forreview,see
expression altered defecation frequency (Fig. 5B). In general, Ref. 62). Recently, a new role for Na(cid:1)/H(cid:1) exchangers has
when young adult worms were subjected to cmd-1 RNAi for emergedfromworkinC.elegansshowingthatNHX-7allows
18h,theybegantoexhibitweakreiterationsoftheDMP.This adjacentcellstocommunicatethroughdirectprotonsignaling
hasbeenobservedpreviouslyinunc-43CaMKIIloss-of-func- (17,18).ThiseventiscoordinatedbyoscillatoryCa2(cid:1)signaling,
tionmutants(20,25),wherethesereiterationswerereferredto which in turn leads to repetitive cellular acidification and
as“shadows”or“echos.”Theintervalbetweentheprincipal,or recovery.Hence,defecationrepresentsabehaviorwhosephys-
strong,DMPandtheshadowswasmuchshorter((cid:2)16–18s)in iologicoutputisintegratedbycross-talkbetweenCa2(cid:1)andpH
cmd-1RNAiwormsthantheaveragedefecationperiod((cid:2)45s), signals. Counterintuitively, apart from its role in signaling,
consistentwithcmd-1normallyfunctioningtosuppressinap- NHX-7contributeslittletore-establishingpHhomeostasisfol-
propriateintracycleexecutionoftheDMP.Surprisingly,how- lowingdefecation.BecauseNa(cid:1)/H(cid:1)exchangersareactivated
ever,anincreasedtimeofexposuretocmd-1RNAiresultedin byallostericprotonbindingandhavebeenshowntoberegu-
notjustasingleshadowoccurringaftertheprincipalDMP,but lated by Ca2(cid:1), it is reasonable to ask whether regulation of
multipleshadowsoccurring(Fig.5B).Theperiodbetweenprin- NHX-7byCa2(cid:1)isadeterminingfactorinitsabilitytofunction
cipalexecutionsoftheDMPincreasedwithlossofcmd-1pri- asasignalingproteinasopposedtoa“pHhousekeeper.”
marily due to an increased number of shadows occurring Apartfromarelativeinsensitivitytotheamiloridederivative
between cycles, whereas the period between shadows, which 5-(N-ethyl-N-isopropyl)amiloride,NHX-7functionsmuchlike
was (cid:2)18 s, did not increase correspondingly. Eventually, the mammalianNHEsandcanfacilitaterecoveryfrominducedaci-
weaker shadows became nearly indistinguishable from the dosiswhentransfectedintocellsinculture(Fig.1).However,
5892 JOURNALOFBIOLOGICALCDHoEwMnIlSoTaRdYed from http://www.jbc.org/ at University of Rochester on JuVnOe L1U7M, 2E012388•NUMBER8•FEBRUARY22,2013
Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
initialattemptstodiscernthesignalingpathwaythroughwhich exchangeactivity,couldhavedirectimplicationsinthetrans-
Ca2(cid:1)mightstimulateNHX-7werehamperedbythefactthat port of ions (65, 66). Moreover, Ca2(cid:1) regulation of NHX-7
recombinantNHX-7activitywasnotresponsivetopharmaco- appears to be uncoupled from the primary regulation by H(cid:1),
logic manipulations that increased intracellular Ca2(cid:1) levels. consistentwiththepHsensorresponsibleforH(cid:1)recognition
AlthoughweconsideredthepossibilitythatNHX-7isnotstim- beinglocatedatthebeginningofhelix9ontheoppositesideof
ulatedbyCa2(cid:1)signaling,wefeelthatitismorelikelyeitherthat theunwoundhelix11(65).
therelevantsignalingpathwayisnotpresentinAP-1cellsor RegulationbyCaMismoredifficulttointerpretmechanisti-
thatitdoesnotrecognizethewormsignalingmotifs.Similarly, cally,asitsbindingoccursfartherdownstreamintheC-termi-
it is possible that overexpression in cell culture could mask nal tail, and the binding site(s) in NHX-7 and NHE1 are not
Ca2(cid:1)regulatoryeffectsonNHX-7. preciselyconservedatthelinearsequencelevel(supplemental
Nevertheless,thesesimpleobservationshelpedtoinformus Fig.S1).AlthoughCaMitselfisnotwellstudiedinC.elegans,
astohowNHX-7maycontributetotheintegrativephysiology CaMKIIhasbeenstudiedextensively,andgeneticapproaches
ofdefecation.Intheworm,protonsareanimportantmeansof have shown that mutations in unc-43, the worm CaMKII
performing work, and a proton gradient formed between the ortholog, cause the DMP to be reiterated, albeit weakly (20).
intestinallumenandcellisessentialfornutrientuptakeacross This ability of CaMKII to normally suppress inappropriate
theapicalmembrane(50,63).Maintenanceofthisgradientis Ca2(cid:1) oscillations between cycles is consistent with cellular
criticalandpresentssomephysiologicchallengesgiventhata memory. We discovered that loss of CaM itself resulted in a
significant portion of the intestine’s luminal contents are qualitativelysimilarresult(Fig.5).However,wealsodiscovered
expulsed every 45 s. To prevent their loss during expulsion, thatthisphenotypewasprogressiveandthatthestrengthand
protonsenterthecellfromthelumenduringdefecation.These occurrenceofthesereiterationsbecamemorepronouncedover
protonsmustthenbequicklyreturnedtothelumenimmedi- time. Astoundingly, after several days of exposure to cmd-1
atelyfollowingdefecationtodrivenutrientuptake.Viewedin RNAi,thewormswereexecutingtheentireDMPevery(cid:2)18s,
thiscontext,sustainedH(cid:1)effluxthroughNHX-7onthebaso- andshortlythereafter,theyexpired.
lateralmembranecouldbecatastrophic,leadingtobothcaloric To understand the physiologic alteration accompanying
deprivationandsystemicacidosis. theseshadowcontractions,wealsowentontoshowthatlate-
Ourdatademonstrateonemechanismforensuringthatpro- stagecmd-1RNAiwormsexhibitedCa2(cid:1)andpHoscillationsat
toneffluxtothepseudocoelomicspaceviaNHX-7isacute.A nearly three times the rate of normal worms (Fig. 5). At this
brief pulse of activity is sufficient to rapidly acidify the small rate,intestinalpHrecoveryprocesses,mostlikelythoseofthe
pseudocoelomtoanadirvalueatwhichwefoundNHX-7was energy-dependent second-phase V-ATPase (63), can not
virtuallyinactive(Fig.1B).Therefore,theinherentpH sensi- accommodatethecyclicacidificationthatoccurs,resultingin
e
tivityofNHX-7appearstoprovideafailsafemethodofensur- sustainedcellularacidosiswellbelowthatofthenormalresting
ingthatH(cid:1)signalingisturnedofffollowingstimulationofthe pHat(cid:2)7.4(Fig.5D).Wespeculatethatcmd-1maycontribute
muscleprotonreceptor. to regulating the inositol 1,4,5-trisphosphate receptor ITR-1,
Atransgenicwormmodelallowedustoperformstructure- whichisthemolecularpacemakerfordefecation.Althoughwe
functionmutagenesisofNHX-7andcomplementationofanull foundtheseresultstobenoteworthyandtodeservereporting,
mutanttoassesstheroleofpotentialCa2(cid:1)regulatorymotifs. thefactthatcmd-1contributedtoCa2(cid:1)pacemakingandthat
OurresultsfrombothbehavioraloutputandinvivopHmea- its loss induced massive cellular acidification derailed our
surementstakentogethersuggestthat1)PIP bindingstimu- attemptstoconfirmCaMregulationofNHX-7throughcmd-1
2
latesbasalNHX-7activity,2)CaMbindingcontributestoCa2(cid:1) RNAi.
activationofNHX-7,and3)stimulatedNHX-7activityexceeds Considered as a whole, our data support a model in which
what is necessary to trigger a “normal” pBoc. Binding of the Ca2(cid:1)activationofNHX-7isnotessentialforitsfunctionbut
CHP ortholog PBO-1 appears to affect NHX-7 trafficking may contribute to proton conservation. Moreover, we have
and/or membrane residence through an unexplored mecha- shown that PIP binding is required for basal function of the
2
nism(datanotshown);hence,wewereunabletodefineitsrole exchangerandsuggestedthatCa2(cid:1)mayalsoplayaroleinsup-
in Ca2(cid:1) activation, although given its EF-hand Ca2(cid:1)-binding pressingtheactivityofNHX-7followingitsactivation.Finally,
domains,wehypothesizethatitmayservedualrolesintrans- althoughitappearslikelythatCaMhelpstocoordinateCa2(cid:1)
portandsignaling.WealsofoundthataT618Amutationofa activationofNHX-7,italsoregulatesoscillatoryCa2(cid:1)signaling
potentialCaMKIIphosphorylationsiteincreasedtheduration frequencyupstreamofNHX-7activity.
of pBoc and the period of extracellular acidification during Inconclusion,theapparentsensitivityofNHX-7withinthe
pBoc (Fig. 3 and Table 1). Although the effect of the T618A integrativecontextofdefecationtochangesinbothCa2(cid:1)and
mutationwasrelativelymodest,phosphorylationbyCaMKIIat pHisconsistentwiththeimportancethatprotonsplayinintes-
this site may provide a “cellular memory” of Ca2(cid:1) signaling, tinalphysiologyinthewormbutmayalsoprovidemechanistic
suchasoccursduringlong-termpotentiationinneurons(64), insights into the regulation of NHE activity in mammals. For
hencecontributingtofeedbackinhibitionofNHEactivity. example,Na(cid:1)/H(cid:1)exchangehasbeenimplicatedintheregula-
Interestingly,crystalstructuredatafrombacterialNhaAand tionofsynaptictransmissionatglutamatergic,GABAergic,and
fromaCHP/NHE1fragmentsuggestapossiblemechanismof dopaminergicsynapses(67–69),andNHE1hasbeenshownto
action for CHP and PIP whereby their binding near helical contributetosynapticacidificationandthecreationofanacidic
2
segment 11, which has been shown to be important for microdomain at the synaptic cleft (70). It is likely not coinci-
FEBRUARY22,2013•VOLUME288•DNoUwMnlBoaEdRe8d from http://www.jbc.org/ at University of Rochester on JunJeO 1U7R, N20A1L3OFBIOLOGICALCHEMISTRY 5893
Ca2(cid:1)SignalingRegulatesNa(cid:1)/H(cid:1)ExchangeinC.elegans
dental that synaptic vesicle release is a Ca2(cid:1)-mediated event. apse.J.Neurosci.25,4108–4117
Alternatively, NHE5 is credited with supporting neuronal 17. Beg,A.A.,Ernstrom,G.G.,Nix,P.,Davis,M.W.,andJorgensen,E.M.
(2008)ProtonsactasatransmitterformusclecontractioninC.elegans.
activity-dependentdendriticspinegrowththroughanovelpH-
Cell132,149–160
mediatednegativefeedbackmechanism(53).Theseresultssug-
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