Functional Association between K -Cl Cotransporter-4 and H ,K -ATPase in the Apical Canalicular ...

17 Pages·2008·1.54 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.284,NO.1,pp.619–629,January2,2009
©2009byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
(cid:1) (cid:2)
Functional Association between K -Cl Cotransporter-4 and
(cid:1) (cid:1)
H ,K -ATPase in the Apical Canalicular Membrane of Gastric
Parietal Cells*□S
Receivedforpublication,August25,2008,andinrevisedform,October15,2008 Published,JBCPapersinPress,November4,2008,DOI10.1074/jbc.M806562200
TakutoFujii‡,YujiTakahashi‡,AkiraIkari§,MagotoshiMorii¶,YoshiakiTabuchi(cid:1),KazuhiroTsukada**,
NoriakiTakeguchi‡,andHidekiSakai‡1
FromtheDepartmentsof‡PharmaceuticalPhysiologyand**SurgeryII,GraduateSchoolofMedicineandPharmaceutical
Sciences,andthe(cid:1)LifeScienceResearchCenter,UniversityofToyama,Toyama930-0194,Japan,the§Departmentof
Pharmaco-Biochemistry,UniversityofShizuoka,Shizuoka422-8526,Japan,andthe¶LaboratoryofPhysicalScience,
FacultyofPharmaceuticalSciences,SuzukaUniversityofMedicalScience,Mie513-8670,Japan
We studied whether K(cid:1)-Cl(cid:2) cotransporters (KCCs) are H(cid:1) secretion, coupled with K(cid:1) uptake, is mediated by the
involved in gastric HCl secretion. We found that KCC4 is gastricprotonpump(H(cid:1),K(cid:1)-ATPase).K(cid:1)recyclingacrossthe
expressed in the gastric parietal cells more abundantly at the luminal membrane is necessary to sustain H(cid:1),K(cid:1)-ATPase
luminalregionoftheglandthanatthebasalregion.KCC4was activityingastricparietalcells.HeteromericKCNQ1/KCNE2
foundinthestimulation-associatedvesicles(SAV)derivedfrom channels(1,2)andtheKir4.1channel(3,4)havebeenpostu-
the apical canalicular membrane but not in the intracellular latedascandidatesforthisK(cid:1)transport.Cl(cid:2)effluxacrossthe
tubulovesicles,whereasH(cid:1),K(cid:1)-ATPasewasexpressedinboth luminal membrane is also necessary for gastric acid (HCl)
ofthem.Incontrast,KCC1,KCC2,andKCC3werenotfoundin secretion.CFTR2(5),CLIC-6(6,7),andSLC26A9(8)havebeen
either SAV or tubulovesicles. KCC4 coimmunoprecipitated postulatedascandidatesforthisCl(cid:2)transport.
with H(cid:1),K(cid:1)-ATPase in the lysate of SAV. Interestingly the IthasbeenassumedthatCl(cid:2)movespassivelydownitselec-
MgATP-dependent uptake of 36Cl(cid:2) into the SAV was sup- trochemical gradient through apical channels. Although the
pressedbyeithertheH(cid:1),K(cid:1)-ATPaseinhibitor(SCH28080)or intracellularCl(cid:2)concentrationoftheparietalcellhasnotbeen
the KCC inhibitor ((R)-((cid:1))-[(2-n-butyl-6,7-dichloro-2-cyclo- reported, it is speculated to be much lower than that of the
pentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The luminalsecretedHCl(160mM).Thus,Cl(cid:2)transportthrough
KCC inhibitor suppressed the H(cid:1) uptake into SAV and the Cl(cid:2)channelswouldrequirealargeelectricalpotentialdiffer-
H(cid:1),K(cid:1)-ATPaseactivityofSAV,buttheinhibitorhadnoeffects
enceacrosstheapicalmembrane(e.g.60mV,insidenegative
ontheseactivitiesinthefreeze-driedleakySAV.Theseresults
againsttheluminalside).Buttodatethereportsofthiselectri-
indicatethattheK(cid:1)-Cl(cid:2)cotransportbyKCC4istightlycoupled
calpotentialdifferenceshavebeenlow,suchas20–25mV(9),
withH(cid:1)/K(cid:1)antiportbyH(cid:1),K(cid:1)-ATPase,resultinginHClaccu- hintingthattheCl(cid:2)secretorymechanismmaybemorecom-
mulationinSAV.Inthetetracycline-regulatedexpressionsys-
plexthanpreviouslyassumed.
tem of KCC4 in the HEK293 cells stably expressing gastric Electroneutral K(cid:1)-Cl(cid:2) cotransporters (KCCs) belong to a
H(cid:1),K(cid:1)-ATPase, KCC4 was coimmunoprecipitated with
cation-chloridecotransportergenefamily(SLC12).KCCscon-
H(cid:1),K(cid:1)-ATPase.TherateofrecoveryofintracellularpHinthe
tributetotransepithelialtransportandtotheregulationofcell
KCC4-expressing cells after acid loading through an ammo-
volume (10–12). At least four KCC isoforms (KCC1–KCC4)
niumpulsewassignificantlyfasterthanthatintheKCC4-non-
havebeenidentifiedtodate.KCC3hasthreesplicingvariants:
expressing cells. Our results suggest that KCC4 and H(cid:1),K(cid:1)-
KCC3a–KCC3c. KCC1 is widely expressed, whereas KCC2 is
ATPasearethemainmachineriesforbasalHClsecretioninthe
restrictedtoneurons.KCC3aandKCC4aremainlyexpressed
apical canalicular membrane of the resting parietal cell. They
inepithelial-typecells(13,14).RecentlywefoundthatKCC3ais
alsomaycontributeinparttomassiveacidsecretioninthestim-
expressedinthebasolateralmembraneofgastricparietalcells
ulatedstate.
locatedattheluminalregionofgastricglandsandcoimmuno-
precipitatedwithNa(cid:1),K(cid:1)-ATPase(15).Exogenousexpression
*ThisworkwassupportedinpartbyGrants-in-aidforScientificResearch of KCC3a in LLC-PK1 cells up-regulates Na(cid:1),K(cid:1)-ATPase
15390062and18390064(toH.S.)andforJSPSFellows20010617(toT.F.)
fromtheJapanSocietyforthePromotionofScienceandGrants-in-aidfor activityinlipidrafts(15).
ScientificResearch16657042,18059012,and20056010(toH.S.)fromthe
MinistryofEducation,Culture,Sports,ScienceandTechnologyofJapan.
Thecostsofpublicationofthisarticleweredefrayedinpartbythepay-
ment of page charges. This article must therefore be hereby marked 2Theabbreviations used are: CFTR, cystic fibrosis transmembrane con-
“advertisement”inaccordancewith18U.S.C.Section1734solelytoindi- ductance regulator; KCC, K(cid:1)-Cl(cid:2) cotransporter; AQP, aquaporin;
catethisfact. SCH28080, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-ace-
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains tonitrile;DIOA,(R)-((cid:1))-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihy-
supplementalFigs.1–5. dro-1-oxo-1H-inden-5-yl)oxy]acetic acid; BCECF, 2(cid:3),7(cid:3)-bis(carboxy-
1Towhomcorrespondenceshouldbeaddressed:Dept.ofPharmaceutical ethyl)-5(6)-carboxyfluorescein; BCECF-AM, acetoxymethyl ester of
Physiology,GraduateSchoolofMedicineandPharmaceuticalSciences, BCECF;SAV,stimulation-associatedvesicles;TV,tubulovesicles;Tet,tet-
UniversityofToyama,2630Sugitani,Toyama930-0194,Japan.Tel.:81-76- racycline;PIPES,1,4-piperazinediethanesulfonicacid;pH,intracellular
434-7575;Fax:[phone];E-mail:[email protected]. pH;(cid:1)1NaK,Na(cid:1),K(cid:1)-ATPase(cid:1)1subunit. i
JANUARY2,2009•VOLUME284•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 619
This is an Open Access article under the CC BY license.
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
IfKCCsarepresentintheluminalmembraneofgastricpari- IsolationofGastricTissuesandCells—Mice,rats,andrabbits
etal cells, they may be involved in the electroneutral cotrans- were humanely killed in accordance with the guidelines pre-
portofK(cid:1)-Cl(cid:2),whichwouldbedrivenbytheelectrochemical sentedbytheAnimalCareandUseCommitteeoftheUniver-
gradient for K(cid:1) across the luminal membrane established by sityofToyama,andtheirgastricmucosaewereisolatedfrom
theH(cid:1),K(cid:1)-ATPase(averylowluminalK(cid:1)concentration)and thestomachs.Thecellsuspensionrichinparietalcellswaspre-
Na(cid:1),K(cid:1)-ATPase(averyhighintracellularK(cid:1)concentration). paredfromisolatedrabbitgastricmucosaasdescribedprevi-
So far, there have been no reports describing the presence of ously(17).Hoggastricmucosawaspreparedfromthestomach
anyKCCsintheluminalmembrane.Inthepresentstudy,we obtainedfromToyamameatcenter(Toyama,Japan).Human
found that KCC4 is predominantly expressed and associated gastric mucosa was obtained from surgical resection of Japa-
withH(cid:1),K(cid:1)-ATPaseintheapicalcanalicularmembraneofgas- nese patients at Toyama University Hospital in accordance
tricparietalcellsandthatKCC4isanimportantmoleculefor withtherecommendationsoftheDeclarationofHelsinkiand
maintaining H(cid:1),K(cid:1)-ATPase activity in the canalicular with ethics committee approval. All of the patients gave
membrane. informedconsent.
PreparationofHogGastricVesicles(Stimulation-associated
EXPERIMENTALPROCEDURES Vesicles(SAV)andTubulovesicles(TV))—Twokindsofgastric
vesicles(heavyandlightvesicles)werepreparedsimultaneously
Materials—Anti-human KCC4 rabbit polyclonal antibody
from hog gastric mucosa as described previously (18). Briefly
wasgeneratedwithkeyholelimpethemocyanin-coupledpep-
thefundicregionofthemucosawasscrapedandhomogenized
tidesagainst16aminoacidscorrespondingtotheN-terminal
in250mMsucrose,1mMEGTA,and5mMTris-HCl(pH7.4).
sequence of KCC4 (DEESRRREAKAPRMGC). This antigen
Thesuspensionwascentrifugedat1,000(cid:4)gfor10min,andthe
peptidehasnohomologywiththesequencesofKCC1,-2,and supernatantwasfurthercentrifugedat13,500(cid:4)gfor30min.
-3.Anti-mouseKCC4rabbitpolyclonalantibodywasobtained
Thepellet,resuspendedinthebuffersolution,wasappliedto
from Alpha Diagnostic (San Antonio, TX). To check the
the top of a 7% Ficoll shelf on a 12% Ficoll step gradient and
expressionofKCCsotherthanKCC4,anti-humanKCC1goat
centrifuged in an RPV-50T rotor (Hitachi Koki Co., Tokyo,
polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Japan)at132,000(cid:4)gfor1h.Heavyvesicleswerecollectedfrom
CA), anti-rat KCC2 rabbit polyclonal antibody (Upstate Bio-
theinterfacebetweenthe7and12%Ficolllayers,andtheywere
technology,LakePlacid,NY),andanti-ratKCC3rabbitpoly-
clonalantibody(15)wereused.ExpressionofgastricH(cid:1),K(cid:1)- wsaamshpeledwwaitshc2en50trimfuMgesducarto1s2e0t,o00r0em(cid:4)ovgeinthaenFSicWol4l.1TThienrotthoer
ATPase was checked by using anti-mouse H(cid:1),K(cid:1)-ATPase
(Beckman)for20hlayingonadiscontinuoussucrosegradient
(cid:1)-subunitmousemonoclonalantibody(1H9)(Medical&Bio-
(10,20,30,and50%sucrose),and10fractionsof1mleachwere
logical Laboratories Co., Nagoya, Japan), anti-mouse H(cid:1),K(cid:1)-
collectedfromthetopofthegradient.Fraction5wasusedas
ATPase(cid:2)-subunitmousemonoclonalantibody(2B6)(Medical
the SAV that contained the apical canalicular membranes of
& Biological Laboratories Co.), and anti-hog H(cid:1),K(cid:1)-ATPase parietal cells (rich in H(cid:1),K(cid:1)-ATPase and least contaminated
(cid:1)-subunitrabbitpolyclonalantibody(Ab1024)(16).Anti-rab- with Na(cid:1),K(cid:1)-ATPase) (supplemental Fig. 1). On the other
bit Na(cid:1),K(cid:1)-ATPase (cid:1)1 subunit mouse monoclonal antibody hand,thesupernatantafter13,500(cid:4)gcentrifugationwascen-
wasobtainedfromUpstateBiotechnology.Anti-humanaqua- trifuged at 100,000 (cid:4) g for 30 min. The samples were then
porin-4 (AQP4) goat polyclonal antibody, anti-rabbit ezrin applied to a 250 mM sucrose and 7% Ficoll step gradient and
mouse monoclonal antibody, anti-human Rab11 goat poly- centrifugedat132,000(cid:4)gfor1h.Light(microsomal)vesicles
clonalantibody,andanti-ratsyntaxin-1mousemonoclonal werecollectedfromtheinterfacebetweenthe250mMsucrose
antibody were obtained from Santa Cruz Biotechnology. and7%Ficolllayers,andthesamplewasusedastheTVthat
Anti-His tag mouse monoclonal antibody (2D8) was from containedtheintracellularmicrosomalmembranesofparietal
Medical & Biological Laboratories Co. Anti-(cid:2)-actin mouse cells.Allprocedureswerecarriedoutat4°C.Whenindicated,
monoclonal antibody, 2-methyl-8-(phenylmethoxy)imi- freeze-dried SAV and TV were prepared by lyophilization,
dazo[1,2-a]pyridine-3-acetonitrile (SCH28080), and (R)- whichincreasedleakinessofthevesicularmembranes.
((cid:1))-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1- NorthernBlotting—Poly(A)(cid:1)RNAofthecellswasprepared
oxo-1H-inden-5-yl)oxy]acetic acid (DIOA) were from by using PolyATtract mRNA isolation system II (Promega,
Sigma-Aldrich.Lipofectamine2000,anti-Xpressmousemono- Madison,WI),and2.5(cid:3)gofitwasseparatedona1%agarose,
clonal antibody, Alexa Fluor 546-conjugated anti-mouse and formaldehyde gel and transferred onto a nylon membrane
anti-goatIgGantibodies,andAlexaFluor488-conjugatedanti- (Zeta-probeGT,Bio-Rad).Northernblottingwasperformedas
rabbit IgG antibody were from Invitrogen. Protein A-agarose described previously (15) using the 32P-labeled rabbit KCC4
beads were from Pierce. Na36Cl and ACS II scintillant were probethatis713bplongandcorrespondstonucleotides682–
obtained from GE Healthcare. 86RbCl was from PerkinElmer 1394oftheKCC4cDNA.
Life Sciences. Hygromycin B and acridine orange were from Western Blotting—Preparation of membrane fractions and
Wako Pure Chemical Industries (Osaka, Japan). Blasticidin S Westernblottingwerecarriedoutasdescribedpreviously(19).
was from Kaken Pharmaceutical Co. (Tokyo, Japan). 2(cid:3),7(cid:3)- The signals were visualized with the ECL Plus system (GE
Bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and ace- Healthcare).Toquantifythechemiluminescencesignalsonthe
toxymethylesterofBCECF(BCECF-AM)werefromDojindo membranes,aFujiFilmLAS-1000systemandMultiGaugesoft-
Laboratories(Kumamoto,Japan). warewereused.Anti-H(cid:1),K(cid:1)-ATPaseantibodieswereusedat
620 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER1•JANUARY2,2009
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
1:5,000 (1H9 and 2B6) or 1:4,000 dilution (Ab1024). Anti- KCl, 4 mM MgSO , 250 mM sucrose, 2 mM ATP, 5 (cid:3)Ci/ml
4
KCC1, anti-KCC2, anti-KCC3, anti-KCC4, and anti-(cid:2)-actin Na36Cl,and40mMPIPES-Tris(pH6.8)for5minat25°C.The
antibodieswereusedat1:1,000dilution.Anti-Na(cid:1),K(cid:1)-ATPase reaction mixtures were rapidly filtered through a 0.45-(cid:3)m
(cid:1)1subunitantibodywasusedat1:10,000dilution.Anti-Xpress HAWPfilter(MilliporeCo.,Bedford,MA).Tocalibratenon-
antibodywasusedat1:5,000dilution.Anti-ezrinandanti-syn- specific binding of Na36Cl to the vesicles and the filter, the
taxin-1antibodieswereusedat1:500dilution.Anti-Rab11anti- experiment was performed in the absence of ATP. The filter
body was used at 1:200 dilution. For the negative control, 1 was washed with solution containing 150 mM KCl, 250 mM
volumeofeachprimaryantibodywaspreincubatedwith5vol- sucrose,and40mMPIPES-Tris(pH6.8);transferredtoacount-
umesofthecorrespondingblockingpeptide.Horseradishper- ingvial;andsolubilizedwith5mlofACSIIscintillant.Thenthe
oxidase-conjugated anti-mouse, anti-rabbit, or anti-goat IgG radioactivityof36Cl(cid:2)wascountered.
wasusedasasecondaryantibody(1:2,500dilution). Measurement of H(cid:1) Uptake into SAV and TV—H(cid:1) uptake
Immunohistochemistry—The gastric mucosa isolated from intohoggastricvesicleswasassessedbymeasuringthequench-
ratstomachwasembeddedintheoptimumcuttingtempera- ing of acridine orange fluorescence (20). The reaction was
turecompound(SakuraFinetechnicalCo.,Tokyo,Japan)and startedbyadditionofATP(350(cid:3)M)tothemixturecontaining
wascutat8(cid:3)m.Thesectionswerefixedinice-coldmethanol SAV(20(cid:3)g/ml)orTV(5(cid:3)g/ml),150mMKCl,2mMMgCl ,5
2
for7minatroomtemperatureandwerepretreatedwith1.5% (cid:3)Macridineorange,10(cid:3)g/mlvalinomycin,and20mMPIPES-
bovine serum albumin for 1 h atroom temperature to block NaOH(pH7.4).Intheexperimentstotestanionselectivity,150
nonspecificbindingofantibody.Thenthesectionswereincu- mMKClwasreplacedwith150mMKX(X(cid:5)Br,I,H PO ,or
2 4
batedwithanti-mouseKCC4,anti-H(cid:1),K(cid:1)-ATPase(1H9),anti- gluconate). When indicated, SCH28080 (20 (cid:3)M), a specific
Na(cid:1),K(cid:1)-ATPase (cid:1)1 subunit, or anti-AQP4 antibody (1:100 inhibitorofH(cid:1),K(cid:1)-ATPase,orDIOA(10(cid:3)M),aninhibitorof
dilution) overnight at 4°C. Alexa Fluor 488-conjugated and KCCs,wasadded.Fluorescenceofacridineorangewasmeas-
Alexa Fluor 546-conjugated anti-IgG antibodies (1:100 dilu- uredinaShimadzuRF-5000spectrofluorometerat25°C(exci-
tion)wereusedassecondaryantibodies.Immunofluorescence tation, 495 nm; emission; 530 nm). The H(cid:1) uptake was
images were visualized using a Zeiss LSM 510 laser scanning expressedaschangeoffluorescenceintensityfrom0to3min
confocalmicroscope. afteradditionofATP.
Immunocytochemistry—HEK293 cells were fixed with ice- Measurement of H(cid:1),K(cid:1)-ATPase Activity—H(cid:1),K(cid:1)-ATPase
coldmethanolfor7minatroomtemperatureandpermeabi- activitiesofSAVandTVweremeasuredinapyruvatekinase-
lized with phosphate-buffered saline containing 0.3% Triton lactatedehydrogenase-linkedsystemwherehydrolysisofATP
X-100and0.1%bovineserumalbuminfor15minatroomtem- iscoupledwithoxidationofNADH(21).Thereactionmixture
perature.Nonspecificbindingwasblockedby3%bovineserum containedTV(10(cid:3)g/ml)orSAV(40(cid:3)g/ml),150mMKCl,3mM
albumin.Thepermeabilizedcellswereincubatedwiththeanti- MgSO ,200(cid:3)MNADH,1mMATP,0.8mMphosphoenolpyru-
4
mouse KCC4 or anti-H(cid:1),K(cid:1)-ATPase (1H9) antibody (1:100 vate,11IU/mllactatedehydrogenase,4IU/mlpyruvatekinase,
dilution)overnightat4°CandthenwiththeAlexaFluor488- 10(cid:3)g/mlvalinomycin,and5mMPIPES-NaOH(pH7.4)inthe
conjugatedandAlexaFluor546-conjugatedanti-IgGantibod- presenceorabsenceof10(cid:3)MSCH28080.Intheexperimentsto
ies(1:100dilution)for1hatroomtemperature.Immunofluo- testanionselectivity,150mMKClwasreplacedwith150mM
rescence images were visualized using a Zeiss LSM 510 laser KX(X(cid:5)Br,I,H PO ,orgluconate).Whenindicated,DIOA(10
2 4
scanningconfocalmicroscope. (cid:3)M) was added. The decrease in the amount of NADH was
Immunoprecipitation—MembranefractionsofSAV(100(cid:3)g measured by a Beckman spectrophotometer in a dual wave-
ofprotein)andtheHEK293cellsstablyexpressingbothKCC4 lengthmodeat340and500nmat25°C.
andH(cid:1),K(cid:1)-ATPase(2mgofprotein)weresolubilizedinlysis H(cid:1),K(cid:1)-ATPase activities of freeze-dried vesicles prepared
buffer (phosphate-buffered saline containing 0.5% Triton fromTVandSAV(10(cid:3)gofprotein)andthemembranefrac-
X-100, 0.1% bovine serum albumin, and 1 mM EDTA) for 30 tionofHEK293cells(30(cid:3)gofprotein)weremeasuredina1-ml
minoniceandcentrifugedat90,000(cid:4)gfor30minat4°C.The solutioncontaining15mMKCl,3mMMgSO ,1mMATP,5mM
4
lysate was precleared with protein A-agarose beads, and the NaN ,2mMouabain,and40mMTris-HCl(pH6.8)inthepres-
3
supernatantwasincubatedinthepresenceandabsenceofanti- enceorabsenceof50(cid:3)MSCH28080.Afterincubationfor10
KCC4antibody(1:50dilution)oranti-Histagantibody(1:100 min(freeze-driedvesicles)or30min(HEK293cells)at37°C,
dilution)for12hat4°Cwithend-over-endrotation.Antibody- the reaction was terminated by addition of the ice-cold stop
antigen complexes were incubated with protein A-agarose solutioncontaining12%perchloricacidand3.6%ammonium
beadsandincubatedfor4hat4°Cwithend-over-endrotation. molybdate, and the inorganic phosphate released was meas-
Thenthebeadswerewashedthreetimeswiththelysisbuffer ured(22).
andsuspendedinSDSsamplebuffer.Thesampleswereused Measurement of 86Rb(cid:1) Uptake into SAV—SAV (200 (cid:3)g of
forWesternblottingtochecktheexpressionofKCC4,H(cid:1),K(cid:1)- protein)wereaddedtothesolutioncontaining150mMKX(X(cid:5)
ATPase(cid:1)-subunit,or(cid:2)-actin. Cl, Br, H PO , or gluconate), 3 mM MgSO , 1 mM ATP, 20
2 4 4
Measurementof36Cl(cid:2)UptakeintoHogGastricVesicles(SAV (cid:3)Ci/ml86RbCl,and5mMPIPES-Tris(pH7.4)andincubated
and TV)—SAV or TV (100 (cid:3)g of protein) were preincubated for10minat25°C.Thereactionmixtureswererapidlyfiltered
with a solution of 150 mM KCl, 250 mM sucrose, and 40 mM througha0.45-(cid:3)mHAWPfilter.Thefilterwaswashedwitha
PIPES-Tris(pH6.8)for5minor20hat4°C.Thenthevesicle solutioncontaining150mMKX(X(cid:5)Cl,Br,H PO ,orgluco-
2 4
(SAVorTV)wasincubatedwithasolutioncontaining150mM nate)and5mMPIPES-Tris(pH7.4),transferredtoacounting
JANUARY2,2009•VOLUME284•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 621
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
vial,andsolubilizedwith5mlofACSIIscintillant.Thenthe
radioactivityof86Rb(cid:1)wascountered.
Plasmid Construction—Full-length cDNA encoding rat
KCC4wasinsertedintothepcDNA4/Hisvector(Invitrogen)by
using EcoRI and XbaI restriction sites (KCC4-pcDNA4/His
vector).TheKCC4cDNA(withXpressepitope)cutfromthe
KCC4-pcDNA4/HisvectorwasinsertedintothepcDNA5/TO
vector by using AflII and XbaI restriction sites (KCC4-
pcDNA5/TOvector).
Tetracycline-regulated Expression System of KCC4 in
HEK293 Cells Stably Expressing H(cid:1),K(cid:1)-ATPase—HEK293
cells stably expressing (cid:1)- and (cid:2)-subunits of the gastric
H(cid:1),K(cid:1)-ATPase were established as described previously
(23). The cells were cotransfected with the KCC4- FIGURE1.ExpressionofKCC4ingastricparietalcells.A,Northernblotting
wasperformedwithpoly(A)(cid:1)RNA(2.5(cid:3)g/lane)fromisolatedrabbitgastric
pcDNA5/TO and pcDNA6/TR vectors (Invitrogen) using
parietalcells.A4.9-kbbandwasdetectedwiththeKCC4cDNAprobe.B,West-
Lipofectamine 2000 and cultured for 24 h. The transfected ern blotting (left panels) was performed with the membrane fractions of
cellswereselectedinthepresenceof400units/mlhygromy- HEK293cellstransfectedwithratKCC4(clonedKCC4)(3(cid:3)gofprotein),mouse
gastricmucosa(10(cid:3)gofprotein),andratgastricmucosa(50(cid:3)gofprotein)
cinBand6(cid:3)g/mlblasticidinS. wheretwosetsofthreesamples(asA-B-C-A-B-C)inamembranewereelec-
MeasurementofIntracellularpH—IntracellularpH(pH)of trophoresed,andthenthemembranewascutintotwopieces,whichwere
i
separatelyprobedwithanti-mouseKCC4antibody(upperpanel)andwiththe
theHEK293cellswasmeasuredbymonitoringthefluorescence
anti-mouseKCC4antibodyplusthecorrespondingblockingpeptide(lower
ofBCECFasdescribedpreviously(24,25).Thecells(1(cid:4)105 panel).Abandof165kDawasobserved(upperpanel).The165-kDabands
cells), seeded on coverslips (13 (cid:4) 7 mm; Matsunami Glass, disappearedinthepresenceoftheblockingpeptide(lowerpanel).Intheright
panels,WesternblottingwasperformedwiththeclonedKCC4(15(cid:3)gofpro-
Osaka,Japan)coatedwithpoly-L-lysineandrattailcollagenI tein),hoggastricmucosa(100(cid:3)gofprotein),andhumangastricmucosa(100
(Invitrogen),wereculturedfor24h.Thenthecellsweretreated (cid:3)gofprotein)wheretwosetsofthreesamplesinamembranewereelectro-
withorwithouttetracycline(2(cid:3)g/ml)foranadditional24h. phoresed,andthenthemembranewascutintotwopieces,whichweresep-
aratelyprobedwithanti-humanKCC4antibody(upperpanel)andwiththe
Expression of KCC4 was confirmed by Western blotting and anti-humanKCC4antibodyplusthecorrespondingblockingpeptide(lower
immunocytochemistry. panel).Abandof165kDawasobserved(upperpanel).The165-kDabands
ThecellswereincubatedwithBCECF-AM(10(cid:3)M)inbuffer disappearedinthepresenceoftheblockingpeptide(lowerpanel).
containing 145 mM NaCl, 5 mM KCl, 1 mM CaCl , 1.2 mM
2
MgSO ,2mMNaH PO ,10.5mMglucose,and32mMHEPES withthe165-kDaproteininmouseandratgastricmucosaand
4 2 4
(pH7.4)for30minat37°C.Whenindicated,DIOA(10(cid:3)M)or thatanti-humanKCC4antibodyreactedwiththe165-kDapro-
SCH28080(10(cid:3)M)wasadded.Thecoverslipwasplacedinthe teininhogandhumangastricmucosa(Fig.1B,upperpanels).
chamber of a spectrofluorometer (RF-5000, Shimadzu) Bothantibodiesreactedwiththe165-kDabandsinthemem-
equipped with a continuous buffer flow system. Intracellular branefractionofHEK293cellstransfectedwithratKCC4(Fig.
BCECFwasalternatelyexcitedwith490and440nmlight,and 1B,clonedKCC4).Thespecificitiesoftheseantibodiesforthe
theemittedlightwasmeasuredat520nm.Thecellswereacid- 165-kDa bands were confirmed by using the corresponding
loadedbysuccessivelyincubatingintheNH /NH (cid:1)-contain- blockingpeptide(Fig.1B,lowerpanels).
3 4
ingbuffercomposedof125mMNaCl,20mMNH Cl,5mMKCl, Intheimmunohistochemistryofisolatedratgastricmucosa,
4
1mMCaCl ,1.2mMMgSO ,2mMNaH PO ,10.5mMglucose, KCC4wasfoundtobecolocalizedwithH(cid:1),K(cid:1)-ATPase,which
2 4 2 4
and32mMHEPES(pH7.4)andtheNa(cid:1)-freebuffercomposed is expressed in the intracellular tubulovesicles and the apical
of145mMN-methyl-D-glucamine-Cl,5mMKCl,1mMCaCl , canalicularmembraneofparietalcells(Fig.2,A–F).Thespec-
2
1.2mMMgSO ,2mMNaH PO ,10.5mMglucose,and32mM ificity of anti-KCC4 antibody for positive staining was con-
4 2 4
HEPES(pH7.4).Whenindicated,DIOA(10(cid:3)M)orSCH28080 firmedbyusingtheblockingpeptide(Fig.2,G–I).Ontheother
(10(cid:3)M)wasadded.ThetimecourseofpHirecoveryafteracid hand,thedistributionpatternofKCC4wasapparentlydifferent
loadingwasmonitored. fromthatoftheNa(cid:1),K(cid:1)-ATPase(cid:1)1subunit(Fig.2,J–L).Ithas
Statistics—Results are shown as means (cid:6) S.E. Differences beenreportedthatyoungerparietalcellsintheluminalregion
betweengroupswereanalyzedbyone-wayanalysisofvariance, of the glands much more actively secrete acid than the older
and correction for multiple comparisons was made by using parietal cells in the basal region (26–28). AQP4 has been
Tukey’s multiple comparison test. Comparison between the reported to be localized in the basolateral membrane of the
twogroupswasmadebyusingStudent’sttest.Statisticallysig- parietalcellsatthebasalregionofthegastricglands(29).Inter-
nificantdifferenceswereassumedatp(cid:7)0.05. estingly the present double immunostaining of KCC4 and
AQP4inthegastricmucosashowedthatKCC4isexpressedin
RESULTS
theparietalcellsmoreabundantlyattheluminalregionofthe
ExpressionofKCC4inGastricParietalCells—Firstweexam- glandsthanatthebasalregion.(Fig.2,M–O).
ined whether KCC4 mRNA was expressed in gastric parietal Expression of KCC4 in the SAV That Are Derived from the
cells.NorthernblottingshowedsignificantexpressionofKCC4 Apical Canalicular Membranes of Gastric Parietal Cells—To
mRNA((cid:8)4.9kb)inrabbitgastricparietalcells(Fig.1A).West- determinewhetherKCC4isexpressedintheintracellulartubu-
ern blotting showed that anti-mouse KCC4 antibody reacted lovesiclesandtheapicalcanalicularmembraneofparietalcells,
622 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER1•JANUARY2,2009
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
FIGURE3.ExpressionofKCC4intheSAV.A,Westernblottingwasper-
formedwithhogtubulovesicles(5(cid:3)gofprotein)andstimulation-associated
vesicles (5 (cid:3)g of protein) with anti-H(cid:1),K(cid:1)-ATPase (cid:1)-subunit (HK(cid:1)), anti-
H(cid:1),K(cid:1)-ATPase(cid:2)-subunit(HK(cid:2)),andanti-Na(cid:1),K(cid:1)-ATPase(cid:1)1subunit(NaK)
antibodies.ThespecificbandsforH(cid:1),K(cid:1)-ATPase(cid:1)-subunit,H(cid:1),K(cid:1)-ATPase
(cid:2)-subunit,andNa(cid:1),K(cid:1)-ATPase(cid:1)1subunitwereobservedat95,80,and100
kDa,respectively.AsapositivecontrolfordetectingNa(cid:1),K(cid:1)-ATPase(cid:1)1sub-
unit,hoggastricmucosa(5(cid:3)gofprotein)wasused(mucosa).B,expressionof
Rab11(27kDa),(cid:2)-actin(45kDa),ezrin(85kDa),syntaxin-1(35kDa),KCNQ1
FIGURE 2. Immunostaining for KCC4 in isolated rat gastric mucosa. (70kDa),KCNE2(60kDa),andCFTR(150kDa)inTVandSAV(30(cid:3)gofprotein).
A–Cshowthesametissueunderamicroscope(asdoD–F,G–I,J–L,andM–O). C,WesternblottingwasperformedwithTV,SAV,andgastricmucosaofhogs
Double immunostaining was performed with isolated rat gastric mucosa (30(cid:3)gofprotein)usinganti-humanKCC4antibody.Abandof165kDawas
usinganti-mouseKCC4andanti-H(cid:1),K(cid:1)-ATPase(cid:1)-subunitantibodies(HK) observedinSAVbutnotinTV(upperpanel).The165-kDabandsdisappeared
(A–I),anti-KCC4andanti-Na(cid:1),K(cid:1)-ATPase(cid:1)1subunitantibodies(NaK)(J–L), inthepresenceofthecorrespondingblockingpeptide((cid:1)BP;lowerpanel).
andanti-KCC4andanti-AQP4antibodies(M–O).A–F,colocalizationofKCC4 Upperandlowerpanelswerederivedfromamembraneblottedwithtwo
withH(cid:1),K(cid:1)-ATPaseisshown.IntheinsetofF,anenlargedimageofaparietal samesetsofthethreesamples.Themembranewascutintotwopiecesand
cellisshown.G–I,anti-KCC4antibodywaspretreatedwiththeblockingpep- usedfortwoseparateimmunoblottingshownintheupperandlowerpanels.
tide.PositiveKCC4stainingdisappeared.J–L,localizationofKCC4isdifferent D,WesternblottingwasperformedwithTV,SAV,andgastricmucosaof
fromthatofNa(cid:1),K(cid:1)-ATPase.IntheinsetofL,anenlargedimageofaparietal hogs(30(cid:3)gofprotein)usingantibodiesforKCC1,KCC2,andKCC3.The
cellisshown.M–O,localizationofKCC4isdifferentfromthatofAQP4.Scale 180-kDa band of KCC3 detected in the mucosa is due to KCC3a in the
bars,10(cid:3)m. basolateralmembraneofthegastricparietalcell(15).Amembranefrac-
tionofpigkidneyLLC-PK1cellswasusedasapositivecontrolforKCC1,
andthatofhogbrainwasusedasapositivecontrolforKCC2(control).The
twotypesofgastricvesicles(SAVandTV)werepreparedfrom specificbandsforKCC1,KCC2,andKCC3wereobservedat130,138,and
hog gastric mucosa. H(cid:1),K(cid:1)-ATPase (cid:1)- and (cid:2)-subunits were 1d8e0tekrDgean,rteespxtercatcivtseloyf.ES,AimVm(1u0n0o(cid:3)pgreocfippitraottieoinn)(IuP)swinagsapnetrif-oKrCmCe4dawntitibhothdey
highlyexpressedinbothTVandSAV(Fig.3A).Theexpression andproteinA-agaroseinthelysateofSAV(IP:KCC4,(cid:1)).Incontrolexperi-
level of the Na(cid:1),K(cid:1)-ATPase (cid:1)1 subunit in TV and SAV was ments,preimmuneseruminsteadoftheantibodywasused(IP:KCC4,(cid:2)).
Thedetergentextracts(input;1⁄33(forKCC4)or1⁄100(forH(cid:1),K(cid:1)-ATPase
muchlowerthanthatinthegastricmucosa(Fig.3A). (cid:1)-subunit(HK(cid:1))and(cid:2)-actin)oftotalprotein)andimmunoprecipitation
It has been reported that Rab11 is present in the H(cid:1),K(cid:1)- samples(IP:KCC4; 1⁄100(forKCC4)and1⁄50(forH(cid:1),K(cid:1)-ATPase(cid:1)-subunit
(HK(cid:1)) and (cid:2)-actin) of the samples) were detected by Western blotting
ATPase-richvesicularmembraneandrelatedtothevesicular (WB)usingantibodiesforKCC4(toppanel),H(cid:1),K(cid:1)-ATPase(cid:1)-subunit(mid-
traffickingmachineryingastricparietalcells(30).(cid:2)-Actinhas dlepanel),and(cid:2)-actin(bottompanel).Topandmiddlepanelswerederived
been reported to be associated with ezrin in the apical mem- fromamembraneblottedwithtwosamesetsofthethreesamples.The
membranewascutintotwopiecesandusedfortwoseparateimmuno-
braneofgastricparietalcells(31,32).Heretheexpressionlevel blottingshowninthetopandmiddlepanels.Theimmunoprecipitation
ofRab11inTVwasmuchhigherthanthatinSAV.Incontrast, shownisrepresentativeofthreeindependentexperiments.
the expression levels of (cid:2)-actin and ezrin in SAV were much
JANUARY2,2009•VOLUME284•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 623
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
higherthanthoseinTV(Fig.3B).Syntaxin-1wasexpressedin
both TV and SAV as reported previously (33) (Fig. 3B).
KCNQ1/KCNE2 K(cid:1) channels and CFTR Cl(cid:2) channel were
predominantly expressed in TV (Fig. 3B). These results con-
firmedthatSAVandTVarederivedfromtheapicalcanalicular
membranes and the intracellular microsomal membranes of
thegastricparietalcell,respectively.
InterestinglyKCC4(165kDa)waspredominantlyexpressed
inSAVbutnotinTV(Fig.3C).Thespecificityoftheantibody
forthe165-kDabandwasconfirmedbyusingthecorrespond-
ing blocking peptide (Fig. 3C, lower panel). No significant
expressionofotherKCCssuchasKCC1,KCC2,andKCC3was
observedineitherTVorSAV(Fig.3D).
TostudywhetherKCC4isassociatedwithH(cid:1),K(cid:1)-ATPasein
SAV,immunoprecipitationwasperformedusingananti-KCC4
antibody.ThesubsequentWesternblottingoftheimmunepel-
lets with an anti-H(cid:1),K(cid:1)-ATPase (cid:1)-subunit antibody gave a
clearbandcorrespondingtotheH(cid:1),K(cid:1)-ATPase(cid:1)-subunit(95
kDa),whereasblottingwithananti-(cid:2)-actinantibodyasaneg-
ative control gave no (cid:2)-actin band (45 kDa) (Fig. 3E). These
resultssuggestthatKCC4isassociatedwithH(cid:1),K(cid:1)-ATPasein
theSAV.
Notethattheapparentdifferenceinaffinityoftheanti-hu-
manKCC4antibodyforKCC4proteinbetweenFig.3,CandE,
originated from the pretreatment of SAV with (Fig. 3E) and
without (Fig. 3C) the lysis buffer. This buffer effect was con-
firmedbyaseparateexperimentshowninsupplementalFig.2.
InhibitionofCl(cid:2)TransportbytheH(cid:1),K(cid:1)-ATPaseInhibitor
inSAV—HerewemeasuredCl(cid:2)uptakeintoTVandSAVusing
36Cl(cid:2)inasolutioncontaining150mMKCl,4mMMgSO ,250
4
mMsucrose,2mMATP,5(cid:3)Ci/mlNa36Cl,and40mMPIPES-
Tris (pH 6.8). DIOA is known as a potent inhibitor of KCCs
(34). Although high concentrations of DIOA suppressed
H(cid:1),K(cid:1)-ATPaseactivity(IC (cid:5)75–97(cid:3)M),noinhibitoryeffect
50
ofthisdrugonH(cid:1),K(cid:1)-ATPasewasobservedatalowerconcen-
tration((cid:7)20–30(cid:3)M)(35).InFig.4,DIOA(10(cid:3)M)significantly
inhibitedCl(cid:2)uptakeinSAV(Fig.4B)butnotinTV(Fig.4A),
reflectingthepresenceofKCC4inSAVanditsabsencefrom
TV.SCH28080(10(cid:3)M),aspecificinhibitorofH(cid:1),K(cid:1)-ATPase,
significantlyinhibitedCl(cid:2)uptakeinbothSAVandTV(Fig.4).
IntheSAV,inhibitoryeffectsofDIOAandSCH28080werenot
FIGURE4.Inhibitionof36Cl(cid:2)uptakeintoSAVbyaninhibitorofH(cid:1),K(cid:1)-
significantlydifferentfromthatofDIOAplusSCH28080(Fig. ATPase(SCH28080).The36Cl(cid:2)uptakeinTV(A)andSAV(B)wasmeasuredas
4B).TheseresultssuggestthattheDIOA-sensitiveCl(cid:2)trans- describedunder“ExperimentalProcedures.”Effectsof10(cid:3)MSCH28080(gray
portinSAVmaybemediatedbytheH(cid:1),K(cid:1)-ATPaseactivity. DbaIOr;A(cid:1)(ShCaHtc),h1e0d(cid:3)bMarD;(cid:1)IOSAC(Hb(cid:1)laDckIObAa)r;o(cid:1)nDtIhOeA3)6,Canl(cid:2)du1p0t(cid:3)akMeSwCHer2e80ex8a0mpilnuesd1.0T(cid:3)hMe
Inhibition of H(cid:1) Transport by the KCC Inhibitor in SAV— valueobtainedintheabsenceofSCH28080andDIOA(openbar;control)is
NexttheinhibitoryeffectsofDIOAonH(cid:1)uptakeintoSAVand normalizedas100%.n(cid:5)4.NS,notsignificantlydifferent(p(cid:9)0.05);**,signif-
icantlydifferent(p(cid:7)0.01).Dataareshownasmeans(cid:6)S.E.
TVbyH(cid:1),K(cid:1)-ATPasewerestudied.SCH28080(20(cid:3)M)mark-
edlyinhibitedtheH(cid:1)uptakeinTVandSAVasexpected(Fig.5,
AandB).Ontheotherhand,noH(cid:1)uptakewasobservedinthe (Fig.6B),whereasithadnosignificanteffectsinTV(Fig.6A).In
freeze-driedTVandSAV(Fig.5,AandB).InterestinglyDIOA the freeze-dried leaky TV and SAV, no significant effects of
(10 (cid:3)M) significantly inhibited the H(cid:1) uptake into SAV, DIOA (10 (cid:3)M) on the H(cid:1),K(cid:1)-ATPase activity were observed
whereas it had no significant effect in TV (Fig. 5, C and D), (supplementalFig.3,AandB).TheseresultssuggestthatDIOA
indicatingthattheinhibitionofKCC4resultedininhibitionof indirectlyinhibitsH(cid:1),K(cid:1)-ATPaseactivityviainhibitionofthe
H(cid:1)uptakebyH(cid:1),K(cid:1)-ATPase. iontransportbyKCC4.
InhibitionofH(cid:1),K(cid:1)-ATPaseActivitybytheKCCInhibitorin Anion Selectivity for the DIOA-sensitive H(cid:1) Transport and
SAV—WestudiedwhetherDIOAaffectstheSCH28080-sensi- H(cid:1),K(cid:1)-ATPaseActivityinSAV—TostudywhethertheDIOA
tive K(cid:1)-ATPase activity (H(cid:1),K(cid:1)-ATPase activity). DIOA (10 (10(cid:3)M)-sensitiveH(cid:1)transportandH(cid:1),K(cid:1)-ATPaseactivityin
(cid:3)M)significantlyinhibitedtheH(cid:1),K(cid:1)-ATPaseactivityinSAV SAVdependsonthespeciesofanion,H(cid:1)transportandenzyme
624 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER1•JANUARY2,2009
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
These orders were similar to that
reported for the anion selectivity
for K(cid:1) transporting activity of
KCC4(Cl(cid:2)(cid:9)Br(cid:2)(cid:9)PO3(cid:2)(cid:5)I(cid:2)(cid:9)
4
gluconate)(37).
Anion Selectivity for the DIOA-
sensitiveK(cid:1)UptakeintotheSAV—
To study whether the K(cid:1) uptake
into SAV by K(cid:1)-Cl(cid:2) cotransport
dependsonanionspecies,wemeas-
ured the DIOA (10 (cid:3)M)-sensitive
86Rb(cid:1)uptakeinsolutionsof150mM
KX (where X (cid:5) Cl, Br, H PO , or
2 4
gluconate). The order of anion
selectivity for 86Rb(cid:1) uptake into
SAVwasCl(cid:2)(cid:9)Br(cid:2)(cid:9)PO3(cid:2)(cid:5)glu-
4
conate (Fig. 7C). This selectivity is
quantitatively the same as those of
the DIOA-sensitive H(cid:1) transport
(Fig.7A)andH(cid:1),K(cid:1)-ATPaseactiv-
ity(Fig.7B),indicatingthatproton
transport by H(cid:1),K(cid:1)-ATPase de-
pends on ion transport by KCC4.
The SCH28080 (10 (cid:3)M)-sensitive
86Rb(cid:1)uptakeinsolutionsof150mM
KX (where X (cid:5) Cl, Br, H PO , or
2 4
gluconate) was also assessed. The
orderofanionselectivitywasCl(cid:2)(cid:9)
Br(cid:2) (cid:9) PO3(cid:2) (cid:5) gluconate (supple-
4
mentalFig.5).
Stable Coexpression of KCC4
and H(cid:1),K(cid:1)-ATPase in the
HEK293 Cells—The tetracycline-
regulated expression system of
KCC4 was constructed in HEK293
cells that stably expressed (cid:1)- and
(cid:2)-subunits of gastric H(cid:1),K(cid:1)-
ATPase. No significant expression
ofendogenousKCC4wasobserved
in control HEK293 cells (data not
shown). In this heterologous ex-
pressionsystem,exogenousexpres-
FIGURE5.InhibitionofH(cid:1)uptakeintoSAVbyaninhibitorofKCCs(DIOA).TheH(cid:1)uptakeinTV(AandC) sion of KCC4 protein was assessed
aandddeSdAVat(BthaentdimDe)winadsiacassteedssbeydabnyamrreoawsu.Erifnfegctthseoqfu20en(cid:3)cMhiSnCgHo2f8a0c8ri0d(in(cid:1)eSoCrHa2n8g0e8f0lu)oanredsc1e0n(cid:3)cMe.DAITOPA(3((cid:1)50D(cid:3)IOMA)w)oans by using an anti-Xpress antibody.
theH(cid:1)uptakewereexamined.Ascontrolexperiments,theH(cid:1)uptakewasmeasuredintheabsenceof ExpressionofKCC4(165kDa)was
SCH28080andDIOA.AlmostnoH(cid:1)uptakewasobservedinthefreeze-driedvesicles(FDV;AandB).Typical observed in the cells treated with
tracesareshownalongwiththequantitativedata.TheH(cid:1)uptakewasexpressedasthechangeoffluorescence
intensityperminduringtheperiodfrom0to3minafteradditionofATP.n(cid:5)4.NS,notsignificantlydifferent tetracycline(Tet-Oncells),whereas
(p(cid:9)0.05);**,significantlydifferent(p(cid:7)0.01)fromthevaluesobtainedintheabsenceofSCH28080andDIOA no significant expression of KCC4
(control).
was observed in the cells treated
without tetracycline (Tet-Off cells)
activityweremeasuredinvariousanionsolutionscontaining (Fig.8A).ExpressionlevelsofH(cid:1),K(cid:1)-ATPase(cid:1)-subunitinthe
150mMKX(whereX(cid:5)Cl,Br,I,H PO ,orgluconate).The Tet-Oncellswerenotsignificantlydifferentfromthoseinthe
2 4
order of selectivity of various anions for H(cid:1) uptake was Tet-Off cells (Fig. 8B). Both KCC4 and H(cid:1),K(cid:1)-ATPase were
Cl(cid:2)(cid:9)Br(cid:2)(cid:9)PO3(cid:2)(cid:5)gluconate(Fig.7Aandsupplemental found to be present in the plasma membrane of the Tet-On
4
Fig.4).WecouldnotexaminetheeffectofI(cid:2)onH(cid:1)uptake cells (Fig. 8C). To check whether KCC4 is associated with
because KI quenches the fluorescence of acridine orange H(cid:1),K(cid:1)-ATPase in the Tet-On cells as is the case in the SAV
(36). The order of anion selectivity for the H(cid:1),K(cid:1)-ATPase (Fig.3E),immunoprecipitationwasperformedbyusingananti-
activitywasCl(cid:2)(cid:9)Br(cid:2)(cid:9)I(cid:2)(cid:9)PO3(cid:2)(cid:5)gluconate(Fig.7B). Histagantibody(forKCC4).ThesubsequentWesternblotting
4
JANUARY2,2009•VOLUME284•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 625
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
FIGURE 6. Inhibition of H(cid:1),K(cid:1)-ATPase activity in SAV by DIOA.
SCH28080 (10 (cid:3)M)-sensitive K(cid:1)-ATPase activity (H(cid:1),K(cid:1)-ATPase activity)
wasmeasuredintheabsence(control)andpresence(DIOA)of10(cid:3)MDIOA
inTV(A)andSAV(B).n(cid:5)5.NS,notsignificantlydifferent(p(cid:9)0.05);**,
significantlydifferent(p(cid:7)0.01).
oftheimmunepelletswithananti-H(cid:1),K(cid:1)-ATPase(cid:1)-subunit
antibodygaveabandforH(cid:1),K(cid:1)-ATPase(cid:1)-subunit(Fig.8D),
indicatingassociationbetweenKCC4andtheH(cid:1),K(cid:1)-ATPase
(cid:1)-subunit.
KCC4-induced Stimulation of H(cid:1) Transport by H(cid:1),K(cid:1)-
ATPase in the Heterologous Expression System—In the mem-
branefractionsofbothTet-OffandTet-Oncells(i.e.cell-free
condition),DIOA(10(cid:3)M)didnotinhibittheH(cid:1),K(cid:1)-ATPase
activity(supplementalFig.3,CandD)asfoundforthefreeze-
driedTVandSAV(supplementalFig.3,AandB).Thiswould
reflectthefactthatthemembranesamplesobtainedfromthe
HEK293cellswerenottightlysealedashasbeendescribedelse-
where(38).InFig.9,westudiedthecapacityoftheacidextru-
sion (H(cid:1) transport activity) in the Tet-On and Tet-Off cells.
Thecapacitywasassessedbymeasuringtherateofrecoveryof
pH inthecellsafteracidloadingthroughanammoniumpulse.
i
The pH recovery was monitored in the absence of Na(cid:1) to
i
exclude the contribution of the Na(cid:1)/H(cid:1) exchanger. The pH
i
recoveryprocessintheTet-Oncells(Fig.9,BandG)wassig-
nificantlyfasterthanthatintheTet-Offcells(Fig.9,AandG).
InterestinglyDIOA(10(cid:3)M)significantlydecreasedtherecov-
eryrateofpH intheTet-Oncells(Fig.9,DandG)butnotinthe
i
Tet-Offcells(Fig.9,CandG).SCH28080(10(cid:3)M)significantly
decreasedtherecoveryrateofpH inboththeTet-OnandTet- FIGURE 7. Anion selectivity for the DIOA-sensitive H(cid:1) transport,
Off cells (Fig. 9, E–G). These resiults suggest that KCC4 may H(cid:1),K(cid:1)-ATPaseactivityand86Rb(cid:1)transportinSAV.A,theH(cid:1)uptake
intoSAVwasassessedbymeasuringthequenchingofacridineorange
stimulate the H(cid:1) transport activity of H(cid:1),K(cid:1)-ATPase in the fluorescence.ATP(350(cid:3)M)wasaddedinsolutionsof150mMKX(X(cid:5)Cl,Br,
Tet-Oncells. H PO ,orgluconate(Gl)),andthetime-dependentfluorescencequench
of2acr4idineorangewasmonitoredasshowninsupplementalFig.4.TheH(cid:1)
DISCUSSION uptakewasmeasuredintheabsenceandpresenceof10(cid:3)MDIOA.The
DIOA-sensitiveH(cid:1)uptakewasexpressedasthechangeoffluorescence
In the present study, we found the following. 1) KCC4 is intensityperminduringtheperiodfrom0to3minafteradditionofATP.
n(cid:5)5.NS,p(cid:9)0.05;**,p(cid:7)0.01fromthevaluesobtainedwiththeKCl
expressedintheapicalcanalicularmembraneofgastricparietal solution(Cl(cid:2)).B,SCH28080(10(cid:3)M)-sensitiveK(cid:1)-ATPaseactivity(H(cid:1),K(cid:1)-
cellsmoreabundantlyattheluminalregionoftheglandsthan ATPaseactivity)inSAVwasassessed.TheDIOA-sensitiveH(cid:1),K(cid:1)-ATPase
atthebasalregion.Incontrast,KCC1,KCC2,andKCC3arenot activitywasthedifferencebetweenthevaluesmeasuredintheabsence
andpresenceof10(cid:3)MDIOA.n(cid:5)5.NS,p(cid:9)0.05;*,p(cid:7)0.05;**,p(cid:7)0.01
significantly expressed in the apical canalicular membrane. fromthevaluesobtainedwiththeKClsolution(Cl(cid:2)).C,the86Rb(cid:1)uptake
KCC4isabsentfromtheintracellulartubulovesicles.2)KCC4 intoSAVwasmeasuredinsolutionsof150mMKX(X(cid:5)Cl,Br,H2PO4,or
is associated with H(cid:1),K(cid:1)-ATPase in the apical canalicular gsiltuivceon86aRteb)(cid:1)asudpetaskceribweadsutnhdeedri“ffEexrpeenrcimebenettwalePernoctehdeuvraelsu.”eTshmeeDaIsOuAre-sdenin-
membrane. 3) In vesicles of the apical canalicular membrane theabsenceandpresenceof10(cid:3)MDIOA.n(cid:5)5.NS,p(cid:9)0.05;**,p(cid:7)0.01
(SAV),theH(cid:1),K(cid:1)-ATPaseinhibitorsuppressestheCl(cid:2)trans- fromthevaluesobtainedwiththeKClsolution(Cl(cid:2)).
portactivity,whichissensitivetoaKCCinhibitor(DIOA;10
626 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER1•JANUARY2,2009
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
FIGURE9.pH recoveryoftheTet-OnandTet-Offcellsafteracidloading
i
throughanammoniumpulse.A–F,thepH recoverywasmonitoredinthe
FIGURE 8. Tetracycline-regulated expression system of KCC4 in the absenceofNa(cid:1)afteranammoniumpulseofitheTet-Offcells(A,C,andE)and
HEK293cellsstablyexpressinggastricH(cid:1),K(cid:1)-ATPase.Thetetracycline- Tet-Oncells(B,D,andF).Representativetracesareshown.Effectsof10(cid:3)M
regulatedexpressionsystemofKCC4wasintroducedtotheHEK293cellsthat DIOA(CandD)and10(cid:3)MSCH28080(SCH)(EandF)onthepHirecoverywere
stablyexpress(cid:1)-and(cid:2)-subunitsofgastricH(cid:1),K(cid:1)-ATPase.Thecellswere examined.Ascontrolexperiments,thepHirecoverywasmeasuredinthe
treatedwith(on)orwithout(off)2(cid:3)g/mltetracycline.A,theexpressionof absenceofDIOAandSCH28080(AandB).Theredbarsshowthefittedslopeof
KbHCy(cid:1)C,WK4(cid:1)ei-nsAtteThrPneamsbeleo(cid:1)mt-tsbinurgabnuuensifitnraignctatiohnneti-oTXefpttr-heOesnscecalenlsltlsi(b3(0oodn(cid:3))yg.wBoa,fstpchroeomteepxinpa)rrewedsasswiocintohnleftihvremalteoidnf ppGH,(cid:9)tihc0eh.0aav5ne;g*rea*,gfpreod(cid:7)mra00t.e0ts1o.o5ftmheinp(Hbilureecboavre)rayf(t(cid:10)erpiHnit/r5omduinc)inagretshheoNwan(cid:1).n-fr(cid:5)ee4b–5u.ffNeSr,.
theTet-Offcells(off).Intheupperpanel,arepresentativepictureofWestern
ibzleodttainsg1.isns(cid:5)ho5w.nN.SI,npt(cid:9)he0lo.0w5e.Crp,aa–ncels,hthoewstchoeresafomrethceelTlsetu-nOdffecreallmsiiscrnoosrcmopale- (cid:3)M). 4) The KCC inhibitor suppresses the H(cid:1),K(cid:1)-ATPase
(asdod–fandg–i).DoubleimmunostainingwasperformedwiththeTet-On activityintheSAVbutnotinthefreeze-driedleakySAVand
cells(a–f)andtheTet-Offcell(g–i)usinganti-KCC4plusanti-H(cid:1),K(cid:1)-ATPase theTV.5)IntheSAV,theanionselectivityoftheDIOA-sensi-
(cid:1)(b-,sue,baunnditha)n,taibnoddKieCsC.4LopclaulsizaHt(cid:1)io,Kn(cid:1)s-oAfTKPCaCse4((am,edr,gaenddigm),aHg(cid:1)es,K;(cid:1)c,-Af,TaPnadsei)(HarKe) tive H(cid:1),K(cid:1)-ATPase activity and the DIOA-sensitive H(cid:1) and
shown.Ind–f,anti-KCC4antibodywaspretreatedwiththeblockingpeptide K(cid:1)transportsaresimilartothatfortheK(cid:1)transportingactiv-
((cid:1)BP).PositiveKCC4stainingdisappeared.Scalebars,10(cid:3)m.D,immunopre-
cipitation(IP)wasperformedwiththedetergentextractsoftheKCC4-ex-
pressingTet-Oncellsusinganti-HistagantibodyforKCC4andproteinA-aga-
rose.Thedetergentextract(input)andtheimmunoprecipitationsamples detecting KCC4 (upper panel) and anti-H(cid:1),K(cid:1)-ATPase (cid:1)-subunit antibody
obtainedwith(IP:His(KCC4),(cid:1))andwithout(IP:His(KCC4),(cid:2))theantibody (HK(cid:1); lower panel). The immunoprecipitation shown is representatives of
were detected by Western blotting (WB) using anti-Xpress antibody for threeindependentexperiments.
JANUARY2,2009•VOLUME284•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 627
(cid:1) (cid:1)
KCC4AssociationwithH ,K -ATPaseinGastricParietalCells
ity of KCC4 (37). 6) The expression of KCC4 stimulates H(cid:1)
transport across the HEK293 cell membranes that coexpress
H(cid:1),K(cid:1)-ATPase.
RecentlywefoundthatKCC3aisexpressedinthebasolateral
membrane of gastric parietal cells, that KCC3a is associated
with Na(cid:1),K(cid:1)-ATPase (cid:1)1 subunit ((cid:1)1NaK), and that KCC3a
up-regulates (cid:1)1NaK activity in the membrane fraction of the
KCC3a-expressing LLC-PK1 cells and rabbit gastric mucosa.
KCC3a may directly activate the (cid:1)1NaK activity in cell-free
conditions(15).Ontheotherhand,inthisstudy,thefunctional
associationofKCC4withH(cid:1),K(cid:1)-ATPasecouldbeseenonlyin
intact tightly sealed SAV and HEK293 cells and not in the
freeze-dried leaky SAV and the leaky membrane fraction of
HEK293cells.OurpresentresultssuggestthatKCC4indirectly
increases H(cid:1),K(cid:1)-ATPase activity by effectively supplying K(cid:1)
totheluminalsurfaceofthisATPase.
Gastric acid secretion by the parietal cells is accompanied
withdramaticmorphologicalchanges.Inrestingparietalcells,
tubulovesicles are present in intracellular compartments
underlyingtheapicalcanalicularmembraneandformareticu-
latedmeshwork.Uponstimulation,thetubulovesiclestranslo-
cateandconnectwiththecanalicularmembrane,resultingin
massive acid secretion (39–41). So far, several Cl(cid:2) and K(cid:1)
channels have been identified in gastric parietal cells (7, 42).
CFTR(5),CLIC-6(parchorin)(6,7),andSLC26A9(8)arecan-
didates that could be involved in the luminal Cl(cid:2) efflux for
gastricacid(HCl)secretion.KCNQ1/KCNE2(1,2)andKir4.1
(3,4)arecandidatesthatcouldbeinvolvedintheluminalK(cid:1)
efflux for the K(cid:1) recycling. In the present study, CFTR was
confirmedtobelocalizedpredominantlyinthetubulovesicles
FIGURE10.Putativemodelsforgastricacid(HCl)secretionintheresting
(Fig. 3B). KCNQ1 was found to be expressed mainly in the andstimulatedstatesofgastricparietalcells.FunctionsofH(cid:1),K(cid:1)-ATPase
tubulovesicles (Fig. 3B) as reported previously (4, 43). It has (yellowellipses),KCC4(blueellipses),K(cid:1)channelssuchasKCNQ1/KCNE2and
Kir4.1(bluerectangles),andCl(cid:2)channelssuchasCFTR,CLIC-6,andSLC26A9
been reported that CLIC-6 is distributed throughout the (redrectangles)intherestingstate(upperpanels)andstimulatedstate(lower
cytosol(6)andthatKir4.1islocalizedinthetubulovesicles(4). panels)areshown.
Therefore,thedistributionpatternoftheseK(cid:1)andCl(cid:2)chan-
nelsisapparentlydifferentfromthatofKCC4thatispredom- associatedwithrenaltubularacidosis(45).Incontrasttothis
inantlyexpressedintheapicalcanalicularmembrane. basolateral localization in renal (cid:1)-intercalated cells, KCC4 is
Onthebasisofourpresentfindings,wesuggestthatthese localizedintheapicalmembraneofgastricparietalcells.Itwill
two membranes may not mix but remain separate and dis- be necessary to clarify the mechanism of the recruitment of
tinctwhenthetubulovesicularmembraneisconnectedwith KCC4intheapicalcanalicularmembraneoftheparietalcells.
the apical canalicular membrane upon stimulation. Fig. 10 Gastric parietal cells migrate from the luminal to the basal
summarizes the putative mechanisms of HCl secretion in regionoftheglands,andtheluminalparietalcellsmoreactively
these two membranes. In the resting state, KCC4 together secreteacidthandothebasalparietalcells(26–28).KCC4is
withH(cid:1),K(cid:1)-ATPasethatispresentintheapicalcanalicular expressedintheparietalcellsmoreabundantlyattheluminal
membraneisinvolvedinthebasalacidsecretion.Uponstim- region of the glands than at the basal region. It is noted that
ulation, CFTR, CLIC-6, SLC26A9, KCNQ1/KCNE2, Kir4.1, KCC3aislocatedintheluminalregion(15).KCC3ainthebaso-
and KCC4 are involved in the KCl transport for massive lateral membrane of the parietal cell increases the Na(cid:1),K(cid:1)-
gastricacidsecretion. ATPase activity. The electrochemical potential gradient for
Interestingly it has been reported that KCC1, KCC2, and K(cid:1) across the apical membrane drives the ion transport by
KCC3 are inhibited at pH (cid:7)7.5 when these are expressed in KCC4 and is established by mainly Na(cid:1),K(cid:1)-ATPase and
Xenopusoocytes,whereasKCC4isactivatedatacidicpH(44). H(cid:1),K(cid:1)-ATPase.Thus,bothKCC3aandKCC4areinvolved
ThisfindingsuggeststhatKCC4maybespecializedtooperate inacidsecretion.
in an acidic environment. In fact, we found that KCC4 is Inconclusion,KCC4isfunctionallyexpressedintheapical
expressedintheapicalcanalicularmembranes,whichfacethe canalicularmembraneofgastricparietalcells,anditsK(cid:1)-Cl(cid:2)
gastricacid. cotransport is coupled with H(cid:1),K(cid:1)-ATPase activity in the
Inthekidney,KCC4hasbeenreportedtobecrucialforCl(cid:2) canalicularmembrane.KCC4mayfunctionasaK(cid:1)-supplying
extrusion across the basolateral membrane of acid-secreting molecule for H(cid:1),K(cid:1)-ATPase and also as a Cl(cid:2)-transporting
(cid:1)-intercalatedcellsandthatthelossofKCC4leadstodeafness moleculeforHClsecretion.Intherestingstateoftheparietal
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