Francois(Verrey,(Zurich( Kidney'amino'acid'transport ... · 13/09/2014(5(tubulus'lumen'...

13/09/2014(

1(

2014.09.12(FV(Nijmegen( 1(

Francois(Verrey,(Zurich(

•  Session(VIII:(•  Integrated(view(of(disorders(of(the(amino(acid(balance(

Kidney'amino'acid'transport(

2(

Na+'

amino'acid''transporters'

Na+(symporter'

anIporter'

uniporter'

1.''Physiology'background'and'overview'of'amino'acid'reabsorp>on'(

Nijmegen(12.09.2014(

2.''Specific'aminoacidurias''(• ( (Hartnup(disorder( ( ( ( ( ( ((((SLC6A19(=(B0AT1)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (((luminal'Na+'co,transporter'(symporter)'

'

• ( (CysInuria( ( ( ( ( ( ( ( ( ( ((((SLC7A9/SLC3A1(=(b0,+AT/rBAT)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (((luminal'obligatory'exchanger'(an9porter)'

'

• ( (Lysinuric(protein(intolerance((((SLC7A7/SLC3A2(=(y+LAT1/4F2hc)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (((basolateral'obligatory'exchanger'(an9porter)'

•(( ('prerenal'(+(renal(aminoW((( (aciduria(in(mouse(model(( ( ((((SLC16A10(=(TAT1)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ('''basolateral'facilitated'diffusion'pathway'(uniporter)(


3(

Na+'


Na+(symporter'

anIporter'

uniporter'




'


'



Amino(acids((AA´s)(are(building(blocks(of(proteins(

Proteins:'~'15%'of'body'mass''(~(11kg(for(70kg(male(with(normal(BMI)(

~'1'g'free''amino'acids'in''blood'plasma'

H2O( dry(

proteins'' ~

'11'kg'

~15%(body((~(70(kg((

13/09/2014(

2(

~'50g/d'

2014.09.12(FV(Nijmegen( 5(H2O( dry(

proteins'' ~

'11'kg'

~15%(body((~(70(kg((

'IC'free'AA's''(~2(g/l)'''~'60g'

'plasma'free'AA's'(~0.25(g/l)('~'1g'''''ECF'free'AA's''(~0.25(g/l)((((((((~'4g''''

(

(Western(reality(in(CH(much(higher)(

''''''''''~'100'g/d''''

Protein'‘daily'reference'value*‘':'50g'(for(people(eaIng(2000(Cal/day)(

(

*DRF'from'FDA','~recommeded'dietary'allowance'

boEleneck'

– COO

NH 3 +

HC' R

– COO

NH 3 +

HC' R

Plasma(membrane(

20(standard(LW(amino(acids(charge side'chain'(R) essen>al nonY

essen>al small Gly'''(G)

Ala'''(A) branched

aroma>c phenyl indole

O phenol((((((((((10.46) Tyr'''(Y) hydroxyl Ser'''(S) amide Asn''(N)

Gln'''(Q) sulfur thiol(((((((((((((((8.37) Cys'''(C)

+ imidazole(((((((6.04) ε W(amino((((((10.54) guanidino((((12.48)

Val''''(V) Leu'''(L) Ile''''''(I) Phe'''(F) Trp'''(W)

Thr'''(T)

Met'''(M) His''''(H) Lys'''(K) Arg'''(R) [Arg''(R)]

_ β W(carboxyl((3.90) γ W(carboxyl((4.07) Asp'''(D)

Glu'''(E) O pyrrolidine Pro'''(P)

α Yamino

Y'NH 2

='NH

Amino(acids(have(diverse(side(chains((and(need(transporters(to(cross(membranes(

Also(for(crossing(cellular(barriers((epithelia,(endothelia)((amino(acids(are(transported(across(cells(

~(100(g/d(

~(50(g/d(

((((Amino(acids:(many(funcIons(besides(protein(metabolism(

13/09/2014(

3(

~'50g/d'

2014.09.12(FV(Nijmegen( 9(

'IC'free'AA's''(~2(g/l)'''~'60g'

'plasma'free'AA's'(~0.25(g/l)('~'1g'''''ECF'free'AA's''(~0.25(g/l)((((((((~'4g''''

Boileneck!'

The'proximal'tubule'is'the'site'of'nutrient'reabsorp>on(

Kidney'Proximal'Tubule:'reabsorbs''~'99%'of''filtered'amino'acids'

Tubular'''Amino'acid'transport'(Purpose(of(AA(transport(in(tubular(cells:(Y'Reabsorp>on'of'filtered'AAs(W(Metabolism(of(AAs((W(Housekeeping(cellular(funcIons(

2014.09.12(FV(Nijmegen( 11(

Amino'acid'reabsorp>on'in''the'proximal'kidney'tubule'

(S1>S2>S3)'

AA'AA' AA'

extracellular''space'

tubulus'lumen'

symport((

anIport(

anIport(

uniport(

Transcellular'transport:'2'membranes' QuesIon(#1 ( ( (((((((Francois(Verrey(

Reuptake(of(amino(acids(in(the(proximal(kidney(tubule(is:(

A.  driven(mostly(by(the(transepithelial(potenIal(difference(B.  driven(by(secondary(acIve(basolateral(amino(acid(

transporters(of(epithelial(cells(C.  mediated(mostly(via(acIve(luminal(uptake(and(basolateral(

efflux(across(epithelial(cells(D.  mostly(paracellular ((((

13/09/2014(

4(

MulIple((~(50)(mammalian(amino(acid(transporters(((AAT's) 'Na+-dependent (excitatory) AAT's [DAACS; 2.A.23] SLC1 X-

AG EAAT1 (GLAST) EAAT2 (GLT1) EAAT3 (EAAC1) EAAT4, EAAT5 ASC ASCT1 ASCT2 (ATB0)

'Na+,(Cl-) -dep. Neurotransm. and AAT's [NSS; 2.A.22] SLC6 BETA GAT1-GAT3 BGT1, TAUT GLY'GLYT1, GLYT2 B0,+,'ATB0,+'IMINO, PROT, SIT1 B0, B0AT1 neurotransm. creatine

'Syst. A and N Na-coupled neutral AAT's [AAAP; 2.A.18] SLC38 A SNAT 1,2,4 (ATA1,2,3) N SNAT3,5 (SN1,2) Ves. GABA tr. [SLC32] (

'Glycoprotein-associated (heteromeric) AAT's [APC; 2.A.3.8] SLC7 + SLC3 L LAT1,2-4F2hc (CD98) asc Asc1-4F2hc; Asc2 y+LAT y+LAT1,2-4F2hc x-

c xCT-4F2hc AGT1-? b0,+, b0,+AT-rBAT

AA'

Na+

(

H+'

'Cationic AAT's (CAT) [APC; 2.A.3.3] SLC7 y+ CAT1-CAT4

'Monocarbox. transporter- related AAT's [MFS; 2.A.1.13] SLC16 T TAT1 XPCT

'POV1/LAT3 [MFS; 2.A.1.44] SLC43

out in L LAT3,4

AA'

AA'

Na+'

H+'

K+'

ClW

AA'

Na+'

ClW

Na

+ AA'

AA'

AA'

AA'

AA'

AA'

H+'

'

'Proton-coupled AAT's [AAAP; 2.A.18] SLC36 H+-dep. PAT1 (LYATT-1) PAT2

Some(A

AT’s(of(ea

ch(of(t

hese(8(SLC(fa

milies(prese

nt(in(p

roxima

l(kidne

y(tubul

e( Major'renal'amino'acid'transporters''(AAT's)'discussed(here 'Na+,(Cl-) -dep. Neurotransm. and AAT's [NSS; 2.A.22] SLC6 BETA GAT1-GAT3 BGT1, TAUT GLY'GLYT1, GLYT2 B0,+,'ATB0,+'IMINO, PROT, SIT1 B0, B0AT1 neurotransm. creatine

'Glycoprotein-associated (heteromeric) AAT's [APC; 2.A.3.8] SLC7 + SLC3 L LAT1,2-4F2hc (CD98) asc Asc1-4F2hc; Asc2 y+LAT y+LAT1,2-4F2hc x-

c xCT-4F2hc AGT1-? b0,+, b0,+AT-rBAT

'Monocarbox. transporter- related AAT's [MFS; 2.A.1.13] SLC16 T TAT1 XPCT

'

out in

AA'

Na+'

ClW

Na

+ AA'

AA'

AA'

Major'renal'amino'acid'transporters''(AAT's)'discussed(here 'Na+,(Cl-) -dep. Neurotransm. and AAT's SLC6 '' GLYT2 '' SIT1 B0, B0AT1 → Hartnup disorder

'Glycoprotein- associated (heteromeric) AAT's SLC7 + SLC3 LAT2-4F2hc y+LAT1-4F2hc → LPI b0,+, b0,+AT-rBAT → Cystinuria

'Monocarbox. transporter- related AAT's SLC16 TAT1

'

out in

AA'

Na+'

ClW

Na

+ AA'

AA'

AA'

symporter' an>porter' uniporter'


Types'of'(amino'acid)'transporters'

16(

Na+'


Na+(symporter'

anIporter'

uniporter'



• (Symporters(use(for(the((secondary)(acIve(import(of(amino(acids(the(((coWtransport(of(ions((e.g.(Na+)(as(driving(force.((→'direc>onal'and'accumula>ve'transport'of'amino'acids((example:'(luminal(B0AT1((SLC6A19)(defecIve(in(Hartnup(disorder(

• (An>porters(require(for(the(transport(of(amino(acids(the(exchange(of((other)((amino(acids(that(may(provide(a(driving(force(or(represent(a(limiIng(factor(

( (→'transport'not'direc>onal,'poten>ally'asymmetric'and'accumula>ve(('examples:''luminal(b0,+ATWrBAT((SLC7A9WSLC3A1)(defecIve(in(Cys>nuria(and((basolateral(y+LAT1W4F2hc((SLC7A7WSLC3A2)(defecIve(in(Lysinuric'protein'intolerance'''(((((

•((Uniporters(transport(amino(acids(along(their(concentraIon(gradient((diffusive)(('→'transport'not'direc>onal,'equilibra>ve'''examples:(no(known(human(aminoaciduria,(mouse(TAT1((SLC16A10)(

13/09/2014(

5(

extracellular'space'tubulus'lumen'

Transepithelial(amino(acid(transport(

1'secondary'/'ter>ary'ac>ve'transporters'+''2'obligatory'exchangers'(ter>ary'ac>ve'transp.)'

AA AA

3'facilitated'diffusion'transporters'+'2'obligatory'exchangers'

AA

AA((

cys>ne,AA+' AA((

2'an>porter'

AA' AA'

3'uniporters'for(essenIal(AA´s(

AA' AA'

2'an>porter'

AA+Na+( AA((

1'symporters'

2014.09.12(FV(Nijmegen( 18(

amino(acid(reabsorpIon:(mostly(>99%(((→(FE(<1%(

extracellular'space'

AAY'

SLC1A1'

SLC6A19'

cys>ne,AA+'

AA+' AA0+Na+(

AA0+Na+(AA0' AA0'

AA0'

AA–(

AA0'

AA–+Na++H+(

tubulus'lumen'

K+(

Pro+Na+(SLC6A20'

Gly,Ala,Pro+H+(

SLC7A8'

SLC7A7'

SLC36A2'

SLC7A9'

Various(AA(transporters(with(specialized(metabolic(and/or(housekeeping(funcIons(

EAAT3'

PAT2'

SIT1'

b0,+AT'

rBAT'

B0AT1''

SLC3A1'

4F2hc'

4F2hc'

TAT1'and'LAT4'

LAT2'

SLC3A2'

y+LAT1'

SLC3A2'

AA0'

AA0'

Pro((

Cys,AA+'

Gly,Ala,Pro(

AA–((

?'

PEPT1/2'SLC15A1/2'

diY/tripep>des+H+(

hydrolysis(

AA'

SLC16A10'and'43A2'

Kidney'amino'acid'transporters'

Tmem27'in(kidney(and(ACE2(in(sm.(intesIne'


AAY'

SLC1A1'

SLC6A19'

cys>ne,AA+'

AA+' AA0+Na+(

AA0+Na+(AA0' AA0'

AA0'

AA–(

AA0'

AA–+Na++H+(

tubulus'lumen'

K+(

Pro+Na+(SLC6A20'

Gly,Ala,Pro+H+(

SLC7A8'

SLC7A7'

SLC36A2'

SLC7A9'

Various(AA(transporters(with(specialized(metabolic(and/or(housekeeping(funcIons(

EAAT3'

PAT2'

SIT1'

b0,+AT'

rBAT'

B0AT1''

SLC3A1'

4F2hc'

4F2hc'

TAT1'and'LAT4'

LAT2'

SLC3A2'

y+LAT1'

SLC3A2'

AA0'

AA0'

Pro((

Cys,AA+'

Gly,Ala,Pro(

AA–((

?'

PEPT1/2'SLC15A1/2'

diY/tripep>des+H+(

hydrolysis(

AA'

SLC16A10'and'43A2'

Kidney'amino'acid'transporters'&'amino'acidurias'

Tmem27'in(kidney(and(ACE2(in(sm.(intesIne'

Lysinuric''protein''intolerance'

'Dicarboxylic''''aminoaciduria''

Iminoglycinuria'

Hartnup''disorder'

Cys>nuria'

13/09/2014(

6(


21(

Na+'


Na+(symporter'

anIporter'

uniporter'




'


'





AA((

cys>ne,AA+' AA((

an>porter'

AA' AA'

uniporters'for(essenIal(AA´s(

AA' AA'

an>porter'

AA+Na+( AA((

symporter'

Hartnup''disorder'

B0AT1(SLC6A19'

Defects(of(SLC6A19/B0AT1(cause(Hartnup(disorder(

Baron'et'al.'Lancet,'1956' Kleta'et'al.'Nature,'2004'

B0AT1(

Small(intesIne(((human(biopsy)(

B0AT1(

Kidney(proximal(tubule((mouse)(

B0AT1((Slc6a19)(is(the(major(luminal(neutral(amino(acid(transporter(of(intesIne(and(kidney((

13/09/2014(

7(

Camargo(et(al.,(Pflugers(Arch(2005(

2Welectrode(voltage(clamp((in(Xenopus(oocytes((expressing(mouse(B0AT1(

10'mM'AA'''1'mM'AA'

broad'(B)'selec>vity'for'neutral'AAs'(0)'

LWleucine(up

take(

Na+Y'(but'not'ClYY)''dependent''

Amino(acid(transporter(B0AT1(expressed(in(Xenopus(oocytes( QuesIon(#3 ( ( (((((((Francois(Verrey(

Hartnup(disorder(leads(potenIally(to:(

A.  vitamin(B1(deficiency((beri(beri)(B.  vitamin(B3(deficiency((pellagra)(C.  vitamin(C(deficiency((scurvy)(D.  vitamin(D(deficiency((ricket)((

((((

In(kidney,(B0AT1(interacts(with(TMEM27/collectrin(for(expession(

Danilczyk(et(al.,(Nature,(2006((

Western:'collectrin'

B0AT1((intesIne(

B0AT1((kidney(

Ace2((genotype(

β-acIn(

β-acIn(

In(small'intes>ne,(B0AT1(interacts(with(ACE2,(not(with(collectrin(

Camargo,(Singer(et(al.,(Gastroenterology,(2009(

B0AT1(needs(to(be(associated(with(TMEM27/collectrin(or(ACE2(to(be(expressed(at(the(luminal(cell(surface(

13/09/2014(

8(

B0AT1(

B0AT1(

Baron'et'al.'Lancet,'1956'Levy,'Metabolic'&'Molecular'Basis'of'Inherited'Disease,'2001''

impaired'AA'reabsorp>on:'neutral'aminoaciduria'

Oral'load'Tyr,'Phe,'Met,'Leu,'Trp'

'''

impaired'absorp>on'

Steakins'&'Ersser,'Arch.'Dis.'Childh.'1967'Navab'&'Asatoor,'Gut,'1970''etc.'

In(some(cases:(• (PellagraWlike(photosensiIve(rash(• (Ataxia(• (Psychiatric(symptoms((((treatment:'niacin)'

but(absorpIon(of((pepIdes((via(PepT1(

but(normal(absorpIon((of(diW(and(triWpepIdes(luminal'

AA B0AT1' AA

Na+(SLC6A19'

AAYAA'AAYAAYAA'

H+(SLC15A1'PEPT1'

DysfuncIon(of(B0AT1(causes(Hartnup(disorder(

Hatnup(syndrome(may(include(pellagraYlike'symptoms'such(as(photosensiIve(skin(rash((and(ataxia.(These(symptoms(can(be(treated(efficiently(with(niacin(supplementaIon.(

I'

ACE2'is(required(for(Na+Ydependent'transport'(of'B0AT1'and(SIT1(substrate(amino(acids(but((not(for(SGLT1'substrate(glucose(in(small(intesIne(

Camargo(et(et(al.,(Gastroenterology(2009(

1(mM(

Singer(et(al(2012(

0.2(mM(1(mM(1(mM( 5(mM(

No'B0AT1'ac>vity'in'small'intes>ne'of'Ace2'null'mouse'

Uptake'in'small,intes9ne'rings'

Singer(et(al.,(2012(

Ace2(null(mice:(decreased(weight'gain'awer(weaning(

weaning(

B0AT1(null(mice(

weaning(

Broer(et(al.,(2011(

Plasma'amino'acids'in(Ace2'null(mice:((decreased(levels(of(B0AT1'substrates,(in(parIcular(of(LYtryptophan'

Singer(et(al.,(2012(

at(8(weeks(

Tryptophan'deficiency'→'pellagra.((W(Treatment:(niacin((=((nicoInic(acid((N),(nicoInamide((NAM)(or(NAM(riboside((NR()((W(Niacin(serves(as(NAD+(precursor.(

13/09/2014(

9(

.(

ACE2'links'amino'acid''malnutri>on'to'microbial'ecology'and'intes>nal'inflamma>on'

Decreased'LYtryptophan'absorp>on''in'Ace2'null'mice'induces'microbiota'change'and'coli>s'

Hashimoto(et(al.,(2012(

ACE2'null'mice'display'↑'inflamma9on,'weight'loss'and'diarrhoea'upon'dextran''sodium'sulphate'(DSS)'treatment.'(((('

DSSYinduced'coli>s'in'Ace3/y'mice'is'prevented'with''nico>namide'or'tryptophan'dipep>des.'

Trp+((=(GlyWTrp(dipepIde(

Hashimoto(et(al.,(2012(

phosphorylated(S6((brown)((in(mice(fed(a(Trp+(or(normal((chow(diet((Control).(((HE(staining:(DSSWtreated((wildWtype(mice(+/W(RAPA(

3'Ace2'null'mice'small'intes>ne:'↓'mTOR'pathway'ac>va>on'Y'mTOR'inhibi>on'by'RAPA'increases'DSSYinduced'coli>s(

Ace2'defect'(a)''and'tryptophanYfree'diet'(b)''decrease'small'intes>nal'an>microbial'pep>de'expression'

The(lack(of(intesInal(B0AT1(leads(in(mice(under(low(protein(diet(to(LWtryptophan(depleIon(in(many(Issues((as(presumably(in(humans)((Tryptophan(depleIon(impacts(in(mice(on(the(producIon(of(intesInal(anImicrobial(pepIdes(and(thereby(on(microbiota(and(favors(coliIs((diarrhea(is(also(a(symptom(of(pellagra).((

Ace2'null'mice'as'model'of''intes>nal''Hartnup'disorder'

13/09/2014(

10(



'


'


•(( ('prerenal'(+(renal(amino((( (aciduria(in(mouse(model(( ( ((((SLC16A10(=(TAT1)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ('''basolateral'facilitated'diffusion'pathway'(uniporter)(

37(

Na+'


Na+(symporter'

anIporter'

uniporter'


Nijmegen(12.09.2014(Amino(acid(exchangers((anIporters)(in(transepithelial(transport( Identical axial distribution of b0,+AT, y+LAT1 and LAT2 in(kidney(proximal(tubule

Bauch et al., J. Biol. Chem. 2003

AA + Na+

AA–

AA

AA

AA

AA AA

AA+

AA

Mastroberardino et al, Nature 395:288, 1998"

heavy chain type II membrane"glycoprotein"4F2hc (CD98)or rBAT"

CSSC

COOH

COOH

NH2 NH2

1 2 3 5 7 9 11 12 10 864

C

cytosol

hc 2

light chain catalytic"polytopic"membrane protein"

Heterodimeric amino acid transporters are obligatory exchangers

out

in

out

in

out

in

Meier et al., EMBO J 2002

b0,+, LAT2 and y+LAT1 are asymmetrical obligatory exchangers (antiporters) they do not perform net amino acid transport but can extend the substrate selectivity of parallel transporters functioning directionally

b0,+AT- rBAT

LAT2- 4F2hc

y+LAT1- 4F2hc


AA((

cys>ne,AA+' AA((an>porter'

AA' AA'


AA' AA'

an>porter'

AA+Na+( AA((

symporter'

B0AT1(SLC6A19'

Cys>nuria'

DysfuncIon(of(b0,+ATY'rBAT(causes(Cys>nuria(

13/09/2014(

11(

rBAT'

b0,+AT'

Heterodimeric amino acid transporter b0,+AT-rBAT (SLC7A9-SLC3A1)

Structure(of(b0,+AT(inferred(from(AdiC(structure(and(of(rBAT(predicted(from(4F2hc(and(prokaryoIc(glycoside(hydrolases.((Stars(indicate(disulfide(bridge(and(numbers(cysInuria(mutaIons.(Chillarón'et'al.,'Nature'Reviews'Nephrology'2010(

QuesIon(#2 ( ( (((((((Francois(Verrey(

CysInuria(is(characterized(by:(

A.  a(homozygos(defect(of(the(distal(nephron(cysIne(transporter(B.  the(urinary(loss(of(caIonic(amino(acids((e.g.(LWlysine,(LW

arginine)(and(homodimers(of(LWcystein(C.  the(urinary(loss(of(LWcystein(D.  the(urinary(loss(of(neutral(amino(acids(((

Defect in either b0,+AT or rBAT (SLC7A9 or SLC3A1) lead to cystinuria

Chillarón'et'al.,'Nature'Reviews'Nephrology'2010(

Cys>nuria''(type'A,(mutaIon(in(SLC3A1(/(rBAT(recessive(

type'B,(mutaIon(in(SLC7A9(/(b0,+AT'autosomal(dominant(with(incomplete((penetrance((

L-cystine L-arginine L-lysine L-ornithine

Cystinuria: ↑↑↑ urinary

H'

H'

ox(red(

2(LWcysteins((((((((1(LWcysIne(

b0,+AT'

cytosol(

tubular(lumen(

rBAT'

cystine = disulfide-bonded dimer of L-cysteine U

rinary cystine Crystals

cystine = disulfide-bonded dimer of L-cysteine

Clinical(problem(of(cysInuria:(cysIne(lithiasis(

13/09/2014(

12(

Chillarón,(J.(et'al.'Nat.'Rev.'Nephrol.'6,'424–434'(2010)(

form(heterodimers(that(are(more(soluble(than(cysIne(

H'

H'

ox(red(

PrevenIon(of(cysIne(lithiasis(in(cysInuria(paIents(


AA((


AA' AA'


AA' AA'

an>porter'

AA+Na+( AA((

symporter'

B0AT1(SLC6A19'

Lysinuric''protein''intolerance'

DysfuncIon(of(y+LAT1Y'4F2hc(causes(Lysinuric'protein'intolerance(

Strong(expression(of((y+Lat1(in(kidney(proximal(tubule(and(small(intesIne((+(lower(levels(in(most((nonWepithelial(Issues(

small(intesIne((jejunum)(

Basolateral(obligatory(exchanger((amino(acid(anIporter)(y+LAT1(is(expressed(also(in(many(other(Issues(

4F2( y+LAT1(

mouse(proximal(tubule(

Pfeiffer(et(al.,(1999,(Dave(et(al.,(2004,(Bauch(et(al.,(2003(

Func>on'of'y+Lat1'(Efflux(of(caIonic(AAs((LWLys,(LWArg,LWOrn)(in((exchange(for(extracellular(large(neutral(AAs(+(Na+(

Lysinuric(protein(intolerance:(defect(of(y+LAT1((SLC7A7)(

Other'symptoms:'W(failure(to(thrive,(intellectual(impairment,(episodes(of(diarrhea(and(hyperammonemia,(((coinciding(with(increased(protein(intake((hence(the(name)((interrupIon(of(urea(cycle).((W(intersIIal(pneumonia(in(form(of(alveolar(proteinosis,(hepatomegaly(and(liver(cirrhosis,((((osteoporosis,(and(bone(marrow(involvement.((W(Renal(insufficiency(can(develop(due(to(glomerulonephriIs.(

Aminoaciduria:'ElevaIons(of(urinary(dibasic(amino(acids,(lysine,(arginine(and(ornithine((more(pronounced(than(in(cysInuria;(urinary(cysIne(levels(almost(normal(Plasma:'In(contrast(to(cysInuria,(plasma(levels(of(lysine,(arginine,(and(ornithine(tend(to(be(below(the(norm.(

Treatment:(symptomaIc(and(consists(of(protein(restricIon,(as(in(other(urea(cycle(disorders,(and(in(this(case(in(supplementaIon(of(citrulline.(

13/09/2014(

13(



'


'



49(

Na+'


Na+(symporter'

anIporter'

uniporter'




AA((

AA' AA'


AA' AA'

an>porter'

tubulus'lumen'


AA+Na+( AA((

symporter'

B0AT1(SLC6A19'


AA+Na+( AA((

symporter'

B0AT1(SLC6A19'

DysfuncIon(of(uniporter(TAT1'(Slc16a10)((knock(out(mouse)(

Trp

Phe

Tyr

DO

PA Thr

Leu

His

Glu

Arg Gln Ala Val

Lys

Ser

Pro

Dop

amin

e-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

upta

ke r

elat

ive

to L

-Trp

Trp

Phe

Tyr

DO

PA Thr

Leu

His

Glu

Arg Gln Ala Va

lLy

sSe

rPr

oD

opam

ine-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

nd ndndefflu

x re

lativ

e to

L-T

rp

Ramadan(et(al.,(2006(

Example(of(*facilitated(diffusion(pathway((uniporter)(involved(in(transepithelial(transport:(((TAT1((MCT10,(SLC16A10)(

Expression(of(TAT1(in(mice( Expression(in(Xenopus(oocytes(

TAT1'

TAT1'

TAT1'

B0AT1'

TAT1'+'pep>de'

4F2hc'

Influx(

Efflux(

*uniporters(for(essenIal(amino(acids:(TAT1(for(araomaIc,(LAT4(for(βWbranched(AAs(

'Tryptophan'biosensor:'Live'Cell'Imaging'FRET'

Leica(Widefield(Inverted(Fluorescence(Microscope(

wt COS cells TAT1 expressing COS cells

13/09/2014(

14(


SLC6A19'/'18'AA0+Na+(

AA0' AA0'

AA0'

tubulus'lumen'

SLC7A8'

B0AT1(/(B0AT3'

Coll'

4F2hc'

TAT1(and(LAT4?(

LAT2'

SLC3A2'

SLC16A10'and(?((AA0'

AA0'

Hypothesis:(basolateral(efflux(of(amino(acids(depends(on(both(uniporters(for(essenIal(amino(acids((i.e.(TAT1)(and(anIporters((i.e.(LAT2)(that(transport(also(nonWessenIal(amino(acids((i.e.(Gln)(

Efflux'of'glutamine'from'oocytes'60(min(w/o(extracellular(amino(acid((

0 60 700

10

20

30

40

50

60

NI4F2LAT24F2LAT2 mTAT1mTAT1

time (minutes)

% g

luta

min

e ef

flux

Efflux(of(LWglutamine(from((Xenopus(oocytes(depends((on(coWexpression(of(TAT1(

Tat1(null(mouse(model:(impact(of(Tat1(deleIon(on(plasma'amino'acids:(increased'aroma9c'AA'concentra9on'independent'of'protein'diet'

aromaIc((AAs(

βWbranched((AAs(

acidic(AAs(

Effect(of(the(diet(on(wt(

amino(acid(concentraIon(in(wild(type(mice((same(diet)(

Mario~a(et(al.,(unpublished(

Lack(of(TAT1((Slc16a10)(prevents(liver(from(controlling(aromaIc(amino(acid(level( Tat1'deleIon(diminishes(basolateral(efflux(of(aromaIc(amino(acids(from(transporIng(epithelia(of(kidney(and(gut('

accumulaIon(of(SPECT(tracer((2W125/123IWLWPhe(in(mouse(kidneys(

accumulaIon(of(LWPhe(in(everted((

small(gut(sacs(

Mario~a(et(al.,(unpublished(

13/09/2014(

15(

'Impact(of'Tat1'deleIon(on(urinary(amino(acid(excreIon:(under'high'protein'diet,'urinary'excre9on'of'Lat2,4F2hc'substrate'amino'acids'

is'increased,'although'their'plasma'concentra9on'is'normal'

Mario~a(et(al.,(unpublished( extracellular'space'tubulus'lumen'


secondary'/'ter>ary'ac>ve'transporters'1'+''obligatory'exchanger'2'

AA' AA'facilitated'diffusion'transporters'3'+'obligatory'exchangers'2'

AA'

AA((

cys>ne,AA+' AA((

an>porter'2'

AA' AA'

uniporters'3'for(essenIal(AA´s(

AA' AA'

an>porter'2'

AA+Na+( AA((

symporters'1'

Gln' Gln'

Gln' Gln'



secondary'/'ter>ary'ac>ve'transporters'1'+''obligatory'exchanger'2'

AA' AA'facilitated'diffusion'transporters'3'+'obligatory'exchangers'2'

AA'

AA((

cys>ne,AA+' AA((

an>porter'2'

AA' AA'

uniporters'3'for(essenIal(AA´s(

AA' AA'

an>porter'2'

AA+Na+( AA((

symporters'1'

Efflux(of(anIporter(Lat2W4F2hc(substrates((is(reduced(by(lack(of(exchange(substrate(recycling(via(TAT1(

Gln' Gln'

Gln' Gln'

QuesIon(#4 ( ( (((((((Francois(Verrey(

Lysinuric(protein(intolerance(is(due(to(a(defect(of:(

A.  kidney(urea(excreIon(B.  the(basolateral(amino(acid(anIporter(of(proximal(tubule(cells(

SLC7A7((y+LAT1)(C.  the(basolateral(associated(protein(of(y+LAT1(called(4F2hc(

(SLC3A2)(D.  the(luminal(transporter(for(caIonic(amino(acids(SLC7A9(

(b0,+AT)(((

((

13/09/2014(

16(


61(

Na+'


Na+(symporter'

anIporter'

uniporter'




'


'



AA

PT/S

I lum

en

extr

acel

lula

r spa

ce AA AA

kidney PT and small intestine epithelia

AA

Amino acid transport across

brai

n in

ters

t.

Simone Camargo

Liviu Vanoaica

Elena Dolgodilina Victoria Makrides Emilia Boiadjieva

Luminal uptake Blood-brain-barrier

Basolateral efflux

Collaborators:(J.'Penninger,'Wien'R.'Kleta,'London'

Lalita Oparija

Samyuktha Pillai

Julia Jando

blood-brain-barrier endothelium

ContribuIons(from(former(coworkers:(Luca'Mastroberardino'Rahel'Pfeiffer'Chris9on'Meier'Mital'Dave'Elisa'Romeo'Luca'MarioEa'Tamara'Ramadan'Alok'Behera'

2014.09.12(FV(Nijmegen( 63(

2014(

h~p://slc.bioparadigms.org/(

Kidney'amino'acid'transport(((2.''Specific'aminoacidurias''(• ( (Hartnup'disorder' ( ( ( ( ( ((((SLC6A19(=(B0AT1)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (((luminal'Na+'co,transporter'(symporter)'

( ( ( ( ( ( ( ( ( ( ( ( ('• ( (Cys>nuria( ( ( ( ( ( ( ( ( ( ((((SLC7A9/SLC3A1(=(b0,+AT/rBAT)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (((luminal'obligatory'exchanger'(an9porter)'

• ( (Lysinuric'protein'intolerance''(SLC7A7/SLC3A2(=(y+LAT1/4F2hc)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (((basolateral'obligatory'exchanger'(an9porter)'

( (Iminoglycinuria' ( ( ( ( ( ( ((((SLC6A19(+(SLC36A2(=(SIT1(+(PAT2)(( (Dicarboxylic'aminoaciduria' ((((SLC1A1(=(EAAT3)(

(( ('prerenal'(amino(aciduria((( ((in(mouse(knockWout(model( ((((SLC16A10(=(TAT1)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ('''basolateral'facilitated'diffusion'pathway'(uniporter)(

64(2014.09.12(FV(Nijmegen(

Na+'


Na+(symporter'

anIporter'

uniporter'

13/09/2014(

17(

EAAs'

TAT1'(SLC16A10)(or'LAT3/4'(SLC43A1/2)(

uniporter'facilitated((diffusion((pathway(

NEAAs'Na+,' EAAs'

LAT1/2'(SLC7A5/8)(

+''4F2hc'(SLC3A2)'

'

SNAT1/2/4'(SLC38A1/2/4)(

symporter'cotransporter(

an>porter'exchanger(

EAAs'NEAAs'

ClY,Na+,'

ATB0+''

(SLC6A14)''


EAAs'NEAAs'

Na+,'

B0AT1''(SLC6A19)'

+''+ACE2(or(TMEM27(


Amino(acid(transporters: transport types & essential amino acid import

Rudnick(et(al.,(2014(

-25

0

25

50100

350

600

850

1100 alone+hColl+mACE2

WT D173N P265L

pmol

/hou

r/oo

cyte

(NI s

ubtr

acte

d)

-90

-50

-10-10

0

10

I L-Ile(nA)

Camargo,(Singer,(et(al.,(Gastroenterology(2009(

Expression(in((Xenopus'laevis''oocytes(

Associa>on'with'ACE2''(or'TMEM27)'increases'

B0AT1'surface'expression'

Increased'surface'expression'&'transport'func>on'with(TMEM27/hColl(or(ACE2(Hartnup(mutants(mostly(“dead”,(some lack activation by associated protein (not shown) Differen>al'amino'acid'transport'ac>va>on'by(kidney(associated(protein(Tmem27/collectrin'or'small(intesIne(associated(protein(ACE2'

Associated'proteins'and'Hartnup'muta>ons'tested'in'Xenopus'laevis'oocytes'

Mastroberardino et al, Nature, 1998"

Heterodimeric(amino(acid(transporters:((glycoprotein(heavy((chain(is(required(for(catalyIc(light(chain(surface(expression(

4F2

light(chains:( (A:(xLAT1((E:(hLAT1(((S:(SPRM1((schistosoma'mansoni')(

Francois(Verrey,(Zurich( Kidney'amino'acid'transport ... · 13/09/2014(5(tubulus'lumen'...

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