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)(
Kidney'amino'acid'transport(
3(
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)(
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'
Kidney'amino'acid'transport(
Types'of'(amino'acid)'transporters'
16(
Na+'
amino'acid''transporters'
Na+(symporter'
anIporter'
uniporter'
1.''Physiology'background'and'overview'of'amino'acid'reabsorp>on'(
Nijmegen(12.09.2014(
• (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'
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'&'amino'acidurias'
Tmem27'in(kidney(and(ACE2(in(sm.(intesIne'
Lysinuric''protein''intolerance'
'Dicarboxylic''''aminoaciduria''
Iminoglycinuria'
Hartnup''disorder'
Cys>nuria'
13/09/2014(
6(
Kidney'amino'acid'transport(
21(
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)(
extracellular'space'tubulus'lumen'
Transepithelial(amino(acid(transport(
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(
Kidney'amino'acid'transport(
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(amino((( (aciduria(in(mouse(model(( ( ((((SLC16A10(=(TAT1)(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ('''basolateral'facilitated'diffusion'pathway'(uniporter)(
37(
Na+'
amino'acid''transporters'
Na+(symporter'
anIporter'
uniporter'
1.''Physiology'background'and'overview'of'amino'acid'reabsorp>on'(
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
extracellular'space'tubulus'lumen'
AA((
cys>ne,AA+' AA((an>porter'
AA' AA'
uniporters'for(essenIal(AA´s(
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(
extracellular'space'tubulus'lumen'
AA((
cys>ne,AA+' AA((an>porter'
AA' AA'
uniporters'for(essenIal(AA´s(
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(
Kidney'amino'acid'transport(
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)(
49(
Na+'
amino'acid''transporters'
Na+(symporter'
anIporter'
uniporter'
1.''Physiology'background'and'overview'of'amino'acid'reabsorp>on'(
Nijmegen(12.09.2014(
extracellular'space'tubulus'lumen'
AA((
AA' AA'
uniporters'for(essenIal(AA´s(
AA' AA'
an>porter'
tubulus'lumen'
cys>ne,AA+' AA((an>porter'
AA+Na+( AA((
symporter'
B0AT1(SLC6A19'
cys>ne,AA+' AA((an>porter'
AA+Na+( AA((
symporter'
B0AT1(SLC6A19'
DysfuncIon(of(uniporter(TAT1'(Slc16a10)((knock(out(mouse)(
Trp
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DO
PA Thr
Leu
His
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Lys
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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(
extracellular'space'
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'
Transepithelial(amino(acid(transport(
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'
extracellular'space'tubulus'lumen'
Transepithelial(amino(acid(transport(
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(
Kidney'amino'acid'transport(
61(
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)(
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+'
amino'acid''transporters'
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)''
symporter'cotransporter(
EAAs'NEAAs'
Na+,'
B0AT1''(SLC6A19)'
+''+ACE2(or(TMEM27(
symporter'cotransporter(
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')(