Mediated Absorption of Free Amino Acids

Molecular and functional properties of proteins capable of biochemically defined free amino acid transport 'system' activities

As indicated in Fig. 3.1, the mediated absorption of free amino acids across both apical and basolateral membranes is critical to the assimilation of luminal proteins. Remarkably, within the last 12 years, cDNA have been generated that encode proteins for six anionic, four cationic, 11 neutral, and five neutral and cationic free amino acid transporters. Except for exclusive proline and hydroxyproline transport by the IMINO system, at least one of these 26 proteins account for each of the major amino acid transport activities expressed by non-embryonic tissues, which have been biochemically defined over the previous 35 years. To facilitate a working knowledge base for understanding which proteins perform which transport activities, the biochemical and molecular properties of free amino acid transport systems and proteins have been collated in Table 3.5. An important understanding from this research is that the activities of seven biochemically defined transport systems are actually performed by more than one amino acid transporter. In addition, a subset of transporters actually function as heterodimer units, in conjunction with one of two glyco-

Free amino acid transporters expressed by intestinal epithelia

To facilitate an abbreviated discussion of which and how these transporters function to support absorption of amino acids across the gastrointestinal epithelium, the site of expression for the functional activities, mRNA, and protein (when reportai) of free amino acid transporters expressed by gastrointestinal epithelia are collated in Table 3.6. Although few of the transporter proteins have actually been detected in apical or basolateral membranes, the matching of membrane-defined transport activity with detection of mRNA expression (Table 3.5) suggests that at least one molecularly defined transporter has been identified for all biochemically defined transport activities reported for apical and basolateral membranes. In terms of comparing specific substrates with the reported function of cloned transporters (Table 3.5) specific transporters are identified for apical membrane absorption of anionic l-glutamate, l-aspar-tate, d-aspartate by EAAT2 and/or EAAT3; cationic l-lysine, l-arginine, l-histidine and l-ornithine by CAT1 and l-arginine, l-lysine and l-histidine by b°-+AT; and neutral amino acids by ASCT2 and b°-+AT. Interestingly, 4F2-lc6 also encodes a protein capable of b0,+ activity, but which associates with 4F2-hc, not RBAT. As with b°-+AT, however, 4F2-lc6 transports cystine and neutral and cationic amino acids in a Na+-independent manner. In terms of both cationic and neutral amino acid transport, »/stem Ba+ activity has long been identified with the apical membrane of intestinal epithelia. However, the

Table 3.5. Molecular and biochemical properties of free «-amino acid transport proteins8.

CLONE

Alternate names

Genbank Acc. no.

Length

Transport system

Substrate specificity130

Anionic EAAT1

EAAT2

EAAT3

EAAT4

EAAT5

xCTef

Cationic CAT1

CAT2

CAT2a

CAT3

Neutral

GlnT

(neuronal A)

ATA2 (classic A)

ATA3

(hepatic A)

GluT, GLAST X63744

G LT, GLAST2, X67857 GLTR

EAAC1

4F2-IC4

ecoR

CAT2ß CAT2a

ATA1

SA1 SAT2

L12411

U18244

U76362

AB022345 AB026891 AF252872

M26687

NM003046

L03290 U70859

AF249673 AF273024 AF173682 AF295535

543 573

523-525

622-629

657-658 657-659 619

AF075704 485

Lys, Arg; Orn, His (when charged) Lys, Arg; Lys, Arg; Orn Arg

Gin, Asn, His, Ala, Met, Ser, Gly; MeAlB, Pro MeAlB, Ala, Gly, Ser, Pro, Met, His, Asn, Gin

Ala, Gly, Ser, Cys, Asn, Thr; Pro, Met, Gin, His; MeAlB, Lys Lys, Arg

Substrate affinityd

Co-substrate coupling

Source

in' out'

Storck et al. (1992)

OH-/HCO-3out

|jlM

Na+in, K+out,

Pines etal. (1992)

0H-/HC03-Dut

in' out'

Kanai and Hediger (1992)

0H-/HC03-Dut

in' out'

Fairman etal. (1995)

0H-/HC03-0ut

(jlM

Na+in, K+out,

Arriza etal. (1997)

0H-/HC03-0ut

(jlM

AA1CssC;AA2G|u

Sato et at. (1999)

Bridges etal. (2001)

|jlM

none

Kim etal. (1991)

Wang etal. (1991)

|jlM

none

Gloss etal. (1993a)

mM

none

Gloss etal. (1993b)

|jlM

none

Hosokawa et at. (1997)

|jlM

Na+,n

Varoqui et at. (2000)

(jlM

Na+in

Sugawara et at. (2000a)

Reimer et al. (2000)

Yao etal. (2000)

mM

Na+in

Hatanaka etal. (2000)

|jlM

none

Sugawara et al. (2000b)

ASCT1 SATT

L14595

ASCT2 ATB" D-85044 553

U53347

Asc-1e1 AB026688 530 asc

LAT1ef 4F2-IC1 A B015432 512 L

AF104032 507

LAT2ef 4F2-IC5 AF171668 531

AF171669 535 AF170106 535

SN1 NM006841 504

NAT AF159856 505

AF244548 504

SN2 AF276870 472

neutral, except for |jlM

Gin at pH 7.5; plus anionic at pH 5.5

Trp, Gly

Ala, Gly, Ser, Thr, Cys; Val, Met, lie, Leu, His D-AA:

Ala, Ser, p-Ala, AIB, Ala-methyl,Cys, Asn, Leu, lie, Val, His; Gin, Met, Phe l-AA:

Gin, His

NS+irMn, Na+jnAAout

Na+ir

AA1 in;AA2

AA1 ]n;AA2

Arriza etal. (1993) Zerangue and Kavanaugh (1996) Utsunomiya-Tate et al. (1996) Kekuda etal. (1996)

Fukasawa et at. (2000)

Rossier etal. (1999)

Thr, Phe, Trp; Ser, Gin, Leu, Ala, Cys, BCH Gin, His Gin, His; Ala Gin, His; Asn, Ala

(jlM

mM mM mM

2Na+inAA0Ut„

Chaudhry etal. (1999) Gu etal. (2000) Fei et al. (2000)

Continued

Table 3.5. Continued.

Alternate

Genbank

Transport

Substrate

Substrate

Co-substrate

CLONE

names

Acc. no.

Length

system

specificitybc

affinity0

coupling

Source

TAT1

AB047324

Tyr, Trp, Phe, l-Dopa, 3-0-methyl-Dopa d-AA: Trp, Phe

mM

none

Kim et ai. (2001)

Neutral and cationic

ATB0+

AF151978

642

B°'+

lie, Leu, Trp, Met, Val,

NR

2Na+in, 2CI~in

Sloan and Mager (1999)

Ser; His, Tyr, Ala, Lys,

Arg, Cys, Gly; Asn, Thr,

Gin; Pro

y+LAT1ef

AmAT-L-lc

AF092032 AJ130718

511

y+L

Leu; Arg, Lys, Gin, His

(jlM

AA1 in;AA2out Na+in or H+

Torrents etal. (1998) Pfeiffer et ai. (1999a)

4F2-IC2

R82979

Lys, Arg, Orn; Met, Leu, His

(for neutral)

Kanai et ai. (2000)

y+LAT2ef

4F2-IC3

D87432

Arg, Lys, Gin, His, Met;

(jlM

AA1 jn;Argout Na+in (for neutral)

Torrents etal. (1998) Broer etal. (2000)

BAT 1eg

b°'+AT

AB029559 AJ24Q198 Aj249199

467

b°'+

Arg, Leu, Lys, Phe, Tyr; CssC, lie, Val, Trp, His, Ala; Met, Gin, Asn, Thr, Cys, Ser

(jlM

neutral AA, dibasic A A exchange

Chairoungdua et al. (1999) Pfeiffer etal. (1999b)

4F2-lc6ef

AF155119

467

b°'+

Leu, Trp, Phe, Met, Ala, Ser, Cys, Thr, Gin, Asn; Gly, CssC, BCH

(jlM

neutral AA, dibasic A A exchange

Rajan etal. (1999)

aDoes not include members of the BGT, GAT, GLYT, TAUT or PRO neurotransmitter transporter families. b';' denotes physiologically significant differences in degree of substrate affinity.

cCssC, l-cystine; Orn, ornithine; MeAIB, 2-methylaminoisobutyrate; AIB, a-aminoisobutyric acid; BCH, 2-aminobiyclo(2,2,1)heptane-2-carboxylic acid; Dopa, l-dihydroxyphenylalanine.

dWhen possible, values are data from overexpression of cDN A by mammalian cells, rather than by Xenopus oocytes. eMember of the glycoprotein-associated amino acid transporter family. 'Associates with 4F2hc glycoprotein. Associates with rBAT glycoprotein.

Table 3.6. Expression of free «-amino acid transporter activities, mRNA and/or protein by mammalian gastrointestinal epithelia.

Transport

Location of

Specific

Epitheliabc

system

activity8

transporter

evaluated

mRNA

Proteind

Source

Anionic

X~AG

Ap

EAAT2

R, 0, D, J, I, Ce, Co

X

CMV

Howell etal. (2001)

EAAT3

R, 0, D, J, I, Ce, Co

X

CMV

Howell etal. (2001)

V

NR

xCT

intestine

X

Bassi etal. (2001)

Cationic

y+

Ap, Bl

CAT1

Small intestine

X

Kim etal. (1991); Wang etal. (1991)

Neutral

A

Bl

ATA2

Small intestine

X

Sugawara et al. (2000a)

Ap

ASCT2

Intestine

X

Kekuda etal. (1996,1997)

asc

Bl

Asc-1

Small intestine

X

Fukasawa et al. (2000)

L

Bl

LAT2

Small intestine

X

Bl

Rossier et al. (1999)

N

Ap or Bl

SN2

Small intestine

X

Nakanishi et al. (2001)

T

Bl

TAT1

J, I, Co

X

Bl

Kim et al. (2001)

IMINO

Ap

Unknown

Neutral and cationic

B°'+

Ap

ATB0,+

Distal I, Ce, Co

X

Hatanaka etal. (2001)

y+L

Bl

y+LAT1

Small intestine

X

Torrents etal. (1998); Pfeiffer etal. (1999a)

Bl

y+LAT2

Small intestine

X

Broer etal. (2000)

b°'+

Ap

BAT1

J, I

X

Chairoungdua etal. (1999)

Ap

b°-+AT

Intestine

X

Pfeiffer etal. (1999b)

Ap

4F2-IC6

Small intestine

X

Rajan et al. (1999)

aAs reviewed by Ganapathy etal. (1994), Mailliard etal. (1995), Palacin etal. (1998), Wagner etal. (2001), Bode (2001) and/or Matthews and Anderson (2002). Ap, apical; Bl, basolateral membrane; NR, not reported. bR, rumen; O, omasum; D, duodenum; J, jejunum; I, ileum; Co, colon; Ce, caecum. cWhen known, expression is reported for farm animal species.

dCMV, crude membrane vesicles isolated from homogenates of scraped epithelial tissues.

limited mRNA tissue distribution profiles for ATB°-+, which encodes a protein capable of B°-+ activity, show« no expression by the duodenum or jejunum and only weak expression by the distal ileum. In contrast, caecal and colonic expression is high. Therefore, it remains to be determined whether ATB°-+ function contributes significantly to small intestinal absorption of amino acids, or whether another, as yet unidentified, ATB°-+ isoform is responsible for system B°-+ activity.

In terms of basolateral transport capacity, cationic amino acids are unilaterally transported by CAT1 uniport in counterexchange for neutral amino acids by y+LATl. In addition, given its intracellular binding preference for l-arginine and the high blood concentrations of l-glutamine, the predominant function of y+LAT2 is to absorb l-glutamine into enter ocytes in exchange for l-arginine. Accordingly, the function of y+LAT2 may well be the mechanical coupling of the high intestinal l-glutamine uptake from, and l-arginine export into, splanchnic blood (Wu, 1998). In contrast, the presence of a basolateral anionic amino acid transporter has yet to be described (although xCT mRNA has been detected by RT-PCR, Bassi et ai, 2001), and may help explain the low arterial uptake of l-glutamate and l-aspartate by small intestinal epithelia. Neutral amino acid transport across the basolateral membrane of enterocytes appears to be achieved by a combination of activities by Na+-dependent (ATA2) and Na+-independent (TAT1) uniporters and ion-independent amino acid exchangers (Asc-1, LAT2, y+LATl, y+LAT2). In addition, SN2 may contribute significantly to l-histidine, l-serine, l-asparagine and L-glutamine absorption by coupled Na+/H+ counter-exchange (Bode, 2001).

Asymmetrical expression, yet coordinated function, of amino acid transporters by polarized intestinal epithelia

In terms of how amino acid flux is mediated across enterocytes, all of the apical transporters are ion-dependent and capable of concentrative transport, except for the two system b0,+ transporters. Consequently, the molar ratio of cationic and neutral amino acids initially absorbed from the lumen by concentrative transporters can be modulated by ATBa+ activity. Of the basolateral transporters, only ATA2 and TAT1 are uniporters. ATA2 (system A activity) activity is Na+-dependent, capable of concentrative transport, and functions to transport amino acids into the cell, not into the blood. Conversely, TAT1 is a Na+-independent system that selectively transports aromatic amino acids, down their concentration gradients. In contrast the other basolateral transporters are all exchangers. As a consequence of this differential expression of apical and basolateral transporters, it is likely that the bulk of amino acids that enter the blood through enterocytes is dependent on the concentration of amino acids in the cytosol of enterocytes.

A pertinent question that arises from the combined understandings gained from localization and functional studies with intestinal amino acid transporters is the degree to which the functions of apical (including PepTl) and basolateral amino acid transporters functions are coordinated. A working model that reflects current understanding of differential localization and identity of specific transporters responsible for mediated flux of amino acids across apical and basolateral membranes of enterocytes is presented in Fig. 3.2. How differential localization of transporters results in ion-dependent and substrate exchange-dependent vectoral transport of amino acids through enterocytes likely is similar to that proposed for renal epithelia presented by Palacin et al. (1998) and Verrey et al. (2000). As discussed above, it is generally accepted that the majority of amino acids are absorbed as small peptides, by PepTl activity. After absorption, the peptide-bound amino acids are readily hydrol-ysed to free amino acids by intracellular peptidases (Fig. 3.1). As a consequence of these PepTl-dependent activities, and the activity of the Na+-dependent X AG and B° (and, perhaps, SN2 and B°-+), an elevated supply of free amino acids exists to drive counterexchange across the apical membrane by BAT1 (and 4F2-lc6) and the counterexchange transport by basolateral transporters (Asc-1, LAT2) into the blood. Whereas the putative coordinated function of these differentially expressed transporters on transepithelial amino acid flux

What Foods Have Amino Acids
Fig. 3.2. Working model for the membrane-specific expression of peptide and free amino acid transporters by intestinal epithelial cells. The locations and predominant direction of substrate flow is derived from functional properties listed in Tables 3.4 and 3.5, and/or described in the text.

has not been evaluated, the influence of api-cally expressed PepTl function on apical neutral and cationic amino acid uptake capacity by polarized Caco-2 cells has been (Wenzel et al., 2001). PepTl uptake of several neutral amino acid-containing dipeptides resulted in a 2.5- to 3.5-fold increased uptake of l-arginine by apical ba+AT activity. As this stimulation was dependent on intracellular hydrolysis of transported amino acids, it appears that PepTl activity stimulated l-arginine by supplying requisite amino acids to drive b°-+AT antiport uptake of arginine.

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