Sequential Amino Acid Exchange across b0,+-like System in Chicken Brush Border Jejunum

Abstract

In the small intestine, cationic amino acids are transported by y(+)-like and b(0,+)-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b(0,+)-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b(0,+)-like system functions for L-arginine as an obligatory exchanger since its transport capacity increases 100-200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Argin efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Argout is higher than that of Argin, suggesting a possible order of binding (Argout first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed.

Effect of pH on l- and d-methionine uptake across the apical membrane of Caco-2 cells

“…In the present study, the inhibition obtained with NEM confirms the participation of system y ϩ on L-Met uptake in the absence of Na ϩ . System b 0,ϩ is a tertiary transport system that exchanges extracellular cationic amino acids and L-cystine for intracellular neutral amino acids in a Na ϩ -independent way (30), with a higher binding affinity for the extracellular substrate (48). Finally, system L is a Na ϩ -independent transporter that preferentially recognizes large neutral amino acids with branched or aromatic side chains, as well as BCH (9).…”

Functional and Structural Characterization of the First Prokaryotic Member of the L-Amino Acid Transporter (LAT) Family

“…Despite the important roles attributed to HATs, only a few studies have addressed the structure-function relationships of these transporters: (i) most HATs are obligate antiporters with a 1:1 stoichiometry (14), and a sequential mode of exchange has been proposed for system b 0,ϩ (15); (ii) light subunits appear to be sufficient for transport activity, as demonstrated for b 0,ϩ AT (16); (iii) using xCT as a model for the light subunits, a membrane topology with 12 transmembrane segments and with a re-entrant loop between transmembrane segments 2 and 3 has been reported (17); and (iv) the xCT residues His 110 and Cys 327 have been shown to be crucial for function (17,18), whereas the cystinuria-specific mutation A354T inactivates b 0,ϩ AT (16). Similarly, structure-function studies on the APC superfamily as a whole have been very limited and primarily related to membrane topology studies and the identification of relevant residues for substrate interaction (19 -21).…”

The Structural and Functional Units of Heteromeric Amino Acid Transporters

“…It has been shown that system b 0,ϩ from the chicken small intestine shows a sequential exchange mechanism compatible with the formation of a ternary complex (46). If this applies to the expressed b 0,ϩ AT-rBAT complex, then export and import pathways should co-exist simultaneously in the proposed functional unit (i.e.…”