The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport.
The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na+ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na+ cotransport occurred with a 1:1 stoichiometry. The concentration of Na+ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na+ cotransport, together with previous report on the glutamine(ex)/glutamine(in) pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na+. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na+.
Oppedisano
Francesca
F
Department of Cell Biology, University of Calabria, Via P. Bucci 4c 87036 Arcavacata di Rende, Italy.
Pochini
Lorena
L
Galluccio
Michele
M
Indiveri
Cesare
C
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D017136:Ion Transport; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D051381:Rats
D026962:Amino Acid Transport System ASC; D005971:Glutamates; D008081:Liposomes; D015778:Minor Histocompatibility Antigens; C498849:Slc1a5 protein, rat; D005973:Glutamine; D000255:Adenosine Triphosphate