Active transport of alanine by the neutral amino-acid exchanger ASCT1.
ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.
Pinilla
J
J
Departamento de Fisiología y Nutrición, Universidad de Navarra, Pamplona, Spain.
Barber
A
A
Lostao
M P
MP
D000409:Alanine / Q000378:metabolism*; D000465:Algorithms; D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism; D012964:Sodium / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D013054:Spectrophotometry, Atomic; D014982:Xenopus laevis
D026962:Amino Acid Transport System ASC; C498848:SLC1A4 protein, human; D018698:Glutamic Acid; D012964:Sodium; D000409:Alanine