Steady-state kinetic characterization of the mouse B(0)AT1 sodium-dependent neutral amino acid transporter.
The members of the neurotransmitter transporter family SLC6A exhibit a high degree of structural homology; however differences arise in many aspects of their transport mechanisms. In this study we report that mouse B(0)AT1 (mouse Slc6a19) mediates the electrogenic transport of a broad range of neutral amino acids but not of the chemically similar substrates transported by other SLC6A family members. Cotransport of L: -Leu and Na(+) generates a saturable, reversible, inward current with Michaelis-Menten kinetics (Hill coefficient approximately 1) yielding a K(0.5) for L: -Leu of 1.16 mM and for Na(+) of 16 mM at a holding potential of -50 mV. Changing the membrane voltage influences both substrate binding and substrate translocation. Li(+) can substitute partially for Na(+) in the generation of L: -Leu-evoked inward currents, whereas both Cl(-) and H(+) concentrations influence its magnitude. The simultaneous measurement of charge translocation and L: -Leu uptake in the same cell indicates that B(0)AT1 transports one Na(+) per neutral amino acid. This appears to be accomplished by an ordered, simultaneous mechanism, with the amino acid binding prior to the Na(+), followed by the simultaneous translocation of both co-substrates across the plasma membrane. From this kinetic analysis, we conclude that the relatively constant [Na(+)] along the renal proximal tubule both drives the uptake of neutral amino acids via B(0)AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cell.
Camargo
Simone M R
SM
Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.
Makrides
Victoria
V
Virkki
Leila V
LV
Forster
Ian C
IC
Verrey
François
F
D026905:Amino Acid Transport Systems / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000502:physiology*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002712:Chlorides / Q000494:pharmacology; D004594:Electrophysiology; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D005942:Gluconates / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007684:Kidney Tubules / Q000201:enzymology; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008094:Lithium / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010649:Phenylalanine / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D002712:Chlorides; D005942:Gluconates; C505021:SLC6A19 protein, mouse; D010649:Phenylalanine; D008094:Lithium; D012964:Sodium; D007930:Leucine; C030691:gluconic acid; D005998:Glycine