Steady-state kinetic characterization of the mouse B0AT1 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 B0AT1 (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 ~1) yielding a K0.5 for L-Leu of 1.16mM and for Na+ of 16mM at a holding potential of −50mV. 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 B0AT1 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 B0AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cel