Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1.
PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.
Foltz
Martin
M
Molecular Nutrition Unit, Institute of Nutritional Sciences, Center of Life and Food Sciences, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Federal Republic of Germany.
Mertl
Manuela
M
Dietz
Veronika
V
Boll
Michael
M
Kottra
Gabor
G
Daniel
Hannelore
H
D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D004553:Electric Conductivity; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D000074011:Peptide Transporter 1; D011522:Protons; D013379:Substrate Specificity; D000076988:Sulfate Transporters; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D026901:Membrane Transport Proteins; D000074011:Peptide Transporter 1; D011522:Protons; C439529:SLC26A6 protein, human; D000076988:Sulfate Transporters; D027981:Symporters