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Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1
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| Zeitschriftentitel: | Biochemical Journal |
|---|---|
| Personen und Körperschaften: | , , , , , |
| In: | Biochemical Journal, 386, 2005, 3, S. 607-616 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Portland Press Ltd.
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| Schlagwörter: |
| author_facet |
FOLTZ, Martin MERTL, Manuela DIETZ, Veronika BOLL, Michael KOTTRA, Gabor DANIEL, Hannelore FOLTZ, Martin MERTL, Manuela DIETZ, Veronika BOLL, Michael KOTTRA, Gabor DANIEL, Hannelore |
|---|---|
| author |
FOLTZ, Martin MERTL, Manuela DIETZ, Veronika BOLL, Michael KOTTRA, Gabor DANIEL, Hannelore |
| spellingShingle |
FOLTZ, Martin MERTL, Manuela DIETZ, Veronika BOLL, Michael KOTTRA, Gabor DANIEL, Hannelore Biochemical Journal Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 Cell Biology Molecular Biology Biochemistry |
| author_sort |
foltz, martin |
| spelling |
FOLTZ, Martin MERTL, Manuela DIETZ, Veronika BOLL, Michael KOTTRA, Gabor DANIEL, Hannelore 0264-6021 1470-8728 Portland Press Ltd. Cell Biology Molecular Biology Biochemistry http://dx.doi.org/10.1042/bj20041519 <jats:p>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 [Km (Michaelis constant) and Imax (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.</jats:p> Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 Biochemical Journal |
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| title |
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_unstemmed |
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_full |
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_fullStr |
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_full_unstemmed |
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_short |
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_sort |
kinetics of bidirectional h+ and substrate transport by the proton-dependent amino acid symporter pat1 |
| topic |
Cell Biology Molecular Biology Biochemistry |
| url |
http://dx.doi.org/10.1042/bj20041519 |
| publishDate |
2005 |
| physical |
607-616 |
| description |
<jats:p>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 [Km (Michaelis constant) and Imax (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.</jats:p> |
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| author | FOLTZ, Martin, MERTL, Manuela, DIETZ, Veronika, BOLL, Michael, KOTTRA, Gabor, DANIEL, Hannelore |
| author_facet | FOLTZ, Martin, MERTL, Manuela, DIETZ, Veronika, BOLL, Michael, KOTTRA, Gabor, DANIEL, Hannelore, FOLTZ, Martin, MERTL, Manuela, DIETZ, Veronika, BOLL, Michael, KOTTRA, Gabor, DANIEL, Hannelore |
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| description | <jats:p>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 [Km (Michaelis constant) and Imax (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.</jats:p> |
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| spelling | FOLTZ, Martin MERTL, Manuela DIETZ, Veronika BOLL, Michael KOTTRA, Gabor DANIEL, Hannelore 0264-6021 1470-8728 Portland Press Ltd. Cell Biology Molecular Biology Biochemistry http://dx.doi.org/10.1042/bj20041519 <jats:p>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 [Km (Michaelis constant) and Imax (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.</jats:p> Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 Biochemical Journal |
| spellingShingle | FOLTZ, Martin, MERTL, Manuela, DIETZ, Veronika, BOLL, Michael, KOTTRA, Gabor, DANIEL, Hannelore, Biochemical Journal, Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1, Cell Biology, Molecular Biology, Biochemistry |
| title | Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_full | Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_fullStr | Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_full_unstemmed | Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_short | Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| title_sort | kinetics of bidirectional h+ and substrate transport by the proton-dependent amino acid symporter pat1 |
| title_unstemmed | Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1 |
| topic | Cell Biology, Molecular Biology, Biochemistry |
| url | http://dx.doi.org/10.1042/bj20041519 |