Kinetics and Specificity of a H+/Amino Acid Transporter from Arabidopsis thaliana

Abstract

The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and electron microscopic methods to characterize the biophysical properties, kinetics, and specificity of the transporter. Uptake of alanine was H+-dependent increasing from 14 pmol/oocyte/h at 0.032 μM H+ to 370 pmol/oocyte/h at 10 μM H+. AAP1 was electrogenic; there was an amino acid-induced depolarization of the oocyte plasma membrane and net inward currents through the transporter due to the transport of amino acids. AAP1 transported a wide spectrum of amino acids favoring neutral amino acids with short side chains. The maximal current (iMAX) for alanine, proline, glutamine, histidine, and glutamate was voltage- and [H+]o-dependent. Similarly, the iMAXH was voltage- and [amino acid]o-dependent. The iMAX for both H+ and amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transporter. The K0.5 of the transporter for amino acids decreased as [H+]o increased and was lower at negative membrane potentials. The K0.5 for H+ was relatively voltage-independent and decreased as [amino acid]o increased. This positive cooperativity suggests that the transporter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were >1, suggesting that the transporter has more than one binding site for both H+ and amino acid. Freeze-fracture electron microscopy was used to estimate the number of transporters expressed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 increased ∼5-fold above water-injected controls and corresponded to a turnover number 350 to 800 s-1. The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and electron microscopic methods to characterize the biophysical properties, kinetics, and specificity of the transporter. Uptake of alanine was H+-dependent increasing from 14 pmol/oocyte/h at 0.032 μM H+ to 370 pmol/oocyte/h at 10 μM H+. AAP1 was electrogenic; there was an amino acid-induced depolarization of the oocyte plasma membrane and net inward currents through the transporter due to the transport of amino acids. AAP1 transported a wide spectrum of amino acids favoring neutral amino acids with short side chains. The maximal current (iMAX) for alanine, proline, glutamine, histidine, and glutamate was voltage- and [H+]o-dependent. Similarly, the iMAXH was voltage- and [amino acid]o-dependent. The iMAX for both H+ and amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transporter. The K0.5 of the transporter for amino acids decreased as [H+]o increased and was lower at negative membrane potentials. The K0.5 for H+ was relatively voltage-independent and decreased as [amino acid]o increased. This positive cooperativity suggests that the transporter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were >1, suggesting that the transporter has more than one binding site for both H+ and amino acid. Freeze-fracture electron microscopy was used to estimate the number of transporters expressed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 increased ∼5-fold above water-injected controls and corresponded to a turnover number 350 to 800 s-1.