Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1.
KAAT1 is a neutral amino acid transporter activated by K+ or by Na+ (9). The protein shows significant homology with members of the Na+/Cl--dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20-30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of Vmax and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na+ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1.
Institute of General Physiology and Biological Chemistry, University of Milano, Via Trentacoste 2, 20134 Milan, Italy. Franca.Sacchi@unimi.it
V Franca
VF
Sacchi
Michela
M
Castagna
Stefania A
SA
Mari
Carla
C
Perego
Elena
E
Bossi
Antonio
A
Peres
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids / Q000493:pharmacokinetics*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D018698:Glutamic Acid / Q000235:genetics; D019476:Insect Proteins; D007700:Kinetics; D018402:Manduca; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010658:Phenylglyoxal / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014982:Xenopus laevis
D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D019476:Insect Proteins; C112249:KAAT1 protein, insect; D008562:Membrane Glycoproteins; D013439:Sulfhydryl Reagents; D018698:Glutamic Acid; D010658:Phenylglyoxal; D005033:Ethylmaleimide