Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1.
To investigate the peculiar ionic specificity of KAAT1, an Na+- and K+-coupled amino acid cotransporter from Lepidoptera, a detailed analysis of membrane topology predictions was performed, together with sequence comparison with strictly Na+-dependent mammalian cotransporters from the same family. The analysis identified aspartate 338, a residue present also in the other cotransporter accepting K+ (CAATCH1), but absent in most mammalian transporters that have, instead, an asparagine in the corresponding position. Mutation of D338 in KAAT1 led either to non-functional transporters (D338G, D338C), or to an altered ionic selectivity (D338E, D338N), observable in uptake experiments and in electrophysiological properties. In particular, in D338E, the transport activity, while persisting in the presence of Na+, appeared to be completely abolished in the presence of K+. D338E also showed uncoupling between transport-associated current and uptake. The opposite mutation in the gamma-aminobutyric acid transporter rGAT-1 (N327D) resulted in complete loss of function. In conclusion, aspartate 338 in KAAT1 appears to be important in allowing K+, in addition to Na+, to drive the transport mechanism, although other residues in different parts of the protein may also play a role in the complete determination of ionic selectivity.
Mari
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Institute of General Physiology and Biological Chemistry Giovanni Esposito, Via Trentacoste 2, 20134 Milano, Italy.
Soragna
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A
Castagna
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M
Bossi
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E
Peres
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A
Sacchi
V F
VF
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D019476:Insect Proteins; D007930:Leucine / Q000378:metabolism; D018402:Manduca / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D019476:Insect Proteins; C112249:KAAT1 protein, insect; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D001224:Aspartic Acid; D007930:Leucine