Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1.
KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K(+) and Li(+) in addition to Na(+). We have previously demonstrated that Asp338 is essential for KAAT1 activation by K(+) and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na(+) and K(+). However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na(+)-dependent transport and the block in K(+)-dependent transport that characterize the D338E mutant. K(+)-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)(3), we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na(+) binding site in the recently solved crystal structure of the NSS transporter LeuT(Aa) (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.
Castagna
M
M
Institute of General Physiology and Biological Chemistry G Esposito, Via Trentacoste 2, 20134, Milan, Italy.
Soragna
A
A
Mari
S A
SA
Santacroce
M
M
Betté
S
S
Mandela
P G
PG
Rudnick
G
G
Peres
A
A
Sacchi
V F
VF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007915:Lepidoptera; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010618:Phenanthrolines / Q000737:chemistry / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D026921:Amino Acid Transport Systems, Neutral; D003432:Cross-Linking Reagents; D019476:Insect Proteins; C112249:KAAT1 protein, insect; D010618:Phenanthrolines; D001224:Aspartic Acid; D014364:Tryptophan; D012964:Sodium; D008239:Lysine; D003545:Cysteine; D011188:Potassium; D004229:Dithiothreitol; C025205:1,10-phenanthroline