Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

Abstract

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

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure–Function Relationships in Mammalian SLC6 Transporters

“…The amino acid sequence considered are indicated and referred to LeuTAa numbering [137] the simultaneous substitution of the specific Lys 102 with Val. The same behaviour was observed in the corresponding mutant (V56K-N264D) of the bacterial tryptophan transporter TnaT from Symbiobacterium thermophilum, thus giving a functional meaning to the structural proximity observed between TMD2 and TMD7 in the LeuTAa 3D structure [116]. A direct interaction between these residues was proved by the thiol crosslinking inhibition observed in the D338C/K102C mutant, and considering that in LeuTAa N286 participates in the coordination of sodium in the Na1 binding site, our analysis outlined as during the transport cycle Lys102, through its positive charge, may maintain D338E in position, maximizing the coordination of Na + and K + and facilitating cation-substrate coupling.…”

“…with the well-studied mammalian GAT1 (SLC6A1). Starting from the pH modulation of the electrophysiological properties, we discovered and investigated many of the determinants involved in substrate and ion binding, chloride and pH regulation and transport steps [102][103][104][112][113][114][115][116][117][118][119][120][121][122].…”

The Rocking Bundle: A Mechanism for Ion-Coupled Solute Flux by Symmetrical Transporters

“…However, this mechanism does not require the four-helix bundle unit to move as a perfectly rigid body, and some internal rearrangements between the helices are no doubt possible, as illustrated by the LeuT-Trp structure, in which the extracellular ends of the helices are swung further outward. Furthermore, accessibility and cross-linking measurements on the related GABA transporter GAT-1 and the amino acid transporter KAAT1 indicate some rearrangement of the bundle helices during transport (7, 43). In any case, this mechanism does not rule out the possibility of additional intermediate and/or occluded states.…”

Threonine 67 is a key component in the coupling of the NSS amino acid transporter KAAT1

“…However, other Cl – -independent neutral amino acid transporters (mammalian B 0 AT1 and B 0 AT2, IMINO, and others) do not appear to have such negative residue in the adjacent regions of the site. Moreover, Asp 338 in KAAT1 has been demonstrated to be involved in the interaction with K + and in the spatial organization of the cation binding site and of the permeation pathway [30,31]. In addition to Asp 338, sequence comparisons highlight two non-conserved residues in the putative sodium binding sites of KAAT1: Ala 66, corresponding to Gly 20 in the Na2 site of LeuT, and Ser 68, corresponding to Ala 22 in the Na1 site (Fig.…”