Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters

Abstract

Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

“…The results of the Javitch group related to the S2 site, the properties of the F253A mutant and the role of the putative S2 site in the mechanism of LeuT and NSSs are, at present, without satisfactory explanation. In contrast, the results reported here are in agreement with previous results from this group and with the conclusion that there is a single highaffinity substrate site in LeuT [6,14,16,17]. Our results cast further doubt on the relevance of the S2 site to the mechanism of LeuT and NSSs.…”

“…By contrast, molecular dynamics, substrate binding and singlemolecule experiments have been interpreted in terms of a model in which there is a second, or S2, high-affinity substrate binding site [18][19][20][21][22]. In an effort to disrupt substrate binding to the S1 site, Javitch and colleagues [18,21] studied the F253A mutant, a residue that lines a portion of the S1 binding site [6,14]. On the basis of their experiments on the F253A mutant, the authors make several assertions.…”

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

“…All members of the family share a similar predicted topology of twelve transmembrane domains, cytoplasmic amino-and carboxy-terminals, a large second extracellular loop with multiple N-linked glycosylation sites, and cytoplasmic phosphorylation sites. Recent crystallographic studies focusing on LeuT Aa , a bacterial SLC6 transporter homolog, support the predicted DAT topology and reveal a dimeric transporter assembly (Yamashita et al, 2005), consistent with FRET and co-immunoprecipitation studies that suggest SLC6 transporter homooligomerization (Schmid et al, 2001;Scholze et al, 2002;Sorkina et al, 2003;Torres et al, 2003b).…”

Asymmetric perturbations of signalling oligomers

“…Similarly to sodium-coupled neurotransmitter transporters (Krishnamoorthy et al, 2009;Focke et al, 2013), BetP possesses protomers with a conserved LeuT-like fold with two inverted-topology structural repeats (Yamashita et al, 2005) and in agreement with an AA mechanism proposed for the monomeric sodium-dependent amino acid symporter LeuT (Krishnamoorthy et al, 2009), betaine translocation is thought to be mediated by the combination of rigid-body movements and individual flexing of symmetry-related helices (Perez et al, 2012).…”