A Comprehensive Structure-Based Alignment of Prokaryotic and Eukaryotic Neurotransmitter/Na⁺Symporters (NSS) Aids in the Use of the LeuT Structure to Probe NSS Structure and Function

Abstract

The recently elucidated crystal structure of a prokaryotic member of the neurotransmitter/sodium symporter (NSS) family (Yamashita et al., 2005) is a major advance toward understanding structure-function relationships in this important class of transporters. To aid in the generalization of these results, we present here a comprehensive sequence alignment of all known prokaryotic and eukaryotic NSS proteins, based on the crystal structure of the leucine transporter from Aquifex aeolicus (LeuT). Regions of low sequence identity between prokaryotic and eukaryotic transporters were aligned with the aid of a number of bioinformatics tools, and the resulting alignments were validated by comparison with experimental data. In a number of regions, including the transmembrane segments 4, 5, and 9 as well as extracellular loops 2, 3, and 4, our alignment differs from the one proposed previously [Nature (Lond) 437: [215][216][217][218][219][220][221][222][223] 2005]. Important similarities and differences among the sequences of NSS proteins in regions likely to determine selectivity in substrate binding and mechanisms of transport regulation are discussed in the context of the LeuT structure and the alignment.

Transmembrane Domain 6 of the Human Serotonin Transporter Contributes to an Aqueously Accessible Binding Pocket for Serotonin and the Psychostimulant 3,4-Methylene Dioxymethamphetamine

“…In LeuT Aa , TMs 1, 3, 6, and 8 form the leucine binding pocket. Biochemical analyses and homology modeling of SERT proteins predict a similar binding pocket for 5-HT (32)(33)(34)(35)(36). Consistent with these models, pre-structure studies identified residues in hSERT TMs 1 and 3 that confer high affinity interactions and ligand selectivity to substrates and antagonists.…”

“…Impact of Na ϩ on MTSET Inactivation-The LeuT Aa crystal structure and hSERT homology models derived from it predict that TM6 is involved in the coordination of a sodium ion (27,(32)(33)(34)36). Interestingly, replacement of sodium with NMDG in the MTS incubation buffer resulted in protection of three TM6 Cys mutants and exposure of a single mutant (Fig.…”

“…Because hSERT TM6 is predicted to lie adjacent to and share topology with TM1 (27,(32)(33)(34)(35)(36), we explored potential contributions of TM6 to the interactions of 5-HT, MDMA, and cocaine via an assessment of their ability to protect hSERT C109A Cys mutants from inactivation by MTSET and biotinylation with MTSEA-biotin. Before initiation of protection experiments, we examined the sensitivity of each Cys mutant to MTSET at 0.1, 1, and 10 mM to ensure we were using a concentration of MTSET appropriate for detecting either protection or exposure.…”

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

“…Immunohistochemistry with a GAT antibody confirmed that all of the mutant proteins localized to the membrane similarly to the wild-type protein (data not shown). Finally, the location of the binding site is in agreement with those of the other GATs (20,53). For example, the mouse GAT-3 (i.e., GAT-4) binding site is almost identical to that of our GAT-2 model (54).…”

Mechanism for alternating access in neurotransmitter transporters

“…The crystal structure of LeuT (9) provides an example of a structure where the binding site is exposed to only one side of the membrane, in this case to the extracellular surface. LeuT has provided a useful structural model for members of the neurotransmitter sodium symporter (NSS) family (9,15). These proteins include transporters mediating the reuptake of neurotransmitters and amino acids.…”