Abstract
The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.
Highlights
The Neurotransmitter:Sodium Symporters (NSSs) are responsible for clearing neurotransmitters, such as dopamine, serotonin, norepinephrine, glycine and GABA from the synaptic cleft
Our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein
The transporters are thereby crucial for the regulation of synaptic transmission in the CNS and alterations in their function have been linked to several psychiatric and neurological disorders such as depression, bipolar disorders, attention deficit hyperactive disorder (ADHD), epilepsy, and Parkinson’s disease (Broer, 2013; Kristensen et al, 2011)
Summary
The Neurotransmitter:Sodium Symporters (NSSs) are responsible for clearing neurotransmitters, such as dopamine, serotonin, norepinephrine, glycine and GABA from the synaptic cleft. Erlendsson, Gotfryd et al have analyzed a bacterial protein in the Neurotransmitter:Sodium Symporter family This transporter takes up an amino acid called leucine into cells, and is commonly used as a model to understand this family of transporter proteins more generally. Using a technique called solid state nuclear magnetic resonance, Erlendsson, Gotfryd et al could detect a single molecule of leucine bound to each transporter, but not a second one. This technique could pinpoint that the leucine was located at the transporter’s central binding site. Characteristic chemical shifts that are expected to be different due to different chemical environments, i.e. interacting residues (Reyes et al, 2011)
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