The Family of Na+/Cl− Neurotransmitter Transporters (original) (raw)
Related papers
Journal of Biological Chemistry, 2003
The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na ؉ -dependent and highly selective. The K m for tryptophan was ϳ145 nM, and tryptophan transport was unchanged in the presence of 100 M concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K I values of 200 and 440 M, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His 6 tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.
Molecular cloning of an orphan transporter A new member of the neurotransmitter transporter family
FEBS Letters, 1995
A complementary DNA clone predicted to encode a novel transporter was isolated from rat brain and the localization of its mRNA was examined. The cDNA, designated rB21a, predicts a protein with 12 putative transmembrane domains that exhibits significant sequence homology with neurotransmitter transporters. Expression studies have not yet identified the endogenous substrate for this transporter, but the presence of rB21a mRNA within the leptomeninges of the brain suggests the transporter may regulate CSF levels of its substrate. The cloning of rB21a provides the means to determine its physiological functions and the potential to design novel, transporter-based therapeutic agents for neurological and psychiatric disorders.
Structure and Gating Dynamics of Na+/Cl– Coupled Neurotransmitter Transporters
Frontiers in Molecular Biosciences
Neurotransmitters released at the neural synapse through vesicle exocytosis are spatiotemporally controlled by the action of neurotransmitter transporters. Integral membrane proteins of the solute carrier 6 (SLC6) family are involved in the sodium and chloride coupled uptake of biogenic amine neurotransmitters including dopamine, serotonin, noradrenaline and inhibitory neurotransmitters including glycine and γ-amino butyric acid. This ion-coupled symport works through a well-orchestrated gating of substrate through alternating-access, which is mediated through movements of helices that resemble a rocking-bundle. A large array of commercially prescribed drugs and psychostimulants selectively target neurotransmitter transporters thereby modulating their levels in the synaptic space. Drug-induced changes in the synaptic neurotransmitter levels can be used to treat depression or neuropathic pain whereas in some instances prolonged usage can lead to habituation. Earlier structural studies of bacterial neurotransmitter transporter homolog LeuT and recent structure elucidation of the Drosophila dopamine transporter (dDAT) and human serotonin transporter (hSERT) have yielded a wealth of information in understanding the transport and inhibition mechanism of neurotransmitter transporters. Computational studies based on the structures of dDAT and hSERT have shed light on the dynamics of varied components of these molecular gates in affecting the uphill transport of neurotransmitters. This review seeks to address structural dynamics of neurotransmitter transporters at the extracellular and intracellular gates and the effect of inhibitors on the ligand-binding pocket. We also delve into the effect of additional factors including lipids and cytosolic domains that influence the translocation of neurotransmitters across the membrane.
Reconstructing a Chloride-binding Site in a Bacterial Neurotransmitter Transporter Homologue
Journal of Biological Chemistry, 2011
In ion-coupled transport proteins, occupation of selective ion-binding sites is required to trigger conformational changes that lead to substrate translocation. Neurotransmitter transporters, targets of abused and therapeutic drugs, require Na ؉ and Cl ؊ for function. We recently proposed a chloride-binding site in these proteins not present in Cl ؊ -independent prokaryotic homologues. Here we describe conversion of the Cl ؊ -independent prokaryotic tryptophan transporter TnaT to a fully functional Cl ؊ -dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in serotonin transporter provide direct evidence for the involvement of each of the proposed residues in Cl ؊ coordination. In both SERT and TnaT-D268S, Cl ؊ and Na ؉ mutually increased each other's potency, consistent with electrostatic interaction through adjacent binding sites. These studies establish the site where Cl ؊ binds to trigger conformational change during neurotransmitter transport.
Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters
Nature, 2005
Na þ /Cl 2 -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.
Neurotransmitter transporters: why dance with so many partners?
Current Opinion in Pharmacology, 2004
Plasma membrane transporters terminate the actions of several small molecule neurotransmitters, including glutamate, g-aminobutyric acid, glycine, dopamine, serotonin and norepinephrine. The fact that anti-depressants, cocaine and amphetamines can have such profound behavioral effects by inhibiting the activity of some of these transporters underscores the importance of these molecules. Recent studies have begun to define the mechanisms that regulate these transporters. As these studies progress, it is becoming clear that the transporters form complexes both with themselves and with many other proteins that can regulate either transporter localization or activity. In most cases, the physiological and/or pathological relevance of these interactions is only beginning to emerge.
A rat brain cDNA encoding the neurotransmitter transporter with an unusual structure
FEBS Letters, 1993
A rat cDNA clone encoding the novel membrane protem of the nemotransmitter transporters family was cloned and sequenced. The cDNA was identified as a transcript of the gene NTT4 of which a partial genomic clone was previously sequenced. Alignment of the amino acid sequence of NTT4 with other members of the neurotransmitter transporter family revealed a marked deviation from the conserved structure of all other members of the family. The largest extracellular loop with a potential glycosylation site was identified between membrane segments 7 and 8. The protem retains the common glycosylated loop between transmembrane helices 3 and 4 in all members of the family. The transcript of NTT4 was found exclusively in the central nervous system and is more abundant in the cerebellum and the cerebral cortex.
Cytoplasmic Permeation Pathway of Neurotransmitter Transporters
Biochemistry, 2011
Ion-coupled solute transporters are responsible for transporting nutrients, ions, and signaling molecules across a variety of biological membranes. Recent high-resolution crystal structures of several transporters from protein families that were previously thought to be unrelated show common structural features indicating a large structural family representing transporters from all kingdoms of life. This review describes studies that led to an understanding of the conformational changes required for solute transport in this family. The first structure in this family showed the bacterial amino acid transporter LeuT, which is homologous to neurotransmitter transporters, in an extracellularly oriented conformation with a molecule of leucine occluded at the substrate site. Studies with the mammalian serotonin transporter identified positions, buried in the LeuT structure, that defined a potential pathway leading from the cytoplasm to the substrate binding site. Modeling studies utilized an inverted structural repeat within the LeuT crystal structure to predict the conformation of LeuT in which the cytoplasmic permeation pathway, consisting of positions identified in SERT, was open for diffusion of the substrate to the cytoplasm. From the difference between the model and the crystal structures, a simple "rocking bundle" mechanism was proposed, in which a four-helix bundle changed its orientation with respect to the rest of the protein to close the extracellular pathway and open the cytoplasmic one. Subsequent crystal structures from structurally related proteins provide evidence supporting this model for transport.