Dynamic role of GlyT1 as glycine sink or source: Pharmacological implications for the gain control of NMDA receptors.

Glycine transporter 1 (GlyT1) mediates the termination of inhibitory glycinergic receptor signaling in the spinal cord and brainstem, and is also present diffusely in the forebrain. Here, it regulates the ambient glycine concentration and influences the 'glycine' site occupancy of N-methyl-d-aspartate receptors (NMDARs). GlyT1 is a reversible transporter with a substantial, but not excessive, sodium-motive force for uphill transport.

Neurotransmitter content heterogeneity within an interneuron class shapes inhibitory transmission at a central synapse.

Neurotransmitter content is deemed the most basic defining criterion for neuronal classes, contrasting with the intercellular heterogeneity of many other molecular and functional features. Here we show, in the adult mouse brain, that neurotransmitter content variegation within a neuronal class is a component of its functional heterogeneity. Golgi cells (GoCs), the well-defined class of cerebellar interneurons inhibiting granule cells (GrCs), contain cytosolic glycine, accumulated by the neuronal transporter GlyT2, and GABA in various proportions.

Flux coupling, not specificity, shapes the transport and phylogeny of SLC6 glycine transporters.

ATB[Formula: see text] () is a member of the amino acid transporter branch of the SLC6 family along with GlyT1 () and GlyT2 (), two glycine-specific transporters coupled to 2:1 and 3:1 Na[Formula: see text]:Cl[Formula: see text], respectively. In contrast, ATB[Formula: see text] exhibits broad substrate specificity for all neutral and cationic amino acids, and its ionic coupling remains unsettled. Using the reversal potential slope method, we demonstrate a 3:1:1 Na[Formula: see text]:Cl[Formula: see text]:Gly stoichiometry for ATB[Formula: see text] that is consistent with its 2.

Heterogeneous Signaling at GABA and Glycine Co-releasing Terminals.

The corelease of several neurotransmitters from a single synaptic vesicle has been observed at many central synapses. Nevertheless, the signaling synergy offered by cotransmission and the mechanisms that maintain the optimal release and detection of neurotransmitters at mixed synapses remain poorly understood, thus limiting our ability to interpret changes in synaptic signaling and identify molecules important for plasticity. In the brainstem and spinal cord, GABA and glycine cotransmission is facilitated by a shared vesicular transporter VIAAT (also named VGAT), and occurs at many immature inhibitory synapses.

Cytosolic transmitter concentration regulates vesicle cycling at hippocampal GABAergic terminals.

Sustained synaptic transmission requires vesicle recycling and refilling with transmitter, two processes considered to proceed independently. Contrary to this assumption, we show here that depletion of cytosolic transmitter at GABAergic synapses reversibly reduces the number of recycling vesicles. Using paired recordings in hippocampal cultures, we show that repetitive activity causes two phases of reduction of the postsynaptic response.

Mutations in the GlyT2 gene (SLC6A5) are a second major cause of startle disease.

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5).

Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin.

Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis.

A critical role for glycine transporters in hyperexcitability disorders.

Defects in mammalian glycinergic neurotransmission result in a complex motor disorder characterized by neonatal hypertonia and an exaggerated startle reflex, known as hyperekplexia (OMIM 149400). This affects newborn children and is characterized by noise or touch-induced seizures that result in muscle stiffness and breath-holding episodes. Although rare, this disorder can have serious consequences, including brain damage and/or sudden infant death.

The glycine transporter GlyT2 controls the dynamics of synaptic vesicle refilling in inhibitory spinal cord neurons.

At inhibitory synapses, glycine and GABA are accumulated into synaptic vesicles by the same vesicular transporter VGAT/VIAAT (vesicular GABA transporter/vesicular inhibitory amino acid transporter), enabling a continuum of glycine, GABA, and mixed phenotypes. Many fundamental aspects of the presynaptic contribution to the inhibitory phenotypes remain unclear. The neuronal transporter GlyT2 is one of the critical presynaptic factors, because glycinergic transmission is impaired in knock-out GlyT2(-/-) mice and mutations in the human GlyT2 gene slc6a5 are sufficient to cause hyperekplexia.

The transporters GlyT2 and VIAAT cooperate to determine the vesicular glycinergic phenotype.

The mechanisms that specify the vesicular phenotype of inhibitory interneurons in vertebrates are poorly understood because the two main inhibitory transmitters, glycine and GABA, share the same vesicular inhibitory amino acid transporter (VIAAT) and are both present in neurons during postnatal development. We have expressed VIAAT and the plasmalemmal transporters for glycine and GABA in a neuroendocrine cell line and measured the quantal release of glycine and GABA using a novel double-sniffer patch-clamp technique. We found that glycine is released from vesicles when VIAAT is coexpressed with either the neuronal transporter GlyT2 or the glial transporter GlyT1.

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease.

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes.

Why glycine transporters have different stoichiometries.

In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through specific transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters differ at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steady-state kinetics.