Mutations within the human GLYT2 (SLC6A5) gene associated with hyperekplexia.
ABSTRACT Hereditary hyperekplexia is a neuromotor disorder characterized by exaggerated startle reflexes and muscle stiffness in the neonate. The disease has been associated with mutations in the glycine receptor subunit genes GLRA1 and GLRB. Here, we describe mutations within the neuronal glycine transporter 2 gene (GLYT2, or SLC6A5, ) of hyperekplexia patients, whose symptoms cannot be attributed to glycine receptor mutations. One of the GLYT2 mutations identified causes truncation of the transporter protein and a complete loss of transport function. Our results are consistent with GLYT2 being a disease gene in human hyperekplexia.
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ABSTRACT: The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na+/Cl−-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na+ coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). InXenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na+-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ ↔ CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na+, Cl−, and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.Journal of Biological Chemistry 05/2001; 276(21):17699-17705. · 4.65 Impact Factor
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ABSTRACT: Glycine's role as an inhibitory neurotransmitter in the adult vertebrate nervous system has been well characterized in a number of different model organisms. However, a full understanding of glycinergic transmission requires a knowledge of how glycinergic synapses emerge and the role of glycinergic signaling during development. Recent literature has provided a detailed picture of the developmental expression of many of the molecular components that comprise the glycinergic phenotype, namely the glycine transporters and the glycine receptor subunits; the transcriptional networks leading to the expression of this important neurotransmitter phenotype are also being elucidated. An equally important focus of research has revealed the critical role of glycinergic signaling in sculpting many different aspects of neural development. This review examines the current literature detailing the expression patterns of the components of the glycinergic phenotype in various vertebrate model organisms over the course of development and the molecular mechanisms governing the expression of the glycinergic phenotype. The review then surveys the recent work on the role of glycinergic signaling in the developing nervous system and concludes with an overview of areas for further research.Frontiers in Molecular Neuroscience 01/2010; 3:11.
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ABSTRACT: SLC6 family members and ABC transporters represent two extremes: SLC6 transporters are confined to the membrane proper and only expose small segments to the hydrophilic milieu. In ABC transporters the hydrophobic core is connected to a large intracellular (eponymous) ATP binding domain that is comprised of two discontiguous repeats. Accordingly, their folding problem is fundamentally different. This can be gauged from mutations that impair the folding of the encoded protein and give rise to clinically relevant disease phenotypes: in SLC6 transporters, these cluster at the protein-lipid interface on the membrane exposed surface. Mutations in ABC-transporters map to the interface between nucleotide binding domains and the coupling helices, which provide the connection to the hydrophobic core. Folding of these mutated ABC-transporters can be corrected with ligands/substrates that bind to the hydrophobic core. This highlights a pivotal role of the coupling helices in the folding trajectory. In contrast, insights into pharmacochaperoning of SLC6 transporters are limited to monoamine transporters - in particular the serotonin transporter (SERT) - because of their rich pharmacology. Only ligands that stabilize the inward facing conformation act as effective pharmacochaperones. This indicates that the folding trajectory of SERT proceeds via the inward facing conformation. Mutations that impair folding of SLC6 family members can be transmitted as dominant or recessive alleles. The dominant phenotype of the mutation can be rationalized, because SLC6 transporters are exported in oligomeric form from the endoplasmic reticulum (ER). Recessive transmission requires shielding of the unaffected gene product from the mutated transporter in the ER. This can be accounted for by a chaperone-COPII (coatomer protein II) exchange model, where proteinaceous ER-resident chaperones engage various intermediates prior to formation of the oligomeric state and subsequent export from the ER. It is likely that the action of pharmacochaperones is contingent on and modulated by these chaperones.Pharmacological Research 12/2013; · 4.35 Impact Factor