Insights Into the Role of the N-Terminus of the Neuronal Glycine Transporter 2

Hyperekplexia is distinguished by exaggerated muscle tension, sudden shock and even death in response to touch/acoustic stimuli. Genetic analysis showed that mutations on glycinergic proteins on the presynaptic neurons are responsible for the attacks. Mutations in the Glycine Transporter 2 (GlyT2) have been suggested to be the second major cause of hyperekplexia. The GlyT2 consist of twelve transmembrane domains, a 201 amino acid long N-terminus and a shorter C-terminus, both intracellular. Dysfunction of this transporter prevents the intake of glycine from the synapse back to the presynaptic neuron. Our goal is to better understand the role of N-terminus on protein stability and function of transporter.  The wild-type (WT) GlyT2 or a mutant devoid of the N-terminus (Δ201) were used for expression into Porcine Aortic Epithelial (PAE) cells lines. PAE expressing WT or Δ201were analyzed by fluorescence microscopy. We observed the WT transporter localized at the plasma membrane in non-stimulated cells; the Δ201 was retained inside the cells within a network-like membranes network around the nucleus. By Western blot we found two immunoreactive bands for the WT lane corresponding a newly synthesized, non-glycosylated transporter in the ER (75 kDa) and a fully glycosylated band (100 kDa) representing the GlyT2 at the plasma membrane. Analysis of the Δ201 identified a single band that migrated with a molecular weight of 60 kDa (non-glycosylated). These results suggest the N-terminus is required for trafficking of GlyT2 from the ER to the plasma membrane. Future experiments will study glycine uptake in the WT and mutant GlyT2.