Resumen de Estudio del tráfico intracelular del transportador neuronal de glicina glyt2: Modulación por lipid rafts, ubiquitinación e interacción con na/k atpasa
Glycine is the main inhibitory neurotransmitter in the caudal areas of the brain, spinal cord and retina and plays an important role in the processing of sensory and motor information. In addition, glycine exerts a positive modulation in the excitatory glutamatergic neurotransmission by acting as a necessary co-agonist of glutamate in NMDA receptors. Glycine-mediated neurotransmission is terminated by neurotransmitter reuptake by plasma membrane glycine transporters GlyT1 and GlyT2. Mutations in the GlyT2 gene (SLC6A5) cause an impairment of glycinergic neurotransmission that produces hyperekplexia in humans because the neurotransmitter cannot be recaptured back to the presynaptic terminal to refill synaptic vesicles. Many of these mutations impair the normal intracellular trafficking of GlyT2, e.g. the substitution Y705C (c.2114A¿G) described by our laboratory. Given the importance of GlyT2 intracellular trafficking, we have analysed the molecular mecanisms underling GlyT2 endocytosis, describing the dependence on dynamin and clathrin pathway of the constitutive and PKC-induced internalization of the transporter. Moreover, we have described that GlyT2 internalizes constitutively associated to lipid rafts whereas in the PKC-induced endocytosis a previous lateral displacement to non-raft domains is needed before GlyT2 internalizes to endosomal compartments. In addition, we have described that ubiquitination in the GlyT2 C-terminal end plays an important role in both internalization process. Whereas PKC-induced endocytosis of GlyT2 needs the ubiquitination of a single lysine in position K791, constitutive endocytosis of the transporter needs the ubiquitination of a cluster of lysines in positions K751, K773 ,K787, K791. Simultaneous mutation of this c-ter lysine cluster reduced basal ubiquitination of the transporter and increased notably its half-life and stability. Also, we have described that GlyT2 ubiquitination status is highly sensitive to ubiquitin homeostasis in neurons, since the reduction of the free monomeric ubiquitin pool achieved by UCHL1 inhibition reduces significantly GlyT2 ubiquitination. Moreover, in these conditions constitutive endocytosis of the transporter is impaired, denoting the importance of the ubiquitination mechanism in the GlyT2 internalization process.
In this work we have also described that Na/K ATPase is a new interacting partner for GlyT2. GlyT2 interacts with ¿ catalytic subunits, especially with ¿3NKA subunit, and this interaction is restricted to lipid raft subdomains. ¿ subunits bind ouabain, an endogenous cardiotonic steroid that induces the activation of many intracellular signaling events. In the presence of ouabain we have observed endocytosis and degradation of the lipid raft associated pool of GlyT2, which represents a drastic new pathway of transporter regulation. Further studies are needed to dilucidate the mechanisms underling this process, but these findings could help designing more effective actions in glycinergic neuromotor disorders including hyperekplexia and myoclonus, or other glycinergic-related dysfunctions as neuropathic pain or epilepsy.
