Finally we investigated if calpain truncation affects GlyT t

Finally, we investigated if calpain truncation affects GlyT1 turnover and trafficking. To simulate calpain cleavage at the T602/T603 and G626/S627 sites we constructed GlyT1 transporters missing the last 12 and 36 amino acids. Since the 36 amino Cy3-UTP truncation also removes all C-terminal antibody epitopes, we used for our analyses GlyT2N-tagged transporters. As shown in Fig. 6 these transporters are still able to reach the cellular surface. Surface expression of shortened transporters is however significantly decreased and the transporter missing the last 36 amino acids seems to be prone to further degradation. This suggests that calpain could affect GlyT1 surface expression. It is also in consistent with the previous results obtained by Gadea et al. (2002) using a calcium ionophore which resulted in calpain activation in retinal Muller glial cells. Calpain cleaves many substrates, however, including PKC (Hofmann, 1997) and calcineurin (Shioda et al., 2006) and makes these proteins constitutively active. This affects several proteins involved in the regulation of GlyT1 and using such an experimental approach would make it difficult to claim that the effect on GlyT1 is related to GlyT1 phosphorylation at the residues examined here. More sophisticated studies will be necessary to further study this topic.
Introduction Inhibitory glycinergic neurotransmission is terminated by the specific glycine transporters GlyTs (GlyT1 and GlyT2) which actively reuptake glycine from the synaptic cleft. GlyTs belong to the neurotransmitter: sodium symporter family (SLC6 gene family), which includes transporters for most of the neurotransmitters, serotonin, dopamine, norepinephrine and GABA, in the central nervous system (CNS) (Aragón and López-Corcuera, 2003). GlyT1 is associated with both glycinergic and glutamatergic pathways and is the main regulator of glycine levels in the synapses. The neuronal transporter GlyT2 recycles the neurotransmitter to the presynaptic terminal, a process that is absolutely necessary to preserve the quantal glycine content inside the synaptic vesicles (Apostolides and Trussell, 2013, Gomeza et al., 2003b, Harvey and Yee, 2013, Rousseau et al., 2008). Mouse gene disruption studies have revealed that constitutive deletion of either GlyT1 or GlyT2 is lethal (primarily as a result of excessive or deficient glycinergic inhibition, respectively), and suggest that alterations in GlyTs may underlie several human disorders (Aragón and López-Corcuera, 2005, Gomeza et al., 2003a, Gomeza et al., 2003b). These studies revealed the role of GlyTs as homeostatic regulators of glycine levels in glycinergic and glutamatergic pathways that controls the balance of neuronal excitation and inhibition within several neural circuits. GlyT1 and GlyT2 have been related to disorders of central and peripheral nervous system, such as schizophrenia, depression, epilepsy, obsessive-compulsive disorders, anxiety disorders, pain, alcohol dependence, breathing disorders and hereditary hyperekplexia (Harvey and Yee, 2013). Indeed, mutations in the gene encoding GlyT2 are the second main cause of hyperekplexia in humans (Carta et al., 2012, Eulenburg et al., 2006, Gimenez et al., 2012, Rees et al., 2006) and produce congenital muscular dystonia type 2 (CMD2) in Belgian Blue cattle (Gill et al., 2012). Moreover GlyT1 inhibitors may improve cognitive deficits of schizophrenia by increasing glycine levels around the NMDA receptors. GlyT1 inhibitors are being developed by the pharmaceutical industry, mostly intended for treatment of cortical NMDA receptor hypofunction in schizophrenia (Javitt, 2008, Pinard et al., 2010). An analysis of knock-out animals proved that the modulation of glycine transporter expression and/or transport activity influenced glycine-mediated neurotransmission and opened a way to find therapeutic applications (Gomeza et al., 2003a, Gomeza et al., 2003b). The levels of active glycine transporters in the plasma membrane are controlled by several factors in a regulated manner (de Juan-Sanz et al., 2011, de Juan-Sanz et al., 2013a, de Juan-Sanz et al., 2013b, Fornes et al., 2008, Geerlings et al., 2001, Nuñez et al., 2008). In the CNS these regulatory pathways must be triggered by physiological stimuli or the activity of appropriate receptors (Jiménez et al., 2011).