br Acknowledgments This work was

Acknowledgments This work was supported by the Slovak grant agency VEGA, Grant 2/0084/13. We would like to thank Dr. J. Bauer for careful reading of the manuscript.
Introduction Glycine is an important neurotransmitter which regulates both G-1 inhibitory neurotransmission and excitatory neurotransmission. The former is done through a chloride permeable glycine receptor (Betz, 1992) while the latter is carried out by the glycine occupation of a glycine coagonist site on NMDA receptor (Johnson and Ascher, 1987). The local concentration of glycine in the central nervous system is controlled by two high affinity, sodium dependent glycine transporters, GlyT1 and GlyT2 (Borowsky et al., 1993, Liu et al., 1993). In the spinal cord these transporters are an integral part of the hindbrain glycinergic inhibitory system and both GlyT1 and GlyT2 gene knockouts are lethal (Gomeza et al., 2003a, Gomeza et al., 2003b). Accordingly, GlyT2 and GlyT1 are highly abundant in the caudal and, to a much lesser degree, are present in the rostral parts of the brain (Zafra et al., 1995, Jursky and Nelson, 1996, Cubelos et al., 2005a). Consistent with the existence of a glycine/d-serine co-agonits site on the NMDA receptor (Johnson and Ascher, 1987, Mothet et al., 2000), GlyT1 has been found to be physically associated with the NMDA receptor complex (Cubelos et al., 2005a). Recently, d-serine has been suggested to be a true physiological co-agonist of NMDA in vivo (Mothet et al., 2000, Panatier et al., 2006, Wolosker, 2007, Fossat et al., 2012), leaving to glycine the role of controlling NMDA-mediated excitotoxicity (Waxman and Lynch, 2005, Barth et al., 2005, Xu et al., 2009, Hardingham, 2009, Papouin et al., 2012, Imamura et al., 2008). However, the exact role and interplay of these two NMDA agonists still remain to be fully elucidated. The two most important functions of NMDA in the brain, neuronal processing of higher brain function and exitotoxicity, are mediated through different subunit composition as well as different levels of receptor activation (Waxman and Lynch, 2005, Liu et al., 2007, Hardingham, 2009). It is well known that pathological NMDA overexitation is associated with increased calcium permeability; calpain activation and higher Ca2+ levels triggered by NMDA overexcitation might lead to the calpain cleavage of several proteins, including NMDA and its associated GlyT1. Calpains are ubiquitous, tissue specific Ca2+-dependent thiol proteases (Campbell and Davies, 2012). They catalyze the limited, usually interdomain cleavage of many cellular proteins, including those involved in cytoskeletal or membrane attachments, signal transduction, the cell cycle, regulation of gene expression, apoptosis and the pathophysiology of spinal cord injury, where GlyT1 is highly abundant (Ray et al., 2003). These facts suggest that some of the most probable calpain targets are the cytosolic regions of GlyT1. The C-terminal cytosolic portion of GlyT1 has been shown to contain clearly defined regulatory elements, including PKC, PDZ and SEC24-dependent signals, which are crucial for its cellular trafficking (Olivares et al., 1994, Cubelos et al., 2005b, Fernández-Sánchez et al., 2008). Calpain mediated modification of GlyT1 C-terminus under certain pathological conditions is therefore likely to cause significant changes in glycinergic neurotransmission. Since the consensus amino-acid target sequences for calpain cleavage site recognition have not been clearly determined (Tompa et al., 2004, Cuerrier et al., 2005, Kelly et al., 2009), we previously screened recombinant fusion proteins containing different cytosolic parts of GlyT1 for the presence of calpain cleavage sites (Baliova and Jursky, 2005, Baliova and Jursky, 2010). A sequence analysis of the cleavage products revealed several potential calpain cleavage sites, however some of them might not actually be cleaved in vivo because of inaccessibility, other protein–protein interactions or posttranslational modifications, including phosphorylation.