We found that most antigenic regions in GlyT C

We found that most antigenic regions in GlyT1C are located in the second half of the GlyT1C-terminus, which is in concert with previously published results (Olivares et al., 1994). We however further show that in contrast to epitopes 554–625 which are affected by calcium only minimally, the epitopes against the last 12 amino acids (626–638) of GlyT1C exhibits up to 95% decrease of immunoreactivity within 15min upon calcium overload in mouse synaptosomal preparations. This time window is within the range of 5–30min observed previously (Bi et al., 1997, Guttmann et al., 2002). Potential close association of transporter C-terminus with NMDA/calpain signaling complexes (Adamec et al., 1998, Cubelos et al., 2005a) as well as the relatively high abundance of both GlyT1 and calpain in spinal cord white matter (Borowsky et al., 1993, Chakrabarti et al., 1989, Jursky and Nelson, 1996, Ray et al., 2002) could be additionally responsible for relatively high extend of GlyT1C-terminal cleavage. Calpain inhibitor I significantly but not completely inhibited calcium-induced loss of distal GlyT1C immunoreactivity. This prompted us to screen if other calcium modulated proteases or kinases inhibitors could affect change of immunoreactivity. Application of general caspase inhibitor Z-VAD-FMK inhibited decrease of immunoreactivity, which was slightly lesser but comparable to that observed with calpain inhibitor, indicating possible involvement of caspases in this process. Since caspases exhibits asparagine specificity in their cleavage site, direct cleavage could occur after asparagine 635 in GlyT1C protein sequence. Such cleavage will remove last three amino CGP 55845 hydrochloride representing PDZ binding motif. This potential caspase cleavage site is located nine amino acids downstream of calpain cleavage site. Calpain and caspase frequently share substrates. Similar distance between one of the calpain cleavage sites and nine amino acid apart caspase cleavage sites is located in α-spectrin molecule (Wang, 2000). It was however previously reported that caspase general inhibitor Z-VAD-FMK could inhibits calpain (Bizat et al., 2005). For this reason we cannot exclude that the effect observed with Z-VAD-FMK is caused by calpain inhibition. Additionally caspase cleaves calpain inhibitor (Porn-Ares et al., 1998) and it could this way indirectly potentiate calpain cleavage. Caspase inhibition could then paradoxically result in inhibition of calpain activity. Above indicate that such cleavage could have complex regulation. Investigated GlyT1C-terminal region contains several potential phosphorylation sites and phosphorylation itself might cause significant decrease of anti-GlyT1C626–638 immunoreactivity. In order to distinguish between the phosphorylation and proteolytic truncation, we isolated synaptosomes in presence of high concentration of both EDTA and EGTA. We expected that this treatment will remove magnesium from ATP-Mg2+ complex, which is the real substrate of kinases and this will separate potential effect of phosphorylation. Subsequent addition of magnesium to calcium-containing samples indeed stimulated calcium mediated decrease of anti-GlyT1C626–638 immunoreactivity and this stimulation was suppressed by PKC inhibitor chelerythrine. Similar results were obtained with other PKC inhibitor bisindolylmaleimide II (not shown). Above indicate that during calcium overload in vivo, when magnesium is present, distal C-terminal region of GlyT1 might undergo calpain mediated truncation as well as PKC mediated phosphorylation. In consent with this, it was previously reported that GlyT1 is mainly regulated by protein kinase Cα in glioma C6 cells (Morioka et al., 2008). Involvement of cysteine proteases was supported also by influence of certain metals on decrease of anti-GlyT1C626–638 immunoreactivity. We used metals typically influencing cysteine proteases such as zinc, manganese and cobalt. When calcium was replaced with these metals, neither of them was able to induce decrease of anti-GlyT1C626–638 immunoreactivity. Additionally, while zinc and cobalt abolished the effect of calcium, manganese did not prevent calcium-induced decrease of immunoreactivity. These effects correspond with the potential involvement of cysteine proteases (Suzuki and Ishiura, 1983, Pontremoli et al., 1985, Perry et al., 1997).