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    • Ki16198 Glycine regulates several brain functions and glycin

    2022-01-20

    Glycine regulates several brain functions and glycine transporters significantly contribute in such processes. In previous work we reported calpain sensitive regions in N-terminal domains of GlyT1a and GlyT1b (Baliova and Jursky, 2005). Here we show that distal part of glycine transporter GlyT1C-terminus is cleaved by calpain protease. Region removed by calpain is involved in high density PDZ clustering of GlyT1, which results in higher capacity for glycine clearance. Artificial construct of GlyT1 transporter missing the last 12 Ki16198 removed by calpain exhibited decrease of glycine transport upon expression in HEK293 cell, which was about half of the Vmax observed with wild type of GlyT1 and truncation in the N-terminal domain even further decreased the uptake (not shown). This is in agreement with previous mutational studies (Cubelos et al., 2005b). Similarly, it has been demonstrated in Muller glia (Gadea et al., 2002), which express the glycine GlyT1 transport system, that while moderate increase of intracellular calcium with ATP or caffeine resulted in stimulation of glycine uptake, the higher calcium levels induced by ionophores, which was inhibited by calpain inhibitor decreased the uptake. Similar effect on endogenous GlyT1 uptake we observed with ionophore induced calpain activation in rat C6 neuroblastoma cells (not shown). We did not make an attempt to study this model in more detail, since nonspecific calpain over activation leads to modification of many cellular substrates. It is therefore difficult to attribute change in GlyT1 uptake to specific GlyT1 calpain cleavage. However taken together above results, during the pathological insult resulting in calcium-induced calpain activation, one of the reasons for change of glycine transporter uptake efficiency might be modification of its cytosolic regions and especially its PDZ binding motif containing distal C-terminus. Pathways activated through calcium permeable NMDA receptor stimulation frequently lead to calpain activation (Waxman and Lynch, 2005). As a matter of fact NMDA induced calpain activation caused its truncation and downregulation providing negative feedback to hamper NMDA receptor mediated toxicity (Yuen et al., 2008). GlyT1 is colocalized with the NMDA receptor in the same protein complex (Cubelos et al., 2005a) and calpain mediated truncation of GlyT1 with subsequent decrease in glycine uptake capacity might cause glycine overload. If such overload increases over the 0.1mM threshold, which is required for rapid NMDA internalization (Nong et al., 2003) it might contribute to receptor downregulation and protection of neurons against NMDA induced neurotoxicity. Protective function of glycine during brain insults has been previously reported (Tijsen et al., 1997, Tonshin et al., 2007). However, local glycine overload could potentionaly reach the millimolar concentrations when glycine exhibits toxic effect on neurons (Barth et al., 2005). Indeed, previously studied effect of various GlyT1 inhibitors showed negative, neutral or positive effects on development of cerebral ischemic infarction (Szabo, 2005). Interestingly treatment with the calpain inhibitor in the zymosan-induced paw inflammation provided anti-inflammatory and anti-hyperalgesic effects (Kunz et al., 2004). Above suggests that the activation of calpain is involved in the sensitization of nociceptive neurons and calpain activity and may present an interesting novel drug target in the treatment of pain and inflammation. It was demonstrated that intravenous or intrathecal administration of GlyT1 and GlyT2 inhibitors produced a profound anti-allodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice mediated through spinal glycine receptor α3 (Morita et al., 2008). Calpain activation and cleavage of C-terminal region of GlyT1 might lead to decrease of GlyT1 mediated glycine uptake. This could lead to local glycine concentration increase, which has virtually the same effect as application of GlyT1 inhibitors. Additionally in sciatic nerve ligation model of neuropathic pain, GAT1 is upregulated and GAT1 inhibitors have analgesic effects (Daemen et al., 2008). Since GAT1 is also truncated by calpain in C-terminal region (Baliova et al., 2009), calpain activation during inflammation or tissue damage with subsequent GlyT1C and GAT1C-terminal cleavage could therefore represent natural analgesic process during healing of damaged tissue.