The regulation of the Glycine Transporter 1 by phosphorylation

The plasma membrane glycine transporters 1 (GlyT1) is the carrier molecule responsible for the fast removal of the inhibitory neurotransmitter glycine from the synaptic cleft back into the presynaptic terminals of glycinergic neurons by an electrogenic, Na+-Cl- transport couple mechanism maintained by the Na+,K+-ATPase. GlyTs belong to SLC6 family that includes other neurotransmitter transporters, such as the dopamine, norepinephrine, serotonin, and GABA transporters. In order to determine the role of PKC-dependent phosphorylation on GlyT1, we have analyzed the effect of PKC activation by phorbol ester (PMA) on the activity, post-translational modifications and the levels of cell surface transporter in mutants where several replacements of Ser/Thr to Ala were made. We found that, similar to other transporters like DAT, NET and SERT, activation of protein kinase C by phorbol ester led to increased phosphorylation and ubiquitination of GlyT1. These modifications were accompanied by a reduction in transport capacity and enhanced transporter endocytosis. Interestingly, replacement of serine/threonine residues to alanine at both tails abolished PKC-dependent phosphorylation but dramatically enhanced ubiquitination and degradation. These results suggest that transporter modifications represent a mechanism of GlyT1 regulation. This work was supported by a grant from NIMH 5SC1MH 086070-04 to MM, AA is a recipient from the bridges program, grant NIH 5R25GM049011-1