Dimerization Of Glycine Transporter 1 And Its Functional Consequences

Glycine transporter 1(Gly T1)is responsible for the reuptake of glycine,which regulates glutamate signaling as a co-agonist with N-methyl-D-aspartic acid(NMDA)receptors in the excitatory syn-apse and has been proposed to be a potential target in the development of therapies for a broad range of disorders of the central nervous system.Despite significant progress in characterizing the structure and transport mechanism of the transporter,the regulation of transport function through oligomerization remains to be understood.In the present work,association of two forms of Gly T1 into dimers and higher order oligomers was detected by coimmunoprecipitation and ligand binding.To investigate the functional consequences of Gly T1 dimerization,we performed oxidative cross-linking of cysteine residues in extracellular loop 3(EL3)near the extracellular end of TM6.By analyzing the effects of copper phenanthroline(Cu P)-induced dimerization on transport properties,Gly T1 dimerization was found to inhibit transport function and the func-tional activity of a Gly T1 natural dimer was estimated to be only 30%higher than a fully active monomeric form.In addition,an intramolecular ion pair Lys286-Glu289 was revealed to be criti-cal for stabilizing EL3 in a conformation that modulates Gly T1 dimerization and transport function.Furthermore,the influences of transporter conformation and several intracellular biological events on Gly T1b dimerization and transport function were investigated.The substrate glycine,in the presence of both Na~+and Cl~-,significantly reduced oxidative cross-linking,suggesting a large-scale rotation of the bundle domain during substrate transport impairs interfacial interactions in a Gly T1dimer.Phosphatidylinositol 4,5-bisphosphate,and palmitic acid were identified to be important modulators in Gly T1b dynamic dimerization by which these signaling molecules regulate the transport activity.The present study provides new insights into structural and functional elements regulating Gly T1 transport activity through its oligomerization.