Identification of Accessory Proteins Interact with Glycine Transporters

The proposed project aims to identify the nature and function of proteins interacting with different isoforms of glycine transporters. Glycine is the major inhibitory neurotransmitter in the brainstem and spinal cord and acts as a co-agonist at the ionotropic glutamate NMDA (N-methyl-D-aspartic acid) receptors throughout the central nervous system. A role for NMDA receptors - and hence for glycine - has been implicated in several disease states including glutamate neuro-toxicity associated with ischaemia and schizophrenia. The primary means of regulating extracellular levels of glycine is by re-uptake through high affinity glycine transporters. Recent evidence demonstrates that NMDA receptors can indeed be modulated by the function of glycine transport. In addition to removing glycine from the extracellular space, calcium-independent glycine release may occur through reversal of the transporter. It is becoming increasingly clear that ion channels as well as neurotransmitter transporters do not exist simply as isolated macro-molecules at the pre- or postsynaptic membrane in vivo. It is, for instance, evident that essential aspects of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and NMDA receptor localisation and regulation are determined by the interaction of the intracellular C-terminus of the receptor with anchoring and regulatory proteins present in the postsynaptic density. The aim of this study is to identify proteins that interact with glycine transporters: Based on the topology of the glycine transporter intracellular domains of the different transporter iso-forms will be used as baits in a Yeast-Two-Hybrid screen, to identify interaction partners. Thus these will be characterised and the effect on the function and the localisation of the transporter will be studied.