Structure/Function Relationship of the Serotonin Transporter

Research project P 14509 Structure/Function Relationship of the Serotonin Transporter Harald H. SITTE 27.11.2000 Neurotransmitter transporters (NT) are membrane proteins responsible for the re-uptake of previously released neurotransmitter from the synaptic cleft following neurotransmission. Prototypical examples include the serotonin and GABA transporter (SERT, GAT-1, resp.). These are to be studied in the current project. Transport is bi- directional, i.e. NT can also mediate efflux of neurotransmitter. Transport is dependent on Na+-ions and is associated with the generation of a current in analogy with ion channels; moreover, NT also form oligomers. This has been shown recently by indirect evidence. In the current project, we intend to visualize the oligomeric state of transporters using fluorescent energy resonance transfer-microscopy (FRET-M). This approach is the method of choice for monitoring protein- protein interactions in living cells. FRET occurs if (i) two fluorophores are in proximity (<100A) and (ii) appropriate orientation allowing an donor fluorophore to transfer energy to a longer- wavelength acceptor fluorophore in a non-radiative manner. The green fluorescent protein (FP) has been mutated recently to create suitable pairs for FRET-M(e.g. cyan and yellow FP). For the study of interactions FP-transporter fusion proteins will be generated by fusing their coding sequences. To test for the specificty of the FRET-M signal obtained co- transfection experiments will be performed (YFP-GATI-CFP-SERT).To validate the FRET-M data, FPLC-and native PAGE-experiments will be employed. In our working hypothesis we assume (i) that transported substrates and non- transported ligands induce different oligomeric states; this will be monitored by FRET-M in intact cells. Subtle conformational changes within the transporter can also be visualized by FRET by fusing a donor and an acceptor to the N- and C-termini of SERT. (ii) Oligomerization is presumed to depend on specific sites of protein-protein interactions (the leucine-zipper-motif). Their functional relevance for oligomerization will be tested by creating chimeric transporters. (iii) Finally, FRET microscopy will be used to visualize dynamic changes in the subcellular distribution of transporters. SERT is subject to internalization after phosphorylation by protein kinases. We will employ FRET microscopy combined with appropriate manipulations to elucidate pathways of transporter recycling. Because SERT is highly relevant to current grant application focuses on visualizing SERT and its functional states in intact cells in order to understand the mechanisms of its operation and regulation.

Neurotransmitter transporters are obligatory to terminate neural transmission: this is achieved by coupling neurotransmitter reuptake to a preexisting sodium gradient. It has been recognized that neurotransmitter transporters are organized in a distinct quatemary structure. However, these results have been obtained with rather harsh methodology and therefore, it was not clear if neurotransmitter transporters form oligomeric structures at the cellular surface of living cells. Hence, the current project was based on the hypothesis that (i) neurotransmitter transporters are organized in an oligomeric structure in living cells, and that (ii) this oligomeric state is required for their activity (i.e. transport). To achieve these goals, we introduced fluorescence resonance energy transfer (FRET) microscopy at our institute and the field of neurotransmitter transporters. By this means, we monitored the constitutive quaternary organization of neurotransmitter transporters at the plasma membrane. Furthermore, we examined several distinct oligomerization domains in the transporters for serotonin and gamma-amino-butyric acid (GABA). We found that a leucine heptad repeat in transmembrane domain 2 plays an important role to support oligomer formation in the rat GABA transporter. Disruption of this leucine repeat motif resulted in loss of oligomerization but not of function. We determined the role of oligomerization in that it supports the passage of the rigid quality control mechanisms in the endoplasmic reticulum: only properly oligomerized transporters are allowed to exit. However, oligomeric structure is not enough to travel to the cell surface. Transporter proteins also need to interact with a component of the vesicular cargo machinery that shuttles newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus (the COPII vesicles). Functionally, transporter do not need the oligomeric state for reuptake; however, they need the oligomerization to function in reverse, i.e. carrier-mediated efflux depends on this specific structural arrangement. And this forms the basis of our contribution to a better understanding of the actions of amphetamines (like methylene-dioxymethamphetamine = ecstasy), illicit drugs that are widely abused.