The Molecular Basis of Transport Protein Oligomerisation

Neurotransmitter:sodium symporters (NSS) share many structural and functional features. The main physiological function of NSS is to terminate the action of neurotransmitters at their respective pre- and postsynaptic receptors. Biochemical and biophysical experiments indicate that these transporters interact in oligomeric quaternary structures. Fluorescence resonance energy transfer (FRET) micros-copy has provided evidence for a constitutive, homotypic (= only one NSS-subtype expressed) physi-cal interaction of NSS at the cell surface and throughout the biosynthetic pathway. Still, the molecular basis for oligomer formation remains largely unknown. Moreover, it has been shown that members of the NSS-subfamily interact in heterotypic complexes (= two members of the NSS- subfamily interact). Strikingly, conclusive evidence for a physiological role of this heterotypic complex formation or the hetero-expression of two unrelated transport proteins in a single neuron remains scant. In the working hypothesis underlying the current grant application we postulate that homo-oligomer formation is a likely event among NSS. Distinct oligomeric interfaces serve as docking domains between two or more NSS in a complex; hetero-complex formation is possible and may serve a distinct physiological role. We propose to concentrate on the molecular basis of both the homotypic as well as the hetero-typic complex formation. As model systems, we will use two members of the NSS-subfamily, the hu-man serotonin transporter and the rat GABA transporter-1; both proteins are highly relevant to clinical medicine. Based on this working hypothesis the following aims can be defined: to elucidate the molecular determinants that drive assembly in homotypic and heterotypic oli-gomeric protein- protein interactions in distinct members of the NSS. to investigate the possibility of transporter hetero-expression in situ to obtain a better physiological understanding of putative hetero-association in the native environment. to determine the importance of different oligomerization motifs for presumable dynamic/static be-haviour within an oligomeric complex. to identify the amino acid residues that serve as hydrogen bond donors in the oligomeric interface. The results of the studies will help to understand the key role that oligomerization of NSS plays in the living cell. We expect the anticipated data to be of relevance also for the other members of the sub-family. We will generate a model how the molecular basis for oligomer formation influences and drives association in a complex of to date unknown stoichiometry which may also serve to understand the role that oligomerization and/or its disturbance play in diseases.

Neurotransmitter:sodium symporters (NSS) share many structural and functional features. The main physiological function of NSS is to terminate the action of neurotransmitters at their respective pre- and postsynaptic receptors. Biochemical and biophysical experiments indicate that these transporters interact in oligomeric quaternary structures. Fluorescence resonance energy transfer (FRET) micros-copy has provided evidence for a constitutive, homotypic (= only one NSS-subtype expressed) physi-cal interaction of NSS at the cell surface and throughout the biosynthetic pathway. Still, the molecular basis for oligomer formation remains largely unknown. Moreover, it has been shown that members of the NSS-subfamily interact in heterotypic complexes (= two members of the NSS- subfamily interact). Strikingly, conclusive evidence for a physiological role of this heterotypic complex formation or the hetero-expression of two unrelated transport proteins in a single neuron remains scant. In the working hypothesis underlying the current grant application we postulate that homo-oligomer formation is a likely event among NSS. Distinct oligomeric interfaces serve as docking domains between two or more NSS in a complex; hetero-complex formation is possible and may serve a distinct physiological role. We propose to concentrate on the molecular basis of both the homotypic as well as the hetero-typic complex formation. As model systems, we will use two members of the NSS-subfamily, the hu-man serotonin transporter and the rat GABA transporter-1; both proteins are highly relevant to clinical medicine. Based on this working hypothesis the following aims can be defined: to elucidate the molecular determinants that drive assembly in homotypic and heterotypic oli-gomeric protein-protein interactions in distinct members of the NSS. to investigate the possibility of transporter hetero-expression in situ to obtain a better physiological understanding of putative hetero-association in the native environment. to determine the importance of different oligomerization motifs for presumable dynamic/static be-haviour within an oligomeric complex. to identify the amino acid residues that serve as hydrogen bond donors in the oligomeric interface. The results of the studies will help to understand the key role that oligomerization of NSS plays in the living cell. We expect the anticipated data to be of relevance also for the other members of the sub-family. We will generate a model how the molecular basis for oligomer formation influences and drives association in a complex of to date unknown stoichiometry which may also serve to understand the role that oligomerization and/or its disturbance play in diseases.