This project explores the molecular mechanism of active transport of neurotransmitters. This process plays an important role in synaptic transmission, both in the termination of the signal (high affinity cytoplasmic transporters) and in the scavenging and storage of the neurotransmitter (subcellular storage organelles). Membrane vesicles derived from rat brain synaptosomes and from chromaffin granules of these bovine adrenal medulla will be used to study the two prototypes chosen: the sodium-chloride couple Gamma-aminobutyric acid transporter and the hydrogen-ion coupled biogenic amine transporter. The studies will be at a mechanistic and at a molecular level. The studies at the molecular level are based on the technologies we have developed to solubilise, identify, purify and reconstitute the transport protein. We will probe the structure of the binding site(s), the architecture of the protein, its oligomeric structure and its symmetry. Binding of the neurotransmitters and the coupling ions will be studied in detail. The former will be done with the aid of substrate derivatives, the latter through the use of chemical modifiers directed to the binding sites. The mechanistic studies will be carried out based on the knowledge that the movements of hydrogen ions (in the case of the biogenic amines transporter), and sodium and chloride ions (in the case of Gamma-aminobutyric acid transporter), are coupled to neurotransmitter accumulation.
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