Structure and Function of Neurotransmitter Transporters

Summary Chemical neurotransmission forms the basis for most neurotransmission and involves the release of neurotransmitter from a presynaptic neuron, the detection of the transmitter on receptors located on both post and presynaptic neurons, and the ‘uptake’ of the transmitter by neurotransmitter sodium symporters (NSSs), thus quenching the neurotransmitter signal. NSSs are sodium-coupled membrane transport proteins that harness the pre- existing ion gradient to ‘pump’ neurotransmitters into cells and ‘up’ their concentration gradient. The removal of transmitter from the synaptic cleft and surrounding extracellular spaces is crucial to neurotransmission and dysfunction of NSSs underpins multiple neurological conditions. Moreover, there are many important therapeutic agents, as well as illicit substances, that target NSSs, including antidepressants, anti-anxiety medications, and amphetamines and cocaine. The goals of the research described in this grant application are to further our understanding of the biochemical mechanism of NSS function, to better understand how small molecule therapeutic agents and illicit substances interact with NSSs, and to provide structure-based understanding of the consequences of selected naturally occurring mutations in NSS genes. The major focus of the research is to study the human serotonin and dopamine transporters using single particle cryo- electron microscopy (cryo-EM) in combination with complementary biochemical, biophysical and computational methods. We are particularly interested in understanding, at the level of molecular detail, how newly discovered small molecules inhibit NSSs via binding to a novel binding site and how these small molecules block isomerization of the transporter to the inward facing conformations required to release transmitter. Moreover, we will elucidate the underpinning molecular interactions between key small molecules and the dopamine transporter to understand the principles of affinity and specificity, and we will also examine how posttranslational modifications, such as phosphorylation, affect transporter conformation. The results from our studies will unveil new small molecule binding sites and cognate small molecules, illuminate structural underpinnings of phosphorylation, and explore the conformational landscape of NSSs in distinct, ligand and ion bound states. Our studies will not only inform the immediate NSS field, but because NSSs are paradigm integral membrane transporter proteins, our studies will enlighten the larger field of membrane transport.