Conformational mechanisms underlying allosteric regulation of the human serotonin transporter - PROJECT SUMMARY The human serotonin transporter (hSERT) plays a critical role in regulating serotonin (5-HT) signaling across nearly all major systems in the body. Dysregulation of hSERT is linked to numerous psychiatric and gastrointestinal disorders, making hSERT a primary target for clinical therapeutics including selective serotonin reuptake inhibitors (SSRIs). While the core ion-coupled transport cycle of hSERT is well characterized, the allosteric mechanisms that fine-tune its activity to meet diverse physiological demands remain poorly understood. This proposal aims to define the structural mechanisms by which 5-HT and the microbial metabolite butyrate allosterically shape hSERT’s conformational landscape to modulate its function. Aim 1 will leverage innovative cryo-EM approaches capable of resolving the full range of conformational states that define hSERT’s transport cycle, enabling the distinct structural effects of ligand binding at the central (S1) and allosteric (S2) substrate- binding sites to be isolated and characterized. These conformational changes will be directly linked to transport activity using complementary 5-HT uptake and electrophysiological assays. Aim 2 will expand our understanding of hSERT allosteric regulation by identifying the binding site of butyrate, characterizing its effects on hSERT’s conformational equilibrium, and determining its impact on transport activity. The training plan outlined in this fellowship is designed to strengthen technical and conceptual expertise in membrane protein biochemistry, single-particle cryo-EM, and electrophysiology. Mentorship and training from Dr. Eric Gouaux, an internationally recognized leader in membrane protein structural biology, and Dr. Michael Kavanaugh, an expert in transporter electrophysiology, will ensure the successful completion of the proposed aims. Together, these studies will advance the fundamental understanding of hSERT regulation and contribute to a broader framework for understanding allosteric modulation in neurotransmitter transporters, informing the development of innovative therapeutic strategies for disorders involving transporter dysfunction.
