Pentameric ligand-gated ion channels (pLGICs), i.e. cys-loop receptors, represent a major family of receptors in the nervous system that mediate rapid excitatory or inhibitory responses in synapses. Cys-loop receptors are targets of clinically important drugs such as sedative hypnotics, cognitive enhancers used in the treatment of neurodegenerative diseases, and anti-emetics used in cancer treatment. Despite their biological and therapeutic importance, atomic structures of many members of this family, including the serotonin receptor (5-HT3), are unknown and the structural mechanisms underlying pLGIC activation remain unresolved. My goal is to break this barrier by determining a series of atomic structures of the 5-HT3 receptor using single-particle cryo-electron microscopy (cryo-EM). My project will rely on the close collaboration between the labs of Yifan Cheng and David Julius here at UCSF, which recently led to the determination of a series of structures of the TRPV1 ion channel at near- atomic resolution (3-4 ) using single particle cryo-EM. Having established a similar collaboration, David Julius' lab will provide training in membrane protein biophysics and biochemistry, enabling me to identify 5-HT3 variants with properties suitable for high resolution structural work. Making use of the Cheng lab's expertise in single particle cryo-EM, we will then determine high resolution structures of the 5-HT3 receptor in various functional states, including apo-, agonist- and antagonist-bound states, and resolve the binding sites and mechanisms of action of various 5-HT3 agonists and antagonists. Finally, experiments will be carried out to determine the arrangement of subunits within heteromeric 5-HT3, probe the consequences of lipid composition on 5-HT3 structure and function, and determine high resolution structures of these complexes. The experiments proposed in this project will show the structural transitions involved in channel gating, providing an atomic resolution model of 5-HT3 activation and also shedding light on the mechanism of Cys-loop receptors in general. Because activation of the 5-HT3 receptor causes many of the emetic side effects associated with chemotherapy, our studies will also provide a structural framework for the rational design of novel antiemetic drugs and chemotherapeutics with reduced side effects.
Neurotransmitters such as serotonin, acetylcholine and GABA (?-aminobotyric acid) send rapid signals throughout the nervous system by interacting with receptors, termed 'ligand-gated ion channels.' The goal of my project is to visualize the structural transitions that take place within one of these receptors, the serotonin- gated ion channel, as it interacts with neurotransmitters and other drugs. The structures we determine will provide atomic-level insights into the mechanism of the serotonin receptor and the broader class of ligand- gated ion channels to which it belongs, the cys-loop receptor family. .