Structural rearrangement in the pore of a ligand gated ion channel during gating

Ligand-gated ion channels (LGIC) are responsible for rapid cellular communication between neurons in the nervous system. The serotonin 3A receptor (5-HT 3AR), a member of the Cys-loop superfamily of LGIC which includes the nicotinic acetylcholine receptors (nAChR), the GABAA and GABA C receptors, and the glycine receptor, is a homopentameric protein formed by the association of five identical 5-HT3A subunits. The work presented in this dissertation seeks to address how Cys-loop LGIC function upon binding neurotransmitter by using the 5-HT3A receptor as a model for this family of proteins.; The structural rearrangements that occur upon LGIC activation (opening) and deactivation (closing) is collectively referred to as receptor "gating". To study how the 5-HT3AR gates, the substituted cysteine accessibility method (SCAM) was employed to determine the conformational rearrangements that occur within the pore upon ligand binding and subsequent receptor activation. By looking at the pattern of cysteine modification using thiol-modifying reagents, I show that the pore-lining M2 transmembrane domain of the 5-HT3AR adopts an alpha helical conformation in the open state of the receptor. By applying these reagents in either the open (in the presence of serotonin) or closed states, I was also able to determine that the gate (the portion of the receptor which acts as a barrier in the closed state and moves to allow for current flow in the open state) resides in the upper half of the M2, close to the extracellular membrane boundary. By contrast, using Cd2+, I provide evidence for minimal movement during receptor activation and deactivation in the bottom half of the M2 domain (close to the intracellular membrane boundary), in the putative selectivity filter of the receptor. The studies presented here thus provide a working model for the structural rearrangements that occur within the LGIC pore during gating. Identifying the molecular and biophysical mechanisms which govern the gating of these receptors will lead to a better understanding of how these proteins shape the complex temporal and spatial codes that underlie the computation of the nervous system.