Natural and non-canonical mutagenesis studies of receptor gating in 5-HT3A receptors

The 5-HT_(3A) receptor is a homopentameric ligand-gated ion channel belonging to the Cys-loop family of receptors, members of which have many functions and are important targets for a variety of diseases. Previously, our lab has elucidated key binding interactions between these receptors and their ligands. However, the "gating" events that link binding of ligand to receptor activation are less well characterized. While obtaining high-resoln. crystal structures of transmembrane proteins can be difficult, a recent crystal structure of the mouse 5-HT_(3A) receptor has guided our search for intramol. interactions involved in gating. Focusing on regions identified to be important for gating in homologous receptors, we have used both conventional and non- canonical amino acid mutagenesis to identify interactions in the m5-HT_(3A) receptor that connect ligand binding to receptor activation. Previous research on Cys-loop receptors has shown that interplay between the M2-M3 loop and the Cys-loop plays a role in gating. Modifying residues in this region, we expressed mutant m5-HT_(3A) receptors in Xenopus laevis oocytes and monitored their function via two- electrode voltage clamp electrophysiol. Years ago our group identified a crit. proline residue in the M2-M3 loop, Pro281, that cis /trans isomerizes during gating. We now show that the adjacent T280 in the M2-M3 loop is involved in gating, and is energetically coupled to E53 in the Cys-loop. Changes to I283 in the M2- M3 loop and P143 in the Cys-loop affect gating as well. Conformational changes in the C-loop of Cys-loop receptors have also been previously identified as important for gating. Mutagenesis studies in the α7 nicotinic acetylcholine receptor identified residues proximal to the C-loop in the β7, β9, and β10 strands that affect channel gating. Surprisingly, these residues are not strictly conserved in m5-HT_(3A). Using double mutant cycle anal., we have investigated the energetic coupling of T152, E209, and N101 to one another as well as to the aforementioned residues in the M2- M3 loop and the Cys- loop in m5-HT_(3A). Efficacy expts. using partial agonists are used to distinguish whether the functional changes we observe are related to gating or ligand binding. This study increases our understanding of gating dynamics in Cys-loop receptors, and has relevance to the design of drugs (esp. allosteric modulators) for diseases involving these receptors