A chemical-scale study on the ligand-binding site of a serotonin-gated ion channel

Signal transmission is a combination of electrical and chemical processes. Upon binding neurotransmitters, ligand-gated ion channels open to allow ion flux, which converts chemical signals to electrical signals. In this thesis, experiments in conjunction with computations are utilized to study the mechanism of the ligand-binding process.; The target receptor is a serotonin-gated chloride channel, the MOD-1 receptor. From the viewpoint of a chemist, we explore the specific orientation of the agonist inside the binding pocket and the specific non-covalent interactions responsible for binding. In Chapter 3, computational chemistry is used to build a homology model of MOD-1 using the acetylcholine binding protein template. We proceed to dock the agonist into the binding pocket. The binding pattern from the model provides guidance for the ensuing experimental studies.; Unnatural amino acid mutagenesis is a powerful tool to modify the structure of the protein at the chemical level. Systematic perturbations can be introduced at a specific amino acid. Therefore, specific non-covalent interactions, such as hydrogen bonding and cation-pi interactions can be probed. In Chapter 2, we prove that cation-pi interactions between the agonist serotonin and Trp 226 in loop C of MOD-1 play a key role in binding the ligand. Surprisingly, this cation-pi site in MOD-1 is different from that in the serotonin type 3 receptor although these two receptors both bind serotonin, and they are highly homologous. In Chapter 4, we further show that hydrogen bonds between serotonin and Gln 228 and Asn 223 in MOD-1 are important in the binding process. Both conventional and unnatural amino acid mutagenesis are used in conjunction with serotonin analogues. The results from these thorough structure-function studies confirm aspects of the hydrogen bond pattern described in the model.; In Chapter 5, we apply another strategy called the tethered agonist approach to further probe the agonist binding site. This is another elegant example of the effectiveness of the nonsense suppression method.