Chemical Scale Investigations of Ligand-Gated Ion Channels Using Unnatural Amino Acids

Citation

Beene, Darren Lee (2005) Chemical Scale Investigations of Ligand-Gated Ion Channels Using Unnatural Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/B6A3-VT59. https://resolver.caltech.edu/CaltechETD:etd-04262005-153907

Abstract

The Cys loop receptors, a family of ligand-gated ion channels, mediate fast synaptic transmission throughout the peripheral and central nervous systems. These are large multisubunit proteins, whose primary function is to transduce a chemical signal, binding of a neurotransmitter, into an electrical signal, ion flux across the cell membrane. These receptors have been implicated in several disease states and represent major therapeutic targets. The work presented in this thesis focuses on the chemical-scale elucidation of Cys loop receptors. The main approach of this work is the structure-function study using in vivo nonsense suppression methods. This technique allows for the site-specific incorporation of an unnatural amino acid into a protein expressed in a living cell.

Nonsense suppression methods were used to incorporate a series of fluorinated tryptophan derivatives into the binding site of the 5-HT3R. This study identified a cation-pi interaction between Trp 183 and the neurotransmitter, serotonin. A similar study using fluorinated phenylalanine derivatives identified a cation-pi binding site at Tyr 198 in the GABAC receptor. These studies build on previous work from our research group and provide further evidence that the cation-pi interaction is a common feature in ligand recognition by Cys loop receptors.

Nonsense suppression was also used to examine the role of several tyrosine residues in the 5-HT3R. Here the findings demonstrated that the side chains of Tyr 143 and 153 make functionally important hydrogen bonds. These data were used to refine several computational models of serotonin docked into the binding site.

Structure-function studies of two conserved prolines in the M2-M3 loop showed that this region of the receptor is involved in the conformational changes associated with receptor activation. The data also provide preliminary evidence that Pro 308 may serve as hinge for the gating movement of the M2 helix.

Thesis Files