Investigating Structure-Function Relationships in Ion Channels Using Unnatural Amino Acids

Citation

Eastwood, Amy Lynn (2009) Investigating Structure-Function Relationships in Ion Channels Using Unnatural Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/86B5-SV57. https://resolver.caltech.edu/CaltechETD:etd-01062009-133603

Abstract

Ion channels are proteins that traverse the cell membrane and form gated pores that open and close in response to various stimuli. In order to experimentally probe aspects of ion channel functionality, we performed subtle structure function studies using the in vivo nonsense suppression method, which allows for the incorporation of synthetically accessible unnatural amino acids and hydroxy acids into an ion channel at a site of interest.

Fluorinated aromatic amino acids are good probes for a cation-π interaction because fluorine substituents reduce the binding affinity of the aromatic for a cation in a linear, step-wise fashion. In collaboration with Professor Richard Horn at the Thomas Jefferson University, we substituted a series of fluorinated phenylalanines for important tyrosines in the Shaker B K⁺ channel and experimentally determined that TEA was binding to the residues through a cation-π interaction. We also determined that Ca²⁺ binds to and blocks the Na_V1.4 channel through a cation-π interaction with a tyrosine at the top of the pore of this channel. We found that tetrodotoxin, another channel blocker, also binds to this same residue through a cation-π interaction. Finally, we proved that lidocaine and other local anesthetics bind to a phenylalanine at the bottom of the pore of this channel through a cation-π interaction.

An important aspect of our work is the development of unnatural amino acids that can be used in the study of ion channels through the in vivo nonsense-suppression methodology. We determined that D-amino acids could not be incorporated into ion channels using this method. We synthesized several novel fluorescent-MTS reagents to be used in FRET studies. We probed the sterics around phenylalanines using the unnatural amino acid 3,5-dimethylphenylalanine. We also attempted to incorporate 4-amino-phenylalanine, but, unfortunately, we never saw the enhanced binding of a cationic ligand that was our expected phenotype.

Finally, we also designed and synthesized two α-hydroxy acids capable of site-specific proteolysis upon UV irradiation. We used a tripeptide model system to isolate and characterize the cleavage fragments, proving that these two residues are indeed capable of site-specific proteolysis through the predicted mechanism.

Thesis Files