Tricking Out the Toolbox: Use of Genetic Code Expansion for the Study of Ion Channels

Abstract

The application of nonsense suppression strategies to membrane proteins has great potential for the study and manipulation of ion channels and receptors. The technique relies on the incorporation of a stop codon that is subsequently ‘suppressed’ by an orthogonal misacylated tRNA. Ion channels in particular are especially amenable to these approaches due to the built-in signal gain from the high conductance of recorded ions. On one end of the scale of possibility, ‘small mutagenesis’ provides the means for single atom replacement within an amino acid or sub-atomic redistribution of pi electrons of aromatic side-chains. Such approaches provide an atomic resolution functional correlates to match high-resolution structural data, and have been key in decoding the interaction energetics of ligands, therapeutics, backbone contributions, salt-bridges and H-bonds in gating of voltage- and ligand-gated channels and receptors. More unconventional side-chains, such as light activated cross-linkers, hold promise for their ability to characterize transient noncovalent protein contacts. Lastly, encoded amino acid fluorophores, while highly valued, have been technically resistant to ion channel application and largely remain on the experimental fringe. Data will be presented on the use of genetic code expansion as a tool to reveal the basis for inactivation mechanisms employed by voltage-gated sodium and potassium channels.