Methods Mol Biol 2018 Jan 01;1684:253-263. doi: 10.1007/978-1-4939-7362-0_19.

Site-Directed Unnatural Amino Acid Mutagenesis to Investigate Potassium Channel Pharmacology in Xenopus laevis Oocytes.

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Unnatural amino acid mutagenesis is a useful tool enabling detailed investigation of ion channel structure-function relationships and pharmacology. Methods have been developed to apply this technique to different heterologous systems for electrophysiological studies, with each system offering unique advantages and limitations. Synthesis of aminoacylated-tRNA followed by injection into Xenopus laevis oocytes is beneficial because it allows for the incorporation of a wide range of unnatural sidechains, including amino acids with subtle structural differences. Here, we describe a protocol for unnatural amino acid mutagenesis implemented in our lab to study the pharmacology of KCNQ voltage-gated potassium channel opener compounds. This protocol should be applicable to other ion channels and receptor types amenable for functional studies in Xenopus laevis oocytes.

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Genes referenced: mt-tr trna