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

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.