Incorporation of Unnatural Amino Acids into Trimeric Ion Channels

Abstract

The incorporation of unnatural amino acid side chains into ion channels has greatly aided the understanding of these physiologically and pharmacologically important membrane proteins. Although the so-called in vivo nonsense suppression method for the incorporation of unnatural amino acids is extremely versatile, not all ion channels appear to be amenable to this experimental approach. In fact, the technique has thus far been primarily applied to pentameric ligand-gated ion channels, and those in the voltage-gated channel family. Here, we show that unnatural derivatives of Phe and Trp can be successfully incorporated into the trimeric ASIC1 cation channel, from the ENaC/degenerin family of ion channels, as well as the P2X2 receptor, an ATP-gated ion channel. Importantly, unnatural derivatives were well tolerated at different positions in both extracellular and transmembrane domains, with rescued channels displaying robust expression and WT-like gating characteristics. The results suggest that these important ion channel families are amenable to novel chemical biology tools such as the incorporation of unnatural amino acids and thus open up new avenues for their study.