A Semi-Synthetic Approach to Engineer Ligand- and Voltage-Gated Ion Channels in Live Cells

Site-directed insertion of non-canonical amino acids (ncAAs) or post-translational modifications (PTMs) is often challenging, particularly in large membrane proteins, such as ion channels. Amber-codon suppression mutagenesis is routinely used for this purpose. However, this approach has limitations regarding the side chain identity that can be introduced, as well as the efficiency of incorporation. For this reason, it would be valuable to develop a method that overcomes these issues. Here, we present the development of a intein-based approach for the incorporation of synthetic peptides into ion channels expressed in live cells. The approach utilizes split inteins, which can seamlessly join selected protein segments, to replace selected peptide segments within ion channels with synthetic peptides carrying the desired ncAAs and/or PTMs. We demonstrate the successful implementation of this approach by inserting non-canonical lysine analogs into the extracellular binding pocket of P2X2 receptors and by inserting PTMs into intracellular linkers of the cardiac voltage-gated sodium channel, Nav1.5. Correct reconstitution of full-length ion channels and the impact of the mutations on channel function were verified by Western blots and electrophysiology. The technology has the potential to complement existing ribosome-dependent methods to incorporate ncAAs and PTMs, especially for those that cannot currently be incorporated using existing methods. Additionally, the approach offers a unique way to introduce combinations of multiple ncAAs and/or PTMs, thus enhancing the precision with which we can study ion channel function and pharmacology