Engineering Subunit and Nonsense Suppression Stoichiometry in Potassium Channels

Abstract

Multi-subunit membrane proteins employ chemically diverse inter- and intramolecular contacts to regulate their function. Potassium channel slow inactivation relies on a hydrogen bond network around the selectivity filter which supports an internal timing mechanism to control the transition between conducting and non-conducting conformations. Previously we used targeted replacement of W434 with the isosteric non-canonical amino acid 2-amino-3-indol-1-yl-propionic acid (Ind) to highlight the importance of the indole H-bond of this highly conserved Trp residue. However, these experiments were unable to nuance the specific contributions of individual channel subunits to channel inactivation. We therefore designed an engineered protein approach whereby complementary intein sequences are incorporated into the N- or C- termini of Shaker monomers, respectively, flanked by previously identified endoplasmic reticulum (ER) retention signals. The data shows that functional channels composed of monomeric ER-intein subunits are restricted from the surface membrane. However, co-expression of complementary ER-intein monomers results in robust expression of functional K+ channels through intein ligation and excision of both the ER retention signals and intein sequences. This technique was used to express Shaker channels with two W434Ind monomers within a single tetramer, producing an intermediate inactivation rate, consistent with the breakage of multiple H-bonds required for full inactivation. In addition, the fidelity of the stoichiometric assembly was assayed by TEA block of channels with zero, two or four T449F mutations. The development of this approach allows for regulation of subunit stoichiometry in multimeric complexes and advances the use of nonsense suppression by demonstrating a solution to incorporate multiple non-canonical amino acids into a single peptide and multimeric complexes. Specifically, the reduction of overall protein expression that is normally compounded at the second suppression site is effectively abrogated because two transcripts are suppressed independently then ligated as proteins.