Date of Original Version
Cell & Molecular Biology
We report the first successful insertion of an engineered, high-affinity alpha-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, KV4.2, using a short, intra-protein embedded sequence (GGWRYYESSLEPYPDGG), derived from a previously described mimotope peptide, HAP. A major benefit to this approach is the ability to live-image the distribution and fate of functional channels on the plasma membrane surface. The Bgtx binding sequence was introduced into the putative extracellular loop between the S1 and S2 transmembrane domains of KV4.2. Following co-expression with KChIP3 in tsA201 cells, S1-S2 HAP-tagged channels express at levels comparable to wild-type KV4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed KV4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged KV4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged KV4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1-S2-tagged KV4.2 channels are minimally affected by the binding of Bgtx, suggesting a limited role if any for the S1-S2 loop in voltage sensing or gating associated conformational changes. Successful functional insertion of the HAP sequence into the S1-S2 linker of KV4.2 suggests that other related channels may similarly be amenable to this tagging strategy.
Leonard Moise, Jing Liu, Evgeny Pryazhnikov, Leonard Khiroug, AndreasJeromin & Edward Hawrot (2010) KV4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxinbinding provide a new tool for studies of channel expression and localization, Channels, 4:2,115-123, DOI: 10.4161/chan.4.2.10878
Available at: https://doi.org/10.4161/chan.4.2.10878
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