K(V)4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization.
We report the first successful insertion of an engineered, high-affinity alpha-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, K(V)4.2, 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 K(V)4.2. Following co-expression with KChIP3 in tsA201 cells, S1-S2 HAP-tagged channels express at levels comparable to wild-type K(V)4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed K(V)4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged K(V)4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged K(V)4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1-S2-tagged K(V)4.2 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 K(V)4.2 suggests that other related channels may similarly be amenable to this tagging strategy.
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA.
Leonard
L
Moise
Jing
J
Liu
Evgeny
E
Pryazhnikov
Leonard
L
Khiroug
Andreas
A
Jeromin
Edward
E
Hawrot
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D006624:Hippocampus / Q000196:embryology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051605:Kv Channel-Interacting Proteins / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D021381:Protein Transport; D011869:Radioligand Assay; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051672:Shal Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D002038:Bungarotoxins; D051605:Kv Channel-Interacting Proteins; D018091:Receptors, AMPA; D011993:Recombinant Fusion Proteins; D051672:Shal Potassium Channels; C104722:glutamate receptor ionotropic, AMPA 2