title: Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.
abstract: Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX-fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.
authors.affiliation: Department of Physiology and Membrane Biology, University of California, Davis, CA 95616;
authors.forename: Drew C
authors.initials: DC
authors.lastname: Tilley
authors.affiliation: Department of Physiology and Membrane Biology, University of California, Davis, CA 95616; Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA 02543;
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Eum
authors.affiliation: Department of Physiology and Membrane Biology, University of California, Davis, CA 95616;
authors.forename: Sebastian
authors.initials: S
authors.lastname: Fletcher-Taylor
authors.affiliation: Department of Physiology and Membrane Biology, University of California, Davis, CA 95616;
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Austin
authors.affiliation: Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA 02543;
authors.forename: Christophe
authors.initials: C
authors.lastname: Dupré
authors.affiliation: Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA 02543;
authors.forename: Lilian A
authors.initials: LA
authors.lastname: Patrón
authors.affiliation: Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and.
authors.forename: Rita L
authors.initials: RL
authors.lastname: Garcia
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616.
authors.forename: Kit
authors.initials: K
authors.lastname: Lam
authors.affiliation: Department of Physiology and Membrane Biology, University of California, Davis, CA 95616; Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Yarov-Yarovoy
authors.affiliation: Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Cohen
authors.affiliation: Department of Physiology and Membrane Biology, University of California, Davis, CA 95616; Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA 02543; jsack@ucdavis.edu.
authors.forename: Jon T
authors.initials: JT
authors.lastname: Sack
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D050091:Dendrimers / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D015640:Ion Channel Gating; D011485:Protein Binding; D051669:Shab Potassium Channels / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013111:Spider Venoms / Q000494:pharmacology*
chemical_list: D060829:Arthropod Proteins; D050091:Dendrimers; D005456:Fluorescent Dyes; C550903:GxTX-1E, Plesiophrictus guangxiensis; C496988:KCNB1 protein, human; D051669:Shab Potassium Channels; D013111:Spider Venoms
keywords: allostery; fluorescence; gating modifier; potassium channel; voltage-gated ion channel