title: Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA.
abstract: Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.
authors.affiliation: LaGuardia Community College, City University of New York, Long Island City, NY 11101, USA.
authors.forename: Iván O
authors.initials: IO
authors.lastname: Rivera-Torres
authors.affiliation: Department of Chemistry, CUNY Graduate Center and Institute for Macromolecular Assemblies, College of Staten Island, City University of New York, Staten Island, NY 10314, USA.
authors.forename: Tony B
authors.initials: TB
authors.lastname: Jin
authors.affiliation: The Rockefeller University, New York, NY 10065, USA.
authors.forename: Martine
authors.initials: M
authors.lastname: Cadene
authors.affiliation: The Rockefeller University, New York, NY 10065, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: Department of Chemistry, CUNY Graduate Center and Institute for Macromolecular Assemblies, College of Staten Island, City University of New York, Staten Island, NY 10314, USA.
authors.forename: Sébastien F
authors.initials: SF
authors.lastname: Poget
mesh_terms: D000494:Allosteric Regulation; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D018999:Charybdotoxin / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D015221:Potassium Channels / Q000378:metabolism*; D011336:Probability; D011485:Protein Binding; D017433:Protein Structure, Secondary; D030162:Reptilian Proteins / Q000737:chemistry*; D012910:Snake Venoms / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014118:Toxins, Biological / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D004220:Disulfides; D004546:Elapid Venoms; D008567:Membranes, Artificial; D010455:Peptides; D015221:Potassium Channels; D030162:Reptilian Proteins; D012910:Snake Venoms; D014118:Toxins, Biological; C000616613:Tx7335 protein, Dendroaspis angusticeps; C419863:bucandin; D018999:Charybdotoxin