Title: Discovery and Characterization of a Novel Toxin from Dendroaspis Angusticeps, Named TX7335, with an Activating Effect on the Potassium Channel KscA
Abstract: Due to their important role in essential physiological processes such as the propagation of the nerve signal or regulation of the heartbeat, potassium channels are common targets for animal toxins. These toxins provide valuable tools for the study of ion channel function and have potential as lead compounds for drug development. Most toxins affecting potassium channels act as pore blockers, thus inhibiting potassium flow. Using a direct pull-down toxin binding assay with immobilized channel (the bacterial potassium channel KcsA) and crude Dendroaspis angusticeps venom, we identified a novel toxin binder of KcsA, which we called Tx7335. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulfide bonds that belongs to the family of the three-finger toxins, but with a unique modification of its disulfide-bridge scaffold. A functional analysis of Tx7335's effect on KcsA revealed a dose-dependent increase in both open probabilities and mean open times, thus leading to an increase in potassium conductance. Functional assays were also performed with a mutant of KcsA that mimicks the sequence of eukaryotic channels in the outer vestibule region and can bind the pore blocker charybdotoxin. This mutant showed the same susceptibility to the toxin, indicating that the binding site for Tx7335 is distinct from that of pore-blocker toxins. Given the fact that the toxin only showed an effect when added to the extracellular side of the channel, the binding site must be far removed from the intracellular pH gate. We therefore propose that Tx7335 increases potassium flow through KcsA by enhancing recovery from the inactivated state.