Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA.

Iván O Rivera-Torres,Sébastien F Poget,Brian T Chait,Tony B Jin,Martine Cadene

doi:10.1038/srep23904

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.

Highlights

Tools and have been used to study functional and regulatory aspects of channel behaviour, but their usefulness is somewhat reduced by the fact that most toxins affecting potassium channels act as pore blockers, inhibiting potassium flow[18]
We have identified and characterized a new mamba toxin Tx7335 from Dendroaspis angusticeps that acts on the bacterial potassium channel KcsA and has a unique channel-activating effect
The three-finger toxin family can be sub-divided based on the sequence length and number of cysteine residues into the short-form toxins with 4 disulphide bonds and the long-form toxins that show an additional disulphide bond in the loop between first and second beta-strand[30]

Summary

Introduction

Tools and have been used to study functional and regulatory aspects of channel behaviour, but their usefulness is somewhat reduced by the fact that most toxins affecting potassium channels act as pore blockers, inhibiting potassium flow[18]. An NMR structure of KcsA bound to the antagonist scorpion toxin charybdotoxin reveals that this toxin binds to KcsA without inducing any structural changes, instead making specific contacts with the extracellular surface of the ion channel that result in pore blockage[19] This lock-and-key mechanism of toxin block has been confirmed in a recent crystal structure of the same toxin in complex with a eukaryotic voltage-gated potassium channel[20]. We present the discovery, isolation and functional characterisation of a distinct channel-opener toxin obtained from eastern green mamba venom This toxin, named Tx7335, increases channel opening events in a dose-dependent manner if added to the extracellular side of a planar bilayer system with reconstituted KcsA single channels. It offers the opportunity for further studying the underlying conformational and dynamic processes involved in potassium channel inactivation through the use of a direct agonist peptide

Methods

Results

Conclusion