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. 2016 Apr 5:6:23904.
doi: 10.1038/srep23904.

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

Iván O Rivera-Torres  1 , Tony B Jin  2 , Martine Cadene  3 , Brian T Chait  3 , Sébastien F Poget  2
Affiliations

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

Iván O Rivera-Torres et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. Pull-down of Tx7335 with immobilised KcsA as shown by MALDI and HPLC analysis.
The main spectrum shows a MALDI mass spectrum of KcsA eluted from a metal affinity resin column after incubation with D. angusticeps venom. The peaks correspond to the triply-charged KcsA species, to the ubiquitin used as an internal calibrant and to the novel toxin Tx7335 that was pulled down by the channel. The insert shows an HPLC chromatogram of the purification of Tx7335 from the affinity column eluate via reverse-phase chromatography.
Figure 2
Figure 2. Sequence alignment of Tx7335 with other three-finger toxins.
The sequence conservation at individual positions in the alignment is indicated by the darkness of the shading. Cysteine residues are printed in bold yellow. The residues differing from the canonical three-finger toxin cysteine scaffold are highlighted in magenta in Tx7335. Toxins are labelled with their Swiss-Prot sequence identifiers except for bucandin from Malayan krait and TxS6C6 from eastern Jameson’s mamba.
Figure 3
Figure 3. Determination of disulphide bond connectivities in Tx7335.
A MALDI-TOF mass spectrum of LysC-cleaved native Tx7335 is shown, and peaks originating from cysteine-containing cleavage products are labelled. The peptide sequences of Tx7335 and bucandin are shown, and cysteine residues are highlighted in yellow. The Tx7335 sequence shows the Cys-containing peptide species in the same colours as the labels of the corresponding peaks in the mass spectrum. The bucandin sequence shows the disulphide connectivities as observed in the crystal structure, and the Tx7335 sequence shows the disulphide bridges as experimentally determined with remaining ambiguities indicated.
Figure 4
Figure 4. Single-channel traces of wild-type and mutant KcsA in artificial bilayers show the activating effect of adding the indicated concentrations of Tx7335 to the outside bath solution.
Shown are 15 s representative traces of 2 minute recordings for each condition. All traces for each channel are from a single bilayer, and recordings were started 2 minutes after each addition of toxin.
Figure 5
Figure 5. Single-channel KcsA traces upon addition of negative control samples (lyophilized HPLC baseline aliquots).
Representative 10 s traces are shown for both WT and pmut3 KcsA bilayers before and after the addition of lyophilized HPLC baseline samples. The same bilayer is shown before and after addition of blank for each channel type.
Figure 6
Figure 6. Single-channel traces and open-/closed-time histograms of A98G KcsA in the absence and presence of varying amounts of Tx7335.
Representative 15 s traces as well as open- and closed-time histograms with their associated MIL fits are shown. For MIL fitting, the long, silent interburst segments as well as areas with 2 simultaneously open channels were removed from the analysis.
Figure 7
Figure 7. Ribbon diagram of the X-ray crystal structure of bucandin.
Cysteines that are conserved between bucandin and Tx7335 are shown in yellow. The extra N-terminal disulphide bridge that characterizes the long-form three-finger toxins is shown in blue. Y25 (which is a cysteine in Tx7335) and C43 (tyrosine in Tx7335) are shown in cyan. N- and C-termini are labelled.
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