Synthesis, structure, and mechanism of association of a spider toxin for Kv4 channels. 

 

Many venomous organisms produce toxins that inhibit potassium channels.  These compounds are valuable probes for understanding channel structure, and offer approaches to rational design of drugs directed at specific potassium channels. We have synthesized a spider toxin called HpTx2 in E. coli, and found that it is active against specific isoforms of Kv4 channels, but not Kv1.4 potassium channels.  Future studies will focus on locating and characterizing the toxinís binding site on Kv4 channels, and understanding the details of its molecular interactions. Because of its unique specificity for an important heart channel, and its unusual mechanism of binding, a compound with properties similar to the toxin would have great promise as a drug for treatment of arrhythmias.


 

 

Rational

Drugs that block potassium channels are used to treat several diseases, including cardiac arrhythmias.  Unfortunately, these compounds have proven to be of limited effectiveness, primarily due to lack of specificity.  The figure on the left is a cartoon of the tetrameric potassium channel.  All known potassium channel blockers bind to either Site A or B, the outer and inner vestibules of the pore, respectively.   The difficulty with these sites is specificity:  potassium channels are a large and diverse family of proteins, however the regions of the protein that make up the pore are highly conserved.

 

While the most obvious approach to blocking a channel is to occlude the pore, an alternative approach would be to prevent the channel's gate from opening.  This approach allows a much greater diversity of potential targets within the channel protein, and might offer the prospect for high specificity.  Several peptide toxins from spiders and other venomous organisms are known to specifically inhibit sodium or calcium channels through modification of gating.  A family of spider toxins has been characterized that inhibit the fast recovering cardiac Ito. These include the heteropodatoxins (HpTxs) from the huntsman spider Heteropoda venatoria.

 

 

 

 

 

Purification

The venom from the Heteropoda spider is difficult to obtain.  Therefore, we have expressed and purified the toxin as a fusion protein from E. coli.  The Figure A shows a chromatogram of the crude E. coli extract.  The peak at 35%B is pure channel fusion protein.  The fusion protein (Figure B, lane 1) is then digested with protease to give the fusion partner, without toxin, and a ~3.5 Kd synthetic toxin (lane 2 *).  This can then be further purified to give pure synthetic toxin (lane 3), and the remaining proteins (lane 4).

 

 

 

The purified toxin inhibits Kv4.3 in a voltage fashion similar to the native toxin.  Kv1.4 channels are insensitive to this toxin.

 

 

 

 

 

 

 

 

There will be much more to come, just as soon as we can collect the data.  Check back for more details!

 

 

 

 

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