IDENTIFYING TOXINS THAT INTERACT WITH VOLTAGE GATED POTASSIUM CHANNELS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The aim of this project is to find and sequence new toxins that modify the gating of voltage gated potassium channels. Towards this goal, several (over 10) crude venoms of different tarantulas were screened in a bilayer set up against the voltage gated potassium channel from Aeropyrum pernix. The crude venom from Acanthogonatus pissi (Chilean zebra spider) showed an atypical outcome. Here the ion current through the channel increases upon exposure to venom as opposed to the more typical observation of current decrease. The crude venom was separated on offline HPLC and the bilayer screening repeated on the HPLC fractions. A fraction was found that mirrors the behaviors of the crude venom. We determined the open probability of the channel with and without the venom fraction of interest and it is clear that the open probability is increased with toxin present and that the current increase is not an artifact. Subsequently the two major components of the active fraction were de novo sequenced. These two toxins contain 6 cysteines and exhibit cysteine spacing similar to known tarantula toxins, but the residues surrounding the cysteine residues is quite different form known tarantula toxins. The de novo sequences were confirmed with synthetic peptides and mass spectrometry. Weare now in the process of trying to fold the two toxins into their native state and repeat the bilayer experiments to test whether the sequenced toxins cause the increase in the channel open probability. Several different folding conditions are currently under investigation that all lead to different HPLC retention profiles.