Purification and cloning of toxins from elapid venoms that target cyclic XXXX -gated ion channels.

@entity1522 @entity1238 @entity535 @entity377 @entity70831
In 1999, we purified pseudechetoxin (PsTx), the first peptide toxin known to block cyclic @entity377 -gated (CNG) ion channels, from the venom of Pseudechis australis [Brown, R. L., Haley, T. L., West, K. A., and Crabb, J. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 754-759]. Here we report the cloning of the cDNA encoding PsTx, as well as the discovery and cloning of pseudecin, a homologous toxin from the venom of Pseudechis porphyriacus. The mature proteins are 211 and 210 @entity1522 in length, and the @entity535 sequences are 96.7% identical, differing in only seven residues. The purified toxins were applied to outside-out patches excised from @entity1238 oocytes expressing CNG channels composed of the rod @entity70831 or olfactory CNGA2 channel subunits. Surprisingly, these patch-clamp studies revealed a 30-fold difference in affinity between PsTx and pseudecin for channels composed of CNGA2 subunits. The apparent K(i) of PsTx was 15 nM, while the affinity of pseudecin was 460 nM. The difference in affinities for the @entity70831 subunit from rod photoreceptors was less pronounced, but the affinity of PsTx was 70 nM, compared with 1000 nM for pseudecin. This difference in affinity may be instructive as we attempt to identify the regions of the toxins that contact CNG channels. As the only known protein blockers of CNG channels, these toxins promise to be valuable tools to study the structure of the external face of these channels.
