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On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: Site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding

Danse, J.M. and Rowan, E.G. and Gasparini, S. and Ducancel, F. and Vatanpour, H. and Young, L.C. and Poorheidari, G. and Lajeunesse, E. and Harvey, A.L. (1994) On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: Site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding. FEBS Letters, 356 (2-3). pp. 153-158. ISSN 0014-5793

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Abstract

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.

Item type: Article
ID code: 17669
Notes: Strathprints' policy is to record up to 8 authors per publication, plus any additional authors based at the University of Strathclyde. More authors may be listed on the official publication than appear in the Strathprints' record.
Keywords: potassium channel, recombinant snake toxin, site directed mutagenesis, Pharmacy and materia medica
Subjects: Medicine > Pharmacy and materia medica
Department: Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences
Related URLs:
    Depositing user: Strathprints Administrator
    Date Deposited: 07 May 2010 11:55
    Last modified: 04 Oct 2012 12:55
    URI: http://strathprints.strath.ac.uk/id/eprint/17669

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