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

J.M. Danse, E.G. Rowan, S. Gasparini, F. Ducancel, H. Vatanpour, L.C. Young, G. Poorheidari, E. Lajeunesse, A.L. Harvey

Research output: Contribution to journalArticle

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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.
LanguageEnglish
Pages153-158
Number of pages5
JournalFEBS Letters
Volume356
Issue number2-3
DOIs
Publication statusPublished - 19 Dec 1994

Fingerprint

Mutagenesis
Potassium Channels
Site-Directed Mutagenesis
Bioactivity
Lysine
Amino Acid Sequence
Protein Isoforms
Pyrrolidonecarboxylic Acid
Synthetic Genes
Elapidae
Amino Acids
Gene encoding
Synaptosomes
Venoms
Electric potential
Viperidae
Glutamine
Escherichia coli
Neurotransmitter Agents
Toxicity

Keywords

  • potassium channel
  • recombinant snake toxin
  • site directed mutagenesis

Cite this

Danse, J.M. ; Rowan, E.G. ; Gasparini, S. ; Ducancel, F. ; Vatanpour, H. ; Young, L.C. ; Poorheidari, G. ; Lajeunesse, E. ; Harvey, A.L. / 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. In: FEBS Letters. 1994 ; Vol. 356, No. 2-3. pp. 153-158.
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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.",
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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.; Rowan, E.G.; Gasparini, S.; Ducancel, F.; Vatanpour, H.; Young, L.C.; Poorheidari, G.; Lajeunesse, E.; Harvey, A.L.

In: FEBS Letters, Vol. 356, No. 2-3, 19.12.1994, p. 153-158.

Research output: Contribution to journalArticle

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T1 - 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

AU - Danse, J.M.

AU - Rowan, E.G.

AU - Gasparini, S.

AU - Ducancel, F.

AU - Vatanpour, H.

AU - Young, L.C.

AU - Poorheidari, G.

AU - Lajeunesse, E.

AU - Harvey, A.L.

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N2 - 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.

AB - 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.

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