Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels

Richard J. Lewis, Christina I. Schroeder, Jenny Ekberg, Katherine J. Nielsen, Marion L. Loughnan, Linda Thomas, Denise A. Adams, Roger Drinkwater, David J. Adams, Paul F. Alewood

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Abstract

μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [ 3H]saxitoxin and 125I-TIIIA from rat brain (Na v1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na + channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na + current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.

Original languageEnglish
Pages (from-to)676-685
Number of pages10
JournalMolecular Pharmacology
Volume71
Issue number3
DOIs
Publication statusPublished - Mar 2007
Externally publishedYes

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Conotoxins
Voltage-Gated Sodium Channels
Tetrodotoxin
Mollusk Venoms
Tulipa
Saxitoxin
Peptides
Membranes
Sodium Channels
Hydroxyproline
Snails
Xenopus laevis
Brain
Spinal Ganglia
Post Translational Protein Processing
Glutamine
Alanine
Inhibitory Concentration 50
Oocytes
Skeletal Muscle

Cite this

Lewis, R. J., Schroeder, C. I., Ekberg, J., Nielsen, K. J., Loughnan, M. L., Thomas, L., ... Alewood, P. F. (2007). Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels. Molecular Pharmacology, 71(3), 676-685. https://doi.org/10.1124/mol.106.028225
Lewis, Richard J. ; Schroeder, Christina I. ; Ekberg, Jenny ; Nielsen, Katherine J. ; Loughnan, Marion L. ; Thomas, Linda ; Adams, Denise A. ; Drinkwater, Roger ; Adams, David J. ; Alewood, Paul F. / Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels. In: Molecular Pharmacology. 2007 ; Vol. 71, No. 3. pp. 676-685.
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title = "Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels",
abstract = "μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35{\%} homology to other μ-conotoxins. TIIIA potently displaced [ 3H]saxitoxin and 125I-TIIIA from rat brain (Na v1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na + channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na + current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.",
author = "Lewis, {Richard J.} and Schroeder, {Christina I.} and Jenny Ekberg and Nielsen, {Katherine J.} and Loughnan, {Marion L.} and Linda Thomas and Adams, {Denise A.} and Roger Drinkwater and Adams, {David J.} and Alewood, {Paul F.}",
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Lewis, RJ, Schroeder, CI, Ekberg, J, Nielsen, KJ, Loughnan, ML, Thomas, L, Adams, DA, Drinkwater, R, Adams, DJ & Alewood, PF 2007, 'Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels' Molecular Pharmacology, vol. 71, no. 3, pp. 676-685. https://doi.org/10.1124/mol.106.028225

Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels. / Lewis, Richard J.; Schroeder, Christina I.; Ekberg, Jenny; Nielsen, Katherine J.; Loughnan, Marion L.; Thomas, Linda; Adams, Denise A.; Drinkwater, Roger; Adams, David J.; Alewood, Paul F.

In: Molecular Pharmacology, Vol. 71, No. 3, 03.2007, p. 676-685.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels

AU - Lewis, Richard J.

AU - Schroeder, Christina I.

AU - Ekberg, Jenny

AU - Nielsen, Katherine J.

AU - Loughnan, Marion L.

AU - Thomas, Linda

AU - Adams, Denise A.

AU - Drinkwater, Roger

AU - Adams, David J.

AU - Alewood, Paul F.

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N2 - μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [ 3H]saxitoxin and 125I-TIIIA from rat brain (Na v1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na + channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na + current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.

AB - μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [ 3H]saxitoxin and 125I-TIIIA from rat brain (Na v1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na + channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na + current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.

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