Unexpected synergism between Tetrodotoxin and μ-conotoxin in blocking voltage-gated sodium channels

Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, μ-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered μ-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain NaV1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded NaV complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in NaV pharmacology.

[1]  D. Tieleman,et al.  Docking of μ-Conotoxin GIIIA in the Sodium Channel Outer Vestibule , 2007, Channels.

[2]  Brian J. Smith,et al.  Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels* , 2007, Journal of Biological Chemistry.

[3]  R. Horn,et al.  A Cation-π Interaction Discriminates among Sodium Channels That Are Either Sensitive or Resistant to Tetrodotoxin Block* , 2007, Journal of Biological Chemistry.

[4]  R. French,et al.  Marine Toxins That Target Voltage-gated Sodium Channels , 2006, Marine Drugs.

[5]  Brian J. Smith,et al.  Structural and functional diversities among mu-conotoxins targeting TTX-resistant sodium channels. , 2006, Biochemistry.

[6]  William A. Catterall,et al.  International Union of Pharmacology. XLVII. Nomenclature and Structure-Function Relationships of Voltage-Gated Sodium Channels , 2005, Pharmacological Reviews.

[7]  Brian J. Smith,et al.  Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels. , 2005, Biochemistry.

[8]  R. French,et al.  Sodium channel toxins--receptor targeting and therapeutic potential. , 2004, Current medicinal chemistry.

[9]  J. Boorman,et al.  Voltage-gated sodium channels and pain pathways. , 2004, Journal of neurobiology.

[10]  S. Heinemann,et al.  Molecular basis for pharmacological differences between brain and cardiac sodium channels , 1992, Pflügers Archiv.

[11]  D. Yoshikami,et al.  Mu-conotoxin SmIIIA, a potent inhibitor of tetrodotoxin-resistant sodium channels in amphibian sympathetic and sensory neurons. , 2002, Biochemistry.

[12]  H. Fozzard,et al.  Electrostatic and Steric Contributions to Block of the Skeletal Muscle Sodium Channel by μ-Conotoxin , 2002, The Journal of general physiology.

[13]  W. Catterall,et al.  Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. , 2000, Biochimie.

[14]  A. Shcherbatko,et al.  Distinction among Neuronal Subtypes of Voltage-Activated Sodium Channels by μ-Conotoxin PIIIA , 2000, The Journal of Neuroscience.

[15]  G M Whitesides,et al.  A trivalent system from vancomycin.D-ala-D-Ala with higher affinity than avidin.biotin. , 1998, Science.

[16]  R. Kallen,et al.  Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis. , 1997, Biophysical journal.

[17]  R. Horn,et al.  Shifts of Macroscopic Current Activation in Partially Blocked Sodium Channels. Interaction between the Voltage Sensor and a μ-Conotoxin , 1997 .

[18]  R. Horn,et al.  Interactions between a Pore-Blocking Peptide and the Voltage Sensor of the Sodium Channel: An Electrostatic Approach to Channel Geometry , 1996, Neuron.

[19]  R. Horn,et al.  Chimeric study of sodium channels from rat skeletal and cardiac muscle , 1992, FEBS letters.

[20]  R. Gordon,et al.  Action of derivatives of mu-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates. , 1992, Biochemistry.

[21]  G. Tomaselli,et al.  Molecular localization of an ion-binding site within the pore of mammalian sodium channels. , 1992, Science.

[22]  D. Kohda,et al.  Active site of mu-conotoxin GIIIA, a peptide blocker of muscle sodium channels. , 1991, The Journal of biological chemistry.

[23]  R. French,et al.  Block of sodium channels in planar lipid bilayers by guanidium toxins and calcium. Are the mechanisms of voltage dependence the same? , 1986, Annals of the New York Academy of Sciences.

[24]  W. Catterall.  Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. , 1980, Annual review of pharmacology and toxicology.