Abstract
Several subtypes of voltage-gated Na+ (NaV) channels are important targets for pain management. μ-Conotoxins isolated from venoms of cone snails are potent and specific blockers of different NaV channel isoforms. The inhibitory effect of μ-conotoxins on NaV channels has been examined extensively, but the mechanism of toxin specificity has not been understood in detail. Here the known structure of μ-conotoxin PIIIA and a model of the skeletal muscle channel NaV1.4 are used to elucidate elements that contribute to the structural basis of μ-conotoxin binding and specificity. The model of NaV1.4 is constructed based on the crystal structure of the bacterial NaV channel, NaVAb. Six different binding modes, in which the side chain of each of the basic residues carried by the toxin protrudes into the selectivity filter of NaV1.4, are examined in atomic detail using molecular dynamics simulations with explicit solvent. The dissociation constants (K d) computed for two selected binding modes in which Lys9 or Arg14 from the toxin protrudes into the filter of the channel are within 2 fold; both values in close proximity to those determined from dose response data for the block of NaV currents. To explore the mechanism of PIIIA specificity, a double mutant of NaV1.4 mimicking NaV channels resistant to μ-conotoxins and tetrodotoxin is constructed and the binding of PIIIA to this mutant channel examined. The double mutation causes the affinity of PIIIA to reduce by two orders of magnitude.
Highlights
Voltage-gated sodium (NaV) channels play a vital role in cell excitability
Many polypeptide toxins isolated from venomous animals such as scorpions, cone snails and spiders selectively interfere with the gating mechanisms and ion conduction properties of certain subtypes of NaV channels
A family of peptide toxins isolated from venoms of cone snails, referred to as m-conotoxins, are potent and selective blockers of NaV channels. m-Conotoxins typically consist of 20–25 residues, six of which are cysteines forming three disulfide bridges, known as the inhibitor cystine knot [4,5]
Summary
Voltage-gated sodium (NaV) channels play a vital role in cell excitability. Several subtypes of NaV channels such as NaV1.3, NaV1.7, NaV1.8 and NaV1.9 are involved in the pain pathway [1], and are important targets for pain management. Many polypeptide toxins isolated from venomous animals such as scorpions, cone snails and spiders selectively interfere with the gating mechanisms and ion conduction properties of certain subtypes of NaV channels. These toxins are promising scaffolds for novel pesticides and analgesics [2,3]. A family of peptide toxins isolated from venoms of cone snails, referred to as m-conotoxins, are potent and selective blockers of NaV channels. Some among the most wellcharacterized m-conotoxins are GIIIA [6], PIIIA [7], SmIIIA [8] and KIIIA [9]. The R14A mutant of KIIIA was found to be 10-fold selective for NaV1.7 over NaV1.2 and NaV1.4 [18], suggesting that in principle selective inhibitors of NaV1.7 can be developed from m-conotoxins
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