Mechanism of μ-Conotoxin PIIIA Binding to the Voltage-Gated Na+ Channel NaV1.4

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.

Systematic Study of Binding of μ-Conotoxins to the Sodium Channel NaV1.4

“…As discussed above for GIIIA, R20 makes contact with the outer D762 residue, and the EEDD ring is not wide enough to accommodate a second arginine on top of R14 and K17, which have established contacts with the EEDD ring. The Na V 1.4-PIIIA complex was also modeled in [ 27 ], where two binding modes with similar binding free energies were proposed. The first involving K9 and R12 is supported neither by the experimental data ( Table 1 ) nor by the arguments based on alignment with GIIIA, which has the best characterized binding mode experimentally.…”

“…For this reason, initial efforts have focused on modeling of Na V 1.4 channel, as a great deal of functional data is available for this channel. These studies include binding of tetrodotoxin [ 25 , 26 ] and µ -conotoxins [ 27 , 28 , 29 ] to the Na V 1.4 channel.…”

Computational Approaches for Designing Potent and Selective Analogs of Peptide Toxins as Novel Therapeutics

“…A similar study has also been performed for binding of μ-conotoxin PIIIA to Na V 1.4 [81], where two distinct binding modes with very similar binding free energies have been found. This situation is very different from toxin binding to potassium channels where the pore-inserting lysine ensures a unique binding mode and a complete blocking of the narrow selectivity filter.…”

“…Thus, one can use the channel-blocking capacity of a toxin to distinguish among the predicted binding modes. Unfortunately, there are limited functional data on binding of μ-PIIIA [82,83], so it is difficult to check the validity of the complex structure and the Na V 1.4 model used in [81]. The possibility of multiple binding modes with similar binding free energies needs to be investigated further using the Na V 1.4 model that has already been validated with the μ-GIIIA data [12].…”

Binding Modes of Two Scorpion Toxins to the Voltage-Gated Potassium Channel Kv1.3 Revealed from Molecular Dynamics

“…However, both experimental and theoretical studies have suggested that these assumptions are invalid in certain systems. For example, a single mutation can induce significant structural changes to a toxin [ 41 , 42 , 43 ] and multiple binding modes of similar energetics are required to describe the action of certain toxins [ 44 , 45 , 46 ]. Therefore, experimental data can be difficult to interpret.…”