Tailoring μ-Conotoxin-KIIIA to Selectively Inhibit NaV1.7

Abstract

Voltage-gated sodium(NaV) channels are critical in initiating and propagating nerve impulses in a variety of tissues including, skeletal(NaV1.4) and cardiac(NaV1.5) muscle, brain(NaV1.2), and peripheral nerve(NaV1.7). Of special interest as a potential drug target is NaV1.7, which mediates propagation of pain signals from receptors to the CNS. Selective blockers of this channel could offer an important analgesic treatment. μ-Conotoxin KIIIA blocks NaV1.2, 1.4 and 1.7, expressed in mammalian cells(Kds: 5, 37, and 97nM respectively). We found two positions in KIIIA, where substitutions alter its selectivity among these channels. KIIIA-H12A shows increased potency for NaV1.7 compared with NaV1.2 and 1.4, but remains more potent against NaV1.2 than NaV1.7(Kds, μM: NaV1.2,10.8; NaV1.4,110 and NaV1.7,19. The second derivative, KIIIA-R14A, inhibits NaV1.7 more strongly than both NaV1.2 and NaV1.4 with Kds (μM): 0.5, 1.1 and 5.7 for NaVs 1.7, 1.2, and 1.4, respectively. These changes in toxin selectivity are associated with differences in the outer ring charges of these channels. The “outer ring” pore-vestibule residues in all NaV channels, except hNaV1.7, are EEDD. In hNaV1.7, the third aspartate is replaced by isoleucine. We have tested reciprocal mutations at this position(NaV1.4D/I and NaV1.7I/D), and these mutations account for the differences in Kd for the wild-type toxin. These channel mutants show no difference from the native channels for block by KIIIA-R14A, suggesting the interaction between the toxin/channel pair has been removed (double mutant cycle analysis yields ΔΔG=∼1.7kT). We further examined these two positions by synthesizing the double mutant, KIIIA-H12Q/R14A, to examine its potency against NaV1.7. Preliminary data indicate that the double mutant(KIIIA-H12Q/R14A) inhibits NaV1.7 more strongly than either NaV1.2 or NaV1.4(Kds:23, 286, and 70 μM, respectively). Thus, interactions with this channel residue could be important in designing blockers that differentially interact with the outer ring to increase their selectively for NaV1.7.