Molecular determinants for the subtype specificity of μ-conotoxin SIIIA targeting neuronal voltage-gated sodium channels

Voltage-gated sodium channels (NaV channels) play a pivotal role in neuronal excitability; they are specifically targeted by μ-conotoxins from the venom of marine cone snails. These peptide toxins bind to the outer vestibule of the channel pore thereby blocking ion conduction through NaV channels. μ-Conotoxin SIIIA from Conus striatus was shown to be a potent inhibitor of neuronal sodium channels and to display analgesic effects in mice, albeit the molecular targets are not unambiguously known. We therefore studied recombinant NaV channels expressed in mammalian cells using the whole-cell patch-clamp method. Synthetic μSIIIA slowly and partially blocked rat NaV1.4 channels with an apparent IC50 of 0.56 ± 0.29 μM; the block was not complete, leaving at high concentration a residual current component of about 10% with a correspondingly reduced single-channel conductance. At 10 μM, μSIIIA potently blocked rat NaV1.2, rat and human NaV1.4, and mouse NaV1.6 channels; human NaV1.7 channels were only inhibited by 58.1 ± 4.9%, whereas human NaV1.5 as well as rat and human NaV1.8 were insensitive. Employing domain chimeras between rNaV1.4 and hNaV1.5, we located the determinants for μSIIIA specificity in the first half of the channel protein with a major contribution of domain-2 and a minor contribution of domain-1. The latter was largely accounted for by the alteration in the TTX-binding site (Tyr401 in rNaV1.4, Cys for NaV1.5, and Ser for NaV1.8). Introduction of domain-2 pore loops of all tested channel isoforms into rNaV1.4 conferred the μSIIIA phenotype of the respective donor channels highlighting the importance of the domain-2 pore loop as the major determinant for μSIIIA’s subtype specificity. Single-site substitutions identified residue Ala728 in rNaV1.4 as crucial for its high sensitivity toward μSIIIA. Likewise, Asn889 at the homologous position in hNaV1.7 is responsible for the channel’s reduced μSIIIA sensitivity. These results will pave the way for the rational design of selective NaV-channel antagonists for research and medical applications.

► μ-Conotoxin SIIIA blocks TTX-sensitive but not TTX-resistant sodium channels.
► The subtype specificity resides in the pore structures of sodium channel domains 1 and 2.
► Residue N889 in NaV1.7 explains the lower sensitivity of that channel compared to NaV1.4 and NaV1.2.
► The results pave the way of a rational design of analgesics targeting NaV1.7 channels.