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

Abstract Voltage-gated sodium channels (Na V 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 Na V 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 Na V channels expressed in mammalian cells using the whole-cell patch-clamp method. Synthetic μSIIIA slowly and partially blocked rat Na V 1.4 channels with an apparent IC 50 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 Na V 1.2, rat and human Na V 1.4, and mouse Na V 1.6 channels; human Na V 1.7 channels were only inhibited by 58.1 ± 4.9%, whereas human Na V 1.5 as well as rat and human Na V 1.8 were insensitive. Employing domain chimeras between rNa V 1.4 and hNa V 1.5, 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 rNa V 1.4, Cys for Na V 1.5, and Ser for Na V 1.8). Introduction of domain-2 pore loops of all tested channel isoforms into rNa V 1.4 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 rNa V 1.4 as crucial for its high sensitivity toward μSIIIA. Likewise, Asn889 at the homologous position in hNa V 1.7 is responsible for the channel’s reduced μSIIIA sensitivity. These results will pave the way for the rational design of selective Na V -channel antagonists for research and medical applications.