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

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.