Molecular Determinants of μ-Conotoxin KIIIA Block of Voltage-Gated Sodium Channels

Abstract

mu-Conotoxin (muCTX) KIIIA is of special interest both functionally and structurally because (1) it blocks neuronal voltage-gated sodium (Nav) channels involved in pain signalling (Zhang et al., 2007, J. Biol. Chem.) and (2) unlike previously discovered muCTXs (most >22 amino acids), KIIIA has only 16 amino acids, missing amino acids in the N-terminal section. We have performed preliminary molecular dynamics simulations of muCTX KIIIA docking to a model of the Nav1.4 outer vestibule (Choudhary et al, 2007, Channels). The results are consistent with a possible binding orientation in Nav1.4 with K7 facing down into the pore, interacting with the outer ring charges (E403 & E758) in domains I and II. To exam this possible orientation, single-channel bilayer recordings from rat brain (preparation includes Nav1.1, 1.2, 1.3 and 1.6) and rat skeletal muscle (muscle, predominantly Nav1.4) preparations demonstrated that when lysine-7 (K7) is neutralized, channels show an increase in fractional residual current (fres) upon KIIIA[K7A] addition (brain, 48±3% & muscle, 45±9%) compared to wild type KIIIA (brain, 19±3%, muscle 19±3%). The wild-type non-zero fres hints that the lack of N-terminal residues or the use of a lysine residue (instead of arginine) to occlude the pore in KIIIA leads to incomplete toxin block, suggesting KIIIA has a “looser” interaction with the channel, with the key basic residue, K7, playing a smaller role in toxin block than in GIIIA and PIIIA. This data is supported by whole-cell experiments looking at KIIIA and KIIIA[K7A] interactions with multiple Nav isoforms. The single-channel and whole-cell data suggest KIIIA binds to the outer vestibule with the lysine at position 7 blocking current through the pore, similar to R13 in GIIIA (skeletal muscle specific) and R14 in PIIIA (blocks both skeletal and neuronal channels).