title: Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity.
abstract: μ-Conotoxin KIIIA from Conus kinoshitai is a 16-residue peptide that acts as a potent pore blocker of several voltage-gated sodium channels (Na(v)). In order to obtain more selective blockers and to investigate the role of Trp at position 8, we substituted this residue with Arg, Gln and Glu. KIIIA and analogues were tested on a range of Na(v) expressed in Xenopus laevis oocytes. The rank order of potency for KIIIA was: rNa(v)1.4 ≥ rNa(v)1.2 > mNa(v)1.6 > rNa(v)1.3, with IC(50) values of 48 ± 6 nm, 61 ± 5 nm, 183 ± 31 nm and 3.6 ± 0.3 μm, respectively, whereas no effect was seen on hNa(v)1.5 and hNa(v)1.8 at a concentration of 10 μm. Replacement of Trp8 resulted in more selective blockers with a preference for neuronal sodium channels over the skeletal sodium channel. The activity on rNa(v)1.4 was reduced about 40-, 70- and 200-fold for [W8R]KIIIA, [W8Q]KIIIA and [W8E]KIIIA, respectively. All analogues showed a completely reversible block of rNa(v)1.2, as opposed to the partial reversibility of KIIIA. At saturating concentrations, complete block of rNa(v)1.2 was never achieved. The residual current was lower than 10%, except for [W8E]KIIIA. KIIIA had no effect on the voltage dependence of activation of rNa(v)1.2, whereas all analogues caused a depolarizing shift. Overall, this study shows that Trp8 is a key residue in the pharmacophore. Replacement of Trp8 enables more selective blockers to be obtained for neuronal sodium channels. Trp is a key determinant for the reversibility of block of rNa(v)1.2.
authors.affiliation: Laboratory of Toxicology, University of Leuven (K U Leuven), Leuven, Belgium.
authors.forename: Annelies
authors.initials: A
authors.lastname: Van Der Haegen
authors.forename: Steve
authors.initials: S
authors.lastname: Peigneur
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D020916:Conotoxins / Q000737:chemistry* / Q000494:pharmacology*; D015195:Drug Design; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018408:Patch-Clamp Techniques; D017510:Protein Folding; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D016415:Sequence Alignment; D026941:Sodium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology*; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics
chemical_list: D020916:Conotoxins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C502265:mu-conotoxin KIIIA, Conus kinoshitai