Neuronally micro-conotoxins from Conus striatus utilize an alpha-helical motif to target mammalian sodium channels.
Mu-conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of mu-conotoxins SIIIA and SIIIB by (125)I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced (125)I-TIIIA from native rat brain Na(v)1.2 (IC(50) values 10 and 5 nm, respectively) and muscle Na(v)1.4 (IC(50) values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Na(v)1.2 and Na(v)1.4. An alanine scan of SIIIA-(2-20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2-20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2-20) had significantly enhanced neuronal affinity (IC(50) 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2-20) showed greatest Na(v)1.2 versus 1.4 selectivity (136-fold). (1)H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and an alpha-helical motif across residues 11-16 that is not found in larger mu-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller mu-conotoxin pharmacophore.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
Christina I
CI
Schroeder
Jenny
J
Ekberg
Katherine J
KJ
Nielsen
Denise
D
Adams
Marion L
ML
Loughnan
Linda
L
Thomas
David J
DJ
Adams
Paul F
PF
Alewood
Richard J
RJ
Lewis
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D020916:Conotoxins / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D020128:Inhibitory Concentration 50; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D015222:Sodium Channels / Q000737:chemistry*
D020916:Conotoxins; D008978:Mollusk Venoms; D011994:Recombinant Proteins; D015222:Sodium Channels; D006639:Histidine