Isolation and structure-activity of mu-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels.
Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.
Lewis
Richard J
RJ
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. r.lewis@imb.uq.edu.au
Schroeder
Christina I
CI
Ekberg
Jenny
J
Nielsen
Katherine J
KJ
Loughnan
Marion
M
Thomas
Linda
L
Adams
Denise A
DA
Drinkwater
Roger
R
Adams
David J
DJ
Alewood
Paul F
PF
D000595:Amino Acid Sequence; D000818:Animals; D020916:Conotoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D052078:Conus Snail; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D026941:Sodium Channel Blockers / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013779:Tetrodotoxin / Q000494:pharmacology*; D014982:Xenopus laevis
D020916:Conotoxins; D008978:Mollusk Venoms; D026941:Sodium Channel Blockers; C518160:mu-conotoxin TIIIA, Conus tulipa; D013779:Tetrodotoxin