Phoneutria nigriventer omega-phonetoxin IIA blocks the Cav2 family of calcium channels and interacts with omega-conotoxin-binding sites.
omega-Phonetoxin IIA (omegaPtxIIA), a peptide from spider venom (Phoneutria nigriventer), inhibits high threshold voltage-dependent calcium currents in neurons. To define its pharmacological specificity, we have used patch-clamp methods in cell lines expressing recombinant Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 channels (P/Q-, N-, and R-type currents, respectively). Calcium currents generated by Ca(v)2.1 and Ca(v)2.2 were blocked almost irreversibly by 3 nm omegaPtxIIA, whereas Ca(v)2.3 showed partial and readily reversible inhibition. Binding assays with mono[(125)I]iodo-omegaPtxIIA indicated that membranes expressing recombinant Ca(v)2.1 or Ca(v)2.2 channels showed a single class of sites with similar affinity (K(D) approximately 50 pm), whereas low affinity interactions were detectable with Ca(v)2.3. Kinetic, saturation, and displacement assays demonstrated that rat brain synaptosomes displayed multiple classes of binding sites for (125)I-omegaPtxIIA. High affinity binding of (125)I-omegaPtxIIA was totally displaced by omegaPtxIIA (K(i) = 100 pm), but only partially by omega-conotoxin GVIA (25% inhibition) and omega-conotoxin MVIIC (50% inhibition at 0.3 microm). (125)I-omegaPtxIIA thus defines a unique high affinity binding site that is predominantly associated with Ca(v)2.1 or Ca(v)2.2 channels.
Dos Santos
Raquel Gouvea
RG
INSERM U464 and CNRS UMR6560, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord, Marseille, France.
Van Renterghem
Catherine
C
Martin-Moutot
Nicole
N
Mansuelle
Pascal
P
Cordeiro
Marta N
MN
Diniz
Carlos Ribeiro
CR
Mori
Yasuo
Y
De Lima
Maria Elena
ME
Seagar
Michael
M
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013112:Spiders; D013574:Synaptosomes / Q000378:metabolism; D020960:omega-Conotoxins / Q000737:chemistry*
D020864:Calcium Channels, N-Type; D008024:Ligands; D011994:Recombinant Proteins; D020960:omega-Conotoxins; C075851:omega-conotoxin-MVIIC