title: The receptor site of the spider toxin PcTx1 on the proton-gated cation channel ASIC1a.
abstract: Acid-sensing ion channels (ASICs) are excitatory neuronal cation channels, involved in physiopathological processes related to extracellular pH fluctuation such as nociception, ischaemia, perception of sour taste and synaptic transmission. The spider peptide toxin psalmotoxin 1 (PcTx1) has previously been shown to inhibit specifically the proton-gated cation channel ASIC1a. To identify the binding site of PcTx1, we produced an iodinated form of the toxin ((125)I-PcTx1Y(N)) and developed a set of binding and electrophysiological experiments on several chimeras of ASIC1a and the PcTx1-insensitive channels ASIC1b and ASIC2a. We show that (125)I-PcTx1Y(N) binds specifically to ASIC1a at a single site, with an IC(50) of 128 pM, distinct from the amiloride blocking site. Results obtained from chimeras indicate that PcTx1 does not bind to ASIC1a transmembrane domains (M1 and M2), involved in formation of the ion pore, but binds principally on both cysteine-rich domains I and II (CRDI and CRDII) of the extracellular loop. The post-M1 and pre-M2 regions, although not involved in the binding site, are crucial for the ability of PcTx1 to inhibit ASIC1a current. The linker domain between CRDI and CRDII is important for their correct spatial positioning to form the PcTx1 binding site. These results will be useful for the future identification or design of new molecules acting on ASICs.
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, CNRS Université de Nice Sophia-Antipolis, UMR-6097, Institut Paul Hamel, Sophia Antipolis, Valbonne, France.
authors.forename: Miguel
authors.initials: M
authors.lastname: Salinas
authors.forename: Lachlan D
authors.initials: LD
authors.lastname: Rash
authors.forename: Anne
authors.initials: A
authors.lastname: Baron
authors.forename: Gérard
authors.initials: G
authors.lastname: Lambeau
authors.forename: Pierre
authors.initials: P
authors.lastname: Escoubas
authors.forename: Michel
authors.initials: M
authors.lastname: Lazdunski
mesh_terms: D062565:Acid Sensing Ion Channels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003545:Cysteine / Q000032:analysis; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013111:Spider Venoms / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D014981:Xenopus
chemical_list: D062565:Acid Sensing Ion Channels; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C413209:PcTX1 protein, Psalmopoeus cambridgei; D010455:Peptides; D011994:Recombinant Proteins; D015222:Sodium Channels; D013111:Spider Venoms; D003545:Cysteine