A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif.
Bottom Line: Animal toxins that inhibit voltage-gated sodium (Na(v)) channel fast inactivation can do so through an interaction with the S3b-S4 helix-turn-helix region, or paddle motif, located in the domain IV voltage sensor.Here, we used surface plasmon resonance (SPR), an optical approach that uses polarized light to measure the refractive index near a sensor surface to which a molecule of interest is attached, to analyze interactions between the isolated domain IV paddle and Na(v) channel-selective α-scorpion toxins.Our SPR analyses showed that the domain IV paddle can be removed from the Na(v) channel and immobilized on sensor chips, and suggest that the isolated motif remains susceptible to animal toxins that target the domain IV voltage sensor.
Affiliation: Centre National de la Recherche Scientifique, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseillle, Unité Mixte de Recherche 7286, Plates-Formes de Recherche en Neurosciences-Centre d'Analyse Protéomique de Marseille, Aix Marseille Université, 13344 Marseille, France.Show MeSH
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Mentions: The four Nav channel–selective α-scorpion toxins we selected for our experiments were AaHI and AaHII from Androctonus australis Hector, LqqV from Leiurus quinquestriatus hebraeus, and BomIV from Buthus occitanus mardochei (Martin-Eauclaire and Rochat, 2000; Bende et al., 2014). As negative control, we applied kaliotoxin (KTX) from Androctonus mauretanicus, which blocks the Kv1.1 and Kv1.3 pore but does not influence Nav channel function (Crest et al., 1992). All toxins were purified to homogeneity as reported previously (Crest et al., 1992; Martin-Eauclaire and Rochat, 2000) and tested for functionality on the rNav1.2a isoform expressed in Xenopus oocytes. In all cases, the application of 100 nM AaHI, AaHII, LqqV, or BomIV inhibits rNav1.2a fast inactivation, resulting in the appearance of a large persistent current at the end of a 50-ms test pulse (Fig. 1 A). Because AaHII has already been shown to bind to the VSD IV paddle motif in rNav1.2a (Bosmans et al., 2008), we verified if this was also the case with AaHI, LqqV, and BomIV. To this end, we tested whether 100 nM of each toxin influenced the function of a previously constructed chimera in which the S3b–S4 region of the homotetrameric Kv2.1 channel was swapped for the corresponding WT region in VSD IV from rNav1.2a. As a result, we observed a robust voltage-dependent K+ current inhibition, whereas WT Kv2.1 is insensitive, suggesting that AaHI, LqqV, and BomIV indeed interact with the transferred VSD IV paddle motif (Fig. 1, B and C).
Affiliation: Centre National de la Recherche Scientifique, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseillle, Unité Mixte de Recherche 7286, Plates-Formes de Recherche en Neurosciences-Centre d'Analyse Protéomique de Marseille, Aix Marseille Université, 13344 Marseille, France.