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A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif.

Martin-Eauclaire MF, Ferracci G, Bosmans F, Bougis PE - J. Gen. Physiol. (2015)

Bottom Line: 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.As such, our results uncover the inherent pharmacological sensitivities of the isolated domain IV paddle motif, which may be exploited to develop a label-free SPR approach for discovering ligands that target this region.

View Article: PubMed Central - HTML - PubMed

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.

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α-Scorpion toxins interact with the rNav1.2a VSD IV paddle motif. (A) Shown is the effect of 100 nM AaHI, AaHII, LqqV, and BomIV on rNav1.2a channel function. Representative sodium currents were elicited by a 50-ms depolarization to a suitable membrane voltage (−20 to −15 mV) before (black) and after toxin addition (green) from a holding voltage of −90 mV. Clearly, toxin application results in a large persistent current component at the end of the test pulse. Fitting the current decay with a single-exponential function before and after toxin application yields fast inactivation time constants (τ) of 3.2 ± 0.1 and 4.7 ± 0.1 (AaHI); 3.6 ± 0.2 and 4.9 ± 0.1 (AaHII); 2.5 ± 0.1 and 4.6 ± 0.1 (LqqV); and 2.9 ± 0.1 and 8.5 ± 0.1 (BomIV), with n = 3 for each value (mean ± SEM). (B) Shown is the effect of 1 µM AaHI, LqqV, and BomIV on WT rKv2.1. For each toxin, K+ currents were elicited by a 300-ms depolarization to 0 mV from a holding voltage of −90 mV (tail voltage was −60 mV). Currents are shown before (black) and in the presence of toxin (green). (C) Effect of 100 nM AaHI, LqqV, and BomIV on the rNav1.2a/Kv2.1 VSD IV paddle chimera. For each toxin, K+ currents (top) were elicited by a 300-ms depolarization near the foot of the voltage–activation curve (bottom) from a holding voltage of −90 mV. Currents are shown before (black) and in the presence of toxin (green). Representative normalized tail current voltage–activation relationships are shown (bottom), where tail current amplitude (I/Imax) is plotted against test voltage (V) before (black) and in the presence of toxins (green). A Boltzmann fit of the obtained data (n = 3; mean ± SEM) reveals a depolarizing shift in midpoint (V1/2) of ∼15 mV for AaHI, >50 mV for LqqV, and ∼26 mV for BomIV. Holding voltage was −90 mV, and the tail voltage was −60 mV.
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fig1: α-Scorpion toxins interact with the rNav1.2a VSD IV paddle motif. (A) Shown is the effect of 100 nM AaHI, AaHII, LqqV, and BomIV on rNav1.2a channel function. Representative sodium currents were elicited by a 50-ms depolarization to a suitable membrane voltage (−20 to −15 mV) before (black) and after toxin addition (green) from a holding voltage of −90 mV. Clearly, toxin application results in a large persistent current component at the end of the test pulse. Fitting the current decay with a single-exponential function before and after toxin application yields fast inactivation time constants (τ) of 3.2 ± 0.1 and 4.7 ± 0.1 (AaHI); 3.6 ± 0.2 and 4.9 ± 0.1 (AaHII); 2.5 ± 0.1 and 4.6 ± 0.1 (LqqV); and 2.9 ± 0.1 and 8.5 ± 0.1 (BomIV), with n = 3 for each value (mean ± SEM). (B) Shown is the effect of 1 µM AaHI, LqqV, and BomIV on WT rKv2.1. For each toxin, K+ currents were elicited by a 300-ms depolarization to 0 mV from a holding voltage of −90 mV (tail voltage was −60 mV). Currents are shown before (black) and in the presence of toxin (green). (C) Effect of 100 nM AaHI, LqqV, and BomIV on the rNav1.2a/Kv2.1 VSD IV paddle chimera. For each toxin, K+ currents (top) were elicited by a 300-ms depolarization near the foot of the voltage–activation curve (bottom) from a holding voltage of −90 mV. Currents are shown before (black) and in the presence of toxin (green). Representative normalized tail current voltage–activation relationships are shown (bottom), where tail current amplitude (I/Imax) is plotted against test voltage (V) before (black) and in the presence of toxins (green). A Boltzmann fit of the obtained data (n = 3; mean ± SEM) reveals a depolarizing shift in midpoint (V1/2) of ∼15 mV for AaHI, >50 mV for LqqV, and ∼26 mV for BomIV. Holding voltage was −90 mV, and the tail voltage was −60 mV.

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).


A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif.

Martin-Eauclaire MF, Ferracci G, Bosmans F, Bougis PE - J. Gen. Physiol. (2015)

α-Scorpion toxins interact with the rNav1.2a VSD IV paddle motif. (A) Shown is the effect of 100 nM AaHI, AaHII, LqqV, and BomIV on rNav1.2a channel function. Representative sodium currents were elicited by a 50-ms depolarization to a suitable membrane voltage (−20 to −15 mV) before (black) and after toxin addition (green) from a holding voltage of −90 mV. Clearly, toxin application results in a large persistent current component at the end of the test pulse. Fitting the current decay with a single-exponential function before and after toxin application yields fast inactivation time constants (τ) of 3.2 ± 0.1 and 4.7 ± 0.1 (AaHI); 3.6 ± 0.2 and 4.9 ± 0.1 (AaHII); 2.5 ± 0.1 and 4.6 ± 0.1 (LqqV); and 2.9 ± 0.1 and 8.5 ± 0.1 (BomIV), with n = 3 for each value (mean ± SEM). (B) Shown is the effect of 1 µM AaHI, LqqV, and BomIV on WT rKv2.1. For each toxin, K+ currents were elicited by a 300-ms depolarization to 0 mV from a holding voltage of −90 mV (tail voltage was −60 mV). Currents are shown before (black) and in the presence of toxin (green). (C) Effect of 100 nM AaHI, LqqV, and BomIV on the rNav1.2a/Kv2.1 VSD IV paddle chimera. For each toxin, K+ currents (top) were elicited by a 300-ms depolarization near the foot of the voltage–activation curve (bottom) from a holding voltage of −90 mV. Currents are shown before (black) and in the presence of toxin (green). Representative normalized tail current voltage–activation relationships are shown (bottom), where tail current amplitude (I/Imax) is plotted against test voltage (V) before (black) and in the presence of toxins (green). A Boltzmann fit of the obtained data (n = 3; mean ± SEM) reveals a depolarizing shift in midpoint (V1/2) of ∼15 mV for AaHI, >50 mV for LqqV, and ∼26 mV for BomIV. Holding voltage was −90 mV, and the tail voltage was −60 mV.
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fig1: α-Scorpion toxins interact with the rNav1.2a VSD IV paddle motif. (A) Shown is the effect of 100 nM AaHI, AaHII, LqqV, and BomIV on rNav1.2a channel function. Representative sodium currents were elicited by a 50-ms depolarization to a suitable membrane voltage (−20 to −15 mV) before (black) and after toxin addition (green) from a holding voltage of −90 mV. Clearly, toxin application results in a large persistent current component at the end of the test pulse. Fitting the current decay with a single-exponential function before and after toxin application yields fast inactivation time constants (τ) of 3.2 ± 0.1 and 4.7 ± 0.1 (AaHI); 3.6 ± 0.2 and 4.9 ± 0.1 (AaHII); 2.5 ± 0.1 and 4.6 ± 0.1 (LqqV); and 2.9 ± 0.1 and 8.5 ± 0.1 (BomIV), with n = 3 for each value (mean ± SEM). (B) Shown is the effect of 1 µM AaHI, LqqV, and BomIV on WT rKv2.1. For each toxin, K+ currents were elicited by a 300-ms depolarization to 0 mV from a holding voltage of −90 mV (tail voltage was −60 mV). Currents are shown before (black) and in the presence of toxin (green). (C) Effect of 100 nM AaHI, LqqV, and BomIV on the rNav1.2a/Kv2.1 VSD IV paddle chimera. For each toxin, K+ currents (top) were elicited by a 300-ms depolarization near the foot of the voltage–activation curve (bottom) from a holding voltage of −90 mV. Currents are shown before (black) and in the presence of toxin (green). Representative normalized tail current voltage–activation relationships are shown (bottom), where tail current amplitude (I/Imax) is plotted against test voltage (V) before (black) and in the presence of toxins (green). A Boltzmann fit of the obtained data (n = 3; mean ± SEM) reveals a depolarizing shift in midpoint (V1/2) of ∼15 mV for AaHI, >50 mV for LqqV, and ∼26 mV for BomIV. Holding voltage was −90 mV, and the tail voltage was −60 mV.
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).

Bottom Line: 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.As such, our results uncover the inherent pharmacological sensitivities of the isolated domain IV paddle motif, which may be exploited to develop a label-free SPR approach for discovering ligands that target this region.

View Article: PubMed Central - HTML - PubMed

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.

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Related in: MedlinePlus