Martin-Eauclaire MF et al. (2015), A surface plasmon resonance approach to monitor...

XB-ART-51378

J Gen Physiol 2015 Feb 01;1452:155-62. doi: 10.1085/jgp.201411268.

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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 .

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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. 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.

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Species referenced: Xenopus
Genes referenced: kcnb1 spr tbx2

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	Figure 1. 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.
	Figure 2. AaHII interacts with the rNav1.2a VSD IV paddle motif. (A) Nav channel cartoon embedded in a lipid membrane. Each domain (DI–IV) consists of six transmembrane segments (S1–S6) of which S1–S4 form the VSD and the S5–S6 segments of each domain form the pore. Paddle motif amino acid sequences are shown for VSD II (red) and IV (green). Underlined residues were mutated as reported in Results. (B) Effect of 100 nM AaHII on the rNav1.2a/Kv2.1 VSD IV chimera containing the R1629A/L1630A substitutions. K+ current (left) was elicited by a 300-ms depolarization to −100 mV (tail voltage was −150 mV) after a 500-ms step to −150 mV from a holding voltage of −10 mV (near the Nernst potential for K+). The data show a clear toxin-induced inhibition of the double mutant channel (black, control; green, 100 nM AaHII). Right panel displays normalized tail current voltage–activation relationships of the rNav1.2a/Kv2.1 VSD IV chimera without (open circles) and with the R1629A/L1630A substitutions (closed circles) where tail current amplitude (I/Imax) is plotted against test voltage before (black) and in the presence of 100 nM AaHII (green). Holding voltage for the mutant was −10 mV, followed by a 500-ms hyperpolarizing step to −150 mV to close all channels. Next, 10-mV step depolarizations of 300 ms (V) were trailed by a 300-ms tail voltage step to −150 mV (I). A Boltzmann fit of the obtained data (n = 3; error bars represent mean ± SEM) reveals a shift in midpoint (V1/2) for the double mutant (−105 ± 1 mV; slope, 17.1 ± 1.0) compared with the rNav1.2a/Kv2.1 VSD IV chimera (7 ± 2 mV; slope, 17.3 ± 1.6). Moreover, 100 nM AaHII strongly inhibits the double mutant (apparent KD = 193 ± 42 nM), whereas the rNav1.2a/Kv2.1 VSD IV chimera is influenced less (apparent KD = 1,008 ± 92 nM), with n = 3 for each value (error bars represent mean ± SEM).
	Figure 3. α-Scorpion toxins interact with the isolated rNav1.2a VSD IV paddle motif. (A) Shown are the CD spectra of the rNav1.2a VSD II and IV paddle peptides used in this paper. Analysis of both spectra revealed the presence of ∼75% structured (α-helix/β-sheet) and ∼25% unstructured peptide. Inset shows the crystal structure of the NavAb voltage sensor (3RVY) (Payandeh et al., 2011) in which the paddle motif is indicated in green; wheat shows S1 and S2 helices, and white indicates the portion of S3 and S4 helices outside the paddle. (B) Representative association and dissociation kinetic curves obtained using SPR after the application of varying concentrations of AaHII (15–2,000 nM) over a sensor chip to which 50 fmol of the rNav1.2a VSD IV paddle peptide was linked. Toxin was applied after obtaining a steady baseline. Colored traces represent toxin binding obtained after subtraction of the signal from the control flow cell. Green dotted lines depict a fit of the data to a heterogeneous surface ligand model, a typical SPR analysis method (Schuck and Zhao, 2010), which yielded a high affinity KD1 of 479 ± 241 nM (RUmax1 = 135) and a lower affinity KD2 of 747 ± 203 nM (RUmax2 = 23; n = 3; all results presented as mean ± SD). The respective contributions of RUmax1 (∼85%) and RUmax2 (∼15%) to the overall RUmax (100%) are reminiscent of the percent structured (∼75%) versus unstructured (∼25%) paddle peptide as observed in the CD spectrum. Toxin concentrations are indicated on the right in a shade of gray corresponding to the sensorgram. (C) Representative SPR traces after the application of 100 nM KTX, AaHI, AaHII, LqqV, BomIV, TsVII, and CssIV over a sensor chip to which the rNav1.2a VSD II (left) or IV (right) paddle peptide (500 fmol) was linked. Toxin was applied after obtaining a steady baseline. (D) Binding capacities of 100 nM α-scorpion toxins AaHI, AaHII, LqqV, the α-like scorpion toxin BomIV, and the β-scorpion toxins TsVII and CssIV to the VSD II and IV paddle motifs using SPR (error bars represent ± SEM). Y-axis represents the maximum RUs obtained after toxin application. Note that 500 fmol paddle peptide was used in C and D as opposed to 50 fmol in B. As a result, RUs differ by a factor of ∼10.
	Figure 4. CssIV interacts with the rNav1.2a VSD II paddle motif. Shown is the effect of 1 µM CssIV on the rNav1.2a/Kv2.1 VSD II and IV paddle chimera. Normalized tail current voltage–activation relationships in which the tail current amplitude (I/Imax) is plotted against test voltage (V) before (black) and in the presence of toxin (red/green) are displayed. A Boltzmann fit of the obtained data (n = 3; error bars represent mean ± SEM) reveals a depolarizing shift in V1/2 of ∼14 mV (from 33 ± 2 mV to 47 ± 1 mV) for the VSD II chimera, whereas 1 µM CssIV does not influence the VSD IV chimera. Holding voltage was −90 mV, and the tail voltage was −60 mV.

References [+] :

Alabi, Portability of paddle motif function and pharmacology in voltage sensors. 2007, Pubmed, Xenbase