Journal: Toxicon: X

Article Title: Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

doi: 10.1016/j.toxcx.2019.100010

Figure Lengend Snippet: Docking of BeKm-1 in a model of hERG channel and fluorescent BeKm-1 analogues used in this study. A) Left, BeKm-1 (pdb code: 1j5, magenta, top) in interaction with hERG channel (in solid ribbon representation showing the solvent surface colored by hydrophobicity). The tetrameric model of the hERG channel was generated from the pdb structure (5VA1) using the Prepare Protein module within Discovery Studio. The transmembrane domain, the cytoplasmic N-terminal Per-Arnt-Sim (PAS) domain, the C-terminal C-linker and the cyclic nucleotide binding domain (CNBD) are indicated. Docking of BeKm-1 was performed using the ZDOCK rigid body docking program and refined with the RDOCK module. The top ranked pose of the most populated cluster from the protein-protein interaction predictions is showed with BeKm-1 docked at the top of the transmembrane domain of hERG. Right, Details of the pose of BeKm-1 (magenta) within the interaction surface of hERG colored according to the Solvent Accessibility Surface (SAS) (see Supplementary Data S2), with the side chain of the N terminal Arg 1 and Arg 27 (red) and the more buried C terminal Phe 36 (orange). B) Chemical structures of fluorescent BeKm-1 analogues used in this study with different types of linkers grafted on the N-ter of Arg 1 or the side chain of Arg 27 mutated for a Lysine. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min.

Techniques: Generated, Binding Assay

Journal: Toxicon: X

Article Title: Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

doi: 10.1016/j.toxcx.2019.100010

Figure Lengend Snippet: The fluorescent BeKm-1 analogues potently inhibit hERG currents. A) Typical traces of currents recorded in Xenopus laevis oocytes that express hERG. Currents were elicited using the protocol depicted at the top in which a pulse from a holding potential of −80 mV to −30 mV precedes a test pulse at −50 mV, in the presence of increasing concentrations of native BeKm-1, Cy5-PEG3-linker4-BeKm-1, Cy5-spacerGS-linker6-BeKm-1, Cy5-PEG5-linker10-BeKm-1 or Cy5-PEG5-BeKm1-Lys 27 . Scale bars, 200 nA. B) Inhibition curves of native BeKm-1 and its four analogues. The peak current amplitude was measured during the test pulse at −50 mV in the presence of toxin (ITx) and normalized to the current recorded in the absence of toxin (ICt). Data are the mean ± SEM of n = 4–10 oocytes.

Article Snippet: Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min.

Techniques: Inhibition

Journal: Toxicon: X

Article Title: Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

doi: 10.1016/j.toxcx.2019.100010

Figure Lengend Snippet: Dose-response curve parameters (IC 50 and hill coefficient (n Hill)) of native BeKm-1 and its four analogues Cy5-PEG3-linker4-BeKm-1, Cy5-spacerGS-linker6-BeKm-1, Cy5-PEG5-linker10-BeKm-1 and Cy5-PEG5-BeKm1-Lys 27 obtained from Xenopus laevis oocytes that express hERG. Data are the mean ± SEM of n oocytes. *p < 0.01 (compared with native BeKm-1).

Article Snippet: Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min.

Techniques:

Journal: Toxicon: X

Article Title: Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

doi: 10.1016/j.toxcx.2019.100010

Figure Lengend Snippet: The fluorescent BeKm-1 analogues do not affect hKv10.1 and hKv1.3 currents. A) Typical traces of currents elicited by a step depolarization to 0 mV or 70 mV, from a holding potential of −80 mV, obtained from Xenopus laevis oocytes that express hKv10.1 or hKv1.3, respectively, in the absence or presence of 200 nM native BeKm-1 and in the absence or presence of 500 nM of Cy5-PEG3-linker4-BeKm-1, of Cy5-spacerGS-linker6-BeKm-1, of Cy5-PEG5-linker10-BeKm-1 and of Cy5-PEG5-BeKm1-Lys 27 . Note the lack of effect of BeKm-1 and of the four analogues on both channels. B) Percentage of current remaining in the presence of a single dose of native BeKm-1 or its four analogues (Cy5-PEG3-linker4-BeKm-1, Cy5-spacerGS-linker6-BeKm-1, Cy5-PEG5-linker10-BeKm-1 and Cy5-PEG5-BeKm1-Lys 27 ). Current amplitude was measured before (ICt) and during (Itx) the perfusion of the different toxins. Data are the mean ± SEM of n = 4–7 oocytes.

Article Snippet: Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min.

Techniques:

Journal: Toxicon: X

Article Title: Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

doi: 10.1016/j.toxcx.2019.100010

Figure Lengend Snippet: Percentage of current remaining after exposure to a single dose of native BeKm-1 or its four analogues Cy5-PEG3-linker4-BeKm-1, Cy5-spacerGS-linker6-BeKm-1, Cy5-PEG5-linker10-BeKm-1 and Cy5-PEG5-BeKm1-Lys 27 obtained from Xenopus laevis oocytes that express wild type (WT) hERG, hK v 10.1 or hK v 1.3, or hERG harboring the S631C mutation. Data are the mean ± SEM of n oocytes. *p < 0.01 (compared with WT herg channel).

Article Snippet: Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min.

Techniques: Mutagenesis

Journal: Toxicon: X

Article Title: Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

doi: 10.1016/j.toxcx.2019.100010

Figure Lengend Snippet: The mutation S631C in hERG decreases the affinity of native BeKm-1 and of its four fluorescent analogues for the channel. Comparison of the inhibition curves of native BeKm-1 and its four analogues Cy5-PEG3-linker4-BeKm-1, Cy5-spacerGS-linker6-BeKm-1, Cy5-PEG5-linker10-BeKm-1 and Cy5-PEG5-BeKm1-Lys 27 obtained in Xenopus laevis oocytes that express wild type (WT) hERG or the mutant S631C. The data obtained with the mutant channel were fitted by hand for the purpose of comparison. Data are the mean ± SEM of n = 3–5 oocytes.

Article Snippet: Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min.

Techniques: Mutagenesis, Inhibition

Journal: The Journal of Neuroscience

Article Title: Erg Potassium Currents of Neonatal Mouse Purkinje Cells Exhibit Fast Gating Kinetics and Are Inhibited by mGluR1 Activation

doi: 10.1523/JNEUROSCI.5523-12.2013

Figure Lengend Snippet: The toxins BeKm-1 and APETx1 reduced the currents mediated by erg1a and erg1a/erg3 concatemers to a similar degree as the erg current of Purkinje cells. A–D, BeKm-1 (100 nm) or APETx1 (1 μm) were applied to HEK 293 cells stably expressing erg1a, erg2, erg3, or concatemeric erg1a/erg3 channels. E, erg currents measured from Purkinje cells (PN) in acute slices of the cerebellum of P7 mice. In all panels, erg currents were elicited in the absence (black traces) and in the presence (red traces) of the toxins and superimposed. Pulse protocol: from a holding potential of −70 mV, a sequence of membrane potential steps was applied to +20 mV (2 s), −20 mV (0.5 s), −120 mV (0.5 s), and back to the holding potential. The time course of the hyperpolarization-activated cation channel current was fitted with an exponential function and extrapolated to t = 0 (dotted curve).

Article Snippet: The scorpion toxin BeKm-1 was purchased from Alomone Labs.

Techniques: Stable Transfection, Expressing, Sequencing