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

Figures

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  Fig. 1. 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 Arg1 and Arg27 (red) and the more buried C terminal Phe36 (orange). B) Chemical structures of fluorescent BeKm-1 analogues…

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  Fig. 2. 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-Lys27. 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.

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  Fig. 3. 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-Lys27. 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-Lys27). 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.

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  Fig. 4. 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-Lys27 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.