The scorpion toxin peptide BeKm-1, originally isolated from Buthus eupeus, was synthesized by FMOC solid phase chemistry and folded by air oxidation. The peptide’s effects on heterologous human ether-a-go-go related gene (HERG) potassium current (I_HERG) and mutant HERG channels expressed in a mammalian cell line (HEK293 cells) were assessed using ‘whole-cell’ patch-clamp and compared with the commercially available recombinant BeKm-1 (rBeKm-1).

Blockade of I_HERG by BeKm-1 was concentration dependent, temperature dependent, and rapid in onset and reversibility. Two protocols, differing only in the duration at depolarized voltage, were adopted to investigate steady-state potency of I_HERG blockade by a range of BeKm-1 concentrations. Fractional block of I_HERG tails elicited at -40 mV following a depolarizing step to +20 mV for either 2000 ms or 400 ms from a holding potential of -80 mV was determined. Data are presented as means ± S.E.M., or mean plus 95 % confidence intervals (CI).

At 37 °C, IC₅₀ values were 63.3 nM, CI 52.7-76.1 nM (2000 ms step) and 15.3 nM, CI 11.2-20.1 nM (400 ms step), Student’s unpaired t test, P < 0.05). These values were not significantly different from IC₅₀ values for rBeKm-1 under identical conditions (unpaired t test, P > 0.05), indicating the synthesis of an active peptide. At room temperature identical experiments yielded significantly lower IC₅₀ values of 9.7 nM, CI 7.2 to 13.0 nM (2000 ms) and 7.6 nM, CI 7.2 to 8.1 nM (400 ms) respectively (unpaired t test, P < 0.05). Blockade also exhibited inverse voltage dependence and reverse use and frequency dependence. During a three-step protocol, cells were held at -80 mV and stepped to zero for 2 s; further depolarization to +80 mV for 4 s resulted in a dramatic attenuation of I_HERG blockade by 25 nM BeKm-1 from 30.0 ± 3.2 % to 2.2 ± 5.5 % (n = 5). On stepping back from +80 mV to 0 mV for 4 s, BeKm-1 blockade re-developed from 2.2 ± 6 % to 24 ± 2 % (ANOVA, P < 0.0001). The attenuated inactivation mutation S631A abolished initial time-dependent relief of BeKm-1 blockade. BeKm-1 blockade of the HERG S6 mutant Y652A was identical in potency to that of wild-type HERG (250 nM BeKm-1; 71 ± 1 % vs. 73 ± 2 % respectively, unpaired t test, P = 0.4).

We conclude that synthetic BeKm-1 toxin blocks HERG with identical potency to rBeKm-1, preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs. The site of action of BeKm-1 differs from the high affinity site described within the vestibule (Mitcheson et al. 2000).

This work was supported by the British Heart Foundation and Wellcome Trust.