Bekm 1

Expressed currents were recorded at room temperature using the two-electrode voltage clamp method. Electrodes were pulled from borosilicate glass and filled with 3 M KCl. Currents were measured with a Geneclamp 500 amplifier (Molecular Devices), and digitized with a Digidata 1200 converter (Molecular Devices) using the Clampex 5 software (Molecular Devices). Oocytes were clamped at −80 mV and leak currents were subtracted online using a P/3 protocol of hyperpolarizing pre-pulses. Native BeKm-1 (Alomone Labs) and toxin analogues were diluted in the external solution NDherg (96 mM NaCl, 3 mM KCl, 0.5 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes, pH 7.4 with NaOH) and perfused through the recording chamber at a rate of 1 mL/min. The concentration to obtain half maximal inhibition (IC₅₀) was determined by measuring the peak tail current recorded during a test pulse at −50 mV in the presence of increasing concentrations of toxin (ITx), and by normalizing the value to the current measured in the absence of toxin (ICt). Data were fitted with a logistic function using Origin 6.0. For human EAG (Kv10.1) and Kv1.3, peak current inhibition by native BeKm-1 and its analogues was determined during step depolarization from a holding potential of −80 mV to 0 mV or +70 mV, respectively, and normalized to the current recorded without toxin (ITx/ICt).