No abstract

The scorpion toxin peptide BeKm-1 was synthesised by £uorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's e¡ects on heterologous human ether-a-go-go-related gene potassium current (I HERG ) in HEK293 cells were assessed using 'whole-cell' patch clamp. Blockade of I HERG by BeKm-1 was concentration-dependent, temperature-dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use-and frequency-dependence. Blockade by BeKm-1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm-1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs. ß

No abstract

4‐Aminopyridine (4‐AP) has been used extensively to study transient outward K+ current (ITO,1) in cardiac cells and tissues. We report here inhibition by 4‐AP of HERG (the human ether‐à‐go‐go‐related gene) K+ channels expressed in a mammalian cell line, at concentrations relevant to those used to study ITO,1. Under voltage clamp, whole cell HERG current (IHERG) tails following commands to +30 mV were blocked with an IC50 of 4.4 ± 0.5 mm. Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 mm 4‐AP inhibited peak IHERG during an applied action potential clamp waveform by ∼59 %. It also significantly prolonged action potentials and inhibited resurgent IK tails from guinea‐pig isolated ventricular myocytes, which lack an ITO,1. We conclude that by blocking the α‐subunit of the IKr channel, millimolar concentrations of 4‐AP can modulate ventricular repolarisation independently of any action on ITO,1.

The mechanism of human ether-à -go-go-related gene (HERG) K + channel blockade by the antifungal agent ketoconazole was investigated using patch-clamp recording from mammalian cell lines. Ketoconazole inhibited whole-cell HERG current (I HERG ) with a clinically relevant half-maximal inhibitory drug concentration (IC 50 ) value of 1.7 lM. The voltageand time-dependent characteristics of I HERG blockade by ketoconazole indicated dependence of block on channel gating, ruling out a significant role for closed-state channel inhibition. The S6 HERG mutations Y652A and F656A produced $4-fold and $21-fold increases in IC 50 for I HERG blockade, respectively. Thus, ketoconazole accesses the HERG channel pore-cavity on channel gating, and the S6 residue F656 is an important determinant of ketoconazole binding.