Block of human ether-a-go-go-related gene (HERG) K+ channels by a variety of medications has been linked to acquired long QT syndrome, a disorder of cardiac repolarisation that predisposes to lethal arrhythmias. Methanesulfonanilide compounds, such as ibutilide, dofetilide and MK499, are potent open channel blockers of HERG. Recently, we identified residues in the inner cavity of HERG, which form the binding site for MK499 (Mitcheson et al. 2000). Two aromatic residues (Y652 and F656) are particularly important structural determinants of drug block for all compounds tested so far, and all have multiple aromatic groups. To determine if multiple aromatic groups were necessary for interactions with Y652 and F656, we investigated the binding sites of clofilium and ibutilide, which each have a long aliphatic chain but only a single aromatic group. In addition, whereas ibutilide has a methanesulfonanilide moiety, clofilium does not, allowing us to investigate the importance of this chemical group.
The two-microelectrode voltage-clamp technique was used to record membrane currents in isolated Xenopus oocytes injected with cRNA. The sensitivity to block by clofilium and ibutilide was investigated in channels in which individual residues in the pore helix (T623 to V625) and S6 (L646 to Y667), predicted to line the inner cavity, had been mutated to alanine. Oocytes were repetitively depolarised from -90 to 0 mV for 5 s, and tail currents elicited by steps to -70 mV. Both ibutilide and clofilium were used at a concentration of 300 nM (10 X IC50), both of which blocked WT HERG to ~20 % of control current. Data are presented as means ± S.E.M.
In most cases mutating residues to alanine had little effect on drug block. However, seven mutants (T623A, S624A, V625A, G648A, Y652A, F656A and V659A) were profoundly insensitive to ibutilide, with 75 to 100 % (n ▓ge│ 6) of current remaining after drug block. Similar results were obtained for clofilium, apart from two mutants within the pore region, T623A and V625A, which were more sensitive to clofilium (60 ± 4 and 69 ± 6 % of control current after drug block, respectively).
These data suggest that seven residues are involved in binding of ibutilide and clofilium. Since mutations of both Y652 and F656 residues, which were previously thought to interact with aromatic groups on the drugs, produce channels which are insensitive to both ibutilide and clofilium, it seems likely that these aromatic residues can interact with both aliphatic and aromatic groups. In addition, the greater sensitivity of the pore helix mutants to clofilium, when compared with ibutilide, suggests that this region may be important for interacting with the methanesulfonanilide moiety.
This work was supported by Pfizer Global Research and Development.
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