Topological Mapping of the Asymmetric Drug Binding to the Human Ether-à-go-go-Related Gene Product (HERG) Potassium Channel by Use of Tandem Dimers

Abstract

The human ether-à -go-go related gene product (HERG) channel is essential for electrical activity of heart cells, and block of this channel by many drugs leads to lethal arrhythmias. Tyr 652 and Phe 656 of the sixth transmembrane helix are candidates for the drug binding site. In the tetrameric HERG channel, a drug with asymmetric structure should interact unevenly with multiple residues from different subunits. To elucidate the topology of the drug-binding site, we constructed tandem dimers of HERG channels and the aromatic Tyr 652 and Phe 656 residues were replaced by alanine singly or doubly. Eight types of HERG channels, including homotetrameric mutants, having different numbers and arrangements of aromatic residues at the blocking site, were studied. Effects of cisapride on channels expressed in Xenopus laevis oocytes were examined electrophysiologically. The inhibition constants (K i ) were increased significantly as the diagonal Tyr 652 were deleted, whereas those for the diagonal Phe 656 -deleted mutant were not changed. These results suggest that Tyr 652 residues from adjacent subunits contributed to the binding. Two types of double mutants of tandem dimers showed significantly distinct affinities, suggesting that the coexistence of Tyr 652 and Phe 656 on a subunit in diagonal position is crucial to having a high affinity. Thermodynamic double-mutant cycle analyses revealed interactions between Tyr 652 and Phe 656 upon binding. The kinetics and voltage-dependence of blocking suggested transitions of the binding site from low to high affinity. These approaches using a set of mutant HERG channels gave a dynamic picture of the spatial arrangements of residues that contribute to the drug-channel interaction.

Drug Binding to the Inactivated State Is Necessary but Not Sufficient for High-Affinity Binding to Human Ether-à-go-go-Related Gene Channels

“…In this hypothesis, not only would the relationship between the drug binding residues on Ser6, Tyr652, and Phe656 alter with respect to drug binding residues at the base of the pore helixes (Lees-Miller et al, 2000;Chen et al, 2002) but also the intersubunit relationships between Tyr652 and Phe656 would change (Myokai et al, 2008). Evidence in favor of a primary role for reorientation of Ser6 in favoring inactivated state drug binding comes from a study investigating the effect of mutating Phe656 to methionine on the binding of droperidol (Luo et al, 2008).…”

Differential hERG ion channel activity of ultrasmall gold nanoparticles

“…Typical hERG blockers such as cisapride, terfenadine, or dofetilide interact with the aromatic amino acids Tyr652 and Phe656 of the hERG (17)(18)(19). As Au1.4MS is stabilized by TPPMS, a sulfonated triphenylphosphine, an inhibitory interaction mediated by the aromatic moieties of the ligand shell is conceivable.…”

Refining Insights into High-Affinity Drug Binding to the Human Ether-à-go-go-Related Gene Potassium Channel: Fig. 1.

“…Given the strong association between I Kr /hERG block, QT interval prolongation, and risk of TdP, an obvious and important question arises: what makes I Kr /hERG uniquely susceptible to pharmacological inhibition by chemically and therapeutically diverse drugs? Considerable insight into the nature of drughERG interactions has emerged since 2000, and in this issue of Molecular Pharmacology, Myokai et al (2008) further this field. The brief historical perspective below aims to place this important new study in context.…”

“…The data are interpreted as suggestive of additive interactions between Tyr652 and Phe656 on the same subunit, but cooperative interactions between these residues on adjacent subunits. The findings of Myokai et al (2008) are also broadly consistent with a recent simulation study involving simulation of cisapride docking to the hERG K ϩ channel tetramer (using a template based on KvAP), which suggested T-shaped -stacking interactions between cisapride and diagonally opposite Tyr652s and a parallel displaced interaction with a Phe656 (Farid et al, 2006). In summary, this important study not only provides unprecedented insight into the nature of cisapride blockade of the hERG channel but also establishes an exciting approach that can be expected to be invaluable in refining ideas as to how drugs may bind to functional tetrameric hERG channels.…”

“…Drug structures may be asymmetric, however, and it follows that such molecules will not necessarily interact equally with particular residues on all four subunits. In this volume of Molecular Pharmacology, Myokai et al (2008) have investigated this issue for cisapride, using tandem dimers of hERG incorporating wild-type (WT) and/or mutant subunits in which one or both of Tyr652 and Phe656 had been mutated to alanine. The use of tandem dimers is not without potential problems, because linking monomers could potentially influence conformational changes during channel gating and alter channel kinetics.…”