Abstract
Blockade of hERG K(+) channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-pi interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K(+) channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site.
Highlights
Introduction of ProVal-Pro Motif into S6 Domain of human ether-a-go-go related gene (hERG) Alters Channel Gating and Reduces Drug Sensitivity—Most Kv channels have a Pro-Val-Pro motif in the S6 domain (Fig. 1b)
Kv channels of the Kv1– 4 families have an Ile or Val residue in the positions equivalent to Tyr-652 or Phe-656 of hERG (Fig. 1b). This suggests a plausible explanation for why hERG and not Kv1– 4 channels are readily blocked by structurally diverse drugs: aromatic residues in S6 are required for high affinity binding
Because most hERG blockers contain a basic nitrogen, it has been suggested that cation- interactions with Tyr-652 or Phe-656 might be required for high affinity binding to the channel. We systematically investigated these hypotheses by mutating Tyr-652 and Phe-656 to several other amino acids and determined the sensitivity of the resulting mutant channels to block by cisapride, MK-499, and terfenadine
Summary
We have used site-directed mutagenesis and voltage clamp analysis of mutant channels expressed in Xenopus oocytes to elucidate the molecular mechanisms of hERG channel block by structurally diverse drugs, including MK-499, cisapride, terfenadine, vesnarinone, chloroquine, and quinidine (10 –13) These studies identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain and predicted to face the central cavity of the channel (Fig. 1a) that are critical for high affinity binding of these drugs. Kv channels of the Kv1– 4 families have an Ile or Val residue in the positions equivalent to Tyr-652 or Phe-656 of hERG (Fig. 1b) This suggests a plausible explanation for why hERG and not Kv1– 4 channels are readily blocked by structurally diverse drugs: aromatic residues in S6 are required for high affinity binding. A quantitative comparison between the IC50 values for the mutant channels and the physicochemical properties of the side chain of the mutant residues was used to provide further insights into the molecular features of the hERG binding site
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