ABSTRACT
Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K(+) current (IKr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.
Subject(s)
ERG1 Potassium Channel/metabolism , Potassium Channel Blockers/metabolism , Binding Sites , ERG1 Potassium Channel/chemistry , Models, Molecular , Patch-Clamp Techniques , Protein BindingABSTRACT
BACKGROUND AND PURPOSE: Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. EXPERIMENTAL APPROACH: hERG and K(IR)2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr . Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. KEY RESULTS: Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K(IR)2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K(IR)2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. CONCLUSIONS AND IMPLICATIONS: Drug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.
Subject(s)
Anti-Arrhythmia Agents/pharmacology , Ether-A-Go-Go Potassium Channels/metabolism , Membrane Transport Modulators/pharmacology , Pentamidine/pharmacology , Phenethylamines/pharmacology , Potassium Channel Blockers/pharmacology , Shab Potassium Channels/metabolism , Sulfonamides/pharmacology , Animals , Anti-Arrhythmia Agents/chemistry , Antiprotozoal Agents/adverse effects , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/metabolism , Cell Membrane/drug effects , Dogs , ERG1 Potassium Channel , Endocytosis/drug effects , Ether-A-Go-Go Potassium Channels/chemistry , Ether-A-Go-Go Potassium Channels/genetics , HEK293 Cells , Humans , Kinetics , Membrane Transport Modulators/adverse effects , Membrane Transport Modulators/chemistry , Mice , Molecular Dynamics Simulation , Pentamidine/adverse effects , Pentamidine/analogs & derivatives , Pentamidine/chemistry , Phenethylamines/chemistry , Potassium Channel Blockers/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Shab Potassium Channels/chemistry , Shab Potassium Channels/genetics , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
Cardiac inward rectifier channels belong to three different classes of the KIR channel protein family. The KIR2.x proteins generate the classical inward rectifier current, IK1, while KIR3 and KIR6 members are responsible for the acetylcholine responsive and ATP sensitive inward rectifier currents IKAch and IKATP, respectively. Aberrant function of these channels has been correlated with severe cardiac arrhythmias, indicating their significant contribution to normal cardiac electrophysiology. A common feature of inward rectifier channels is their dependence on the lipid phosphatidyl-4,5-bisphospate (PIP2) interaction for functional activity. Cationic amphiphilic drugs (CADs) are one of the largest classes of pharmaceutical compounds. Several widely used CADs have been associated with inward rectifier current disturbances, and recent evidence points to interference of the channel-PIP2 interaction as the underlying mechanism of action. Here, we will review how six of these well known drugs, used for treatment in various different conditions, interfere in cardiac inward rectifier functioning. In contrast, KIR channel inhibition by the anionic anesthetic thiopental is achieved by a different mechanism of channel-PIP2 interference. We will discuss the latest basic science insights of functional inward rectifier current characteristics, recently derived KIR channel structures and specific PIP2-receptor interactions at the molecular level and provide insight in how these drugs interfere in the structure-function relationships.
