Abstract
The hERG channel is a key player in repolarization of the cardiac action potential. Pharmacological blockade of hERG channels depletes the cardiac repolarization reserve, increasing the risk of cardiac arrhythmias. The promiscuous nature of drug interactions with hERG presents a therapeutic challenge for drug design and development. Despite considerable effort, the mechanisms of drug binding remain incompletely understood. One proposed mechanism is that high-affinity drug binding preferentially occurs when channels are in the inactivated state. However, this has been difficult to test, since inactivation is rapid in hERG and access to the drug binding site is limited by slower opening of the activation gate. Here, we have directly assessed the role of inactivation in cisparide and terfenadine drug binding in mutant (I663P) hERG channels where the activation gate is trapped-open. We firstly demonstrate the utility of this approach by showing that inactivation, ion selectivity and high affinity drug binding are preserved in I663P mutant channels. We then assess the role of inactivation by applying cisapride and terfenadine at different membrane voltages, which induce varying degrees of inactivation. We show that the extent of block does not correlate with the extent of inactivation. These data suggest that inactivation is not a major determinant of cisapride or terfenadine binding in hERG channels.
Highlights
The human-ether-a-go-go-related gene encodes the pore-forming α-subunit of the voltage-gated K+ (Kv) channel that underlies the cardiac rapid delayed rectifier current, IKr
We have utilized trapped-open human-ether-a-go-go-related gene (hERG) channels as a novel paradigm with which to study the role of inactivation in drug binding. hERG I663P channels are trapped in the open state over a wide range of voltages yet exhibit voltage-dependent inactivation that is similar to Wild type (WT) channels
We have previously shown that the I663P mutation produces constitutively active hERG channels that do not appear to deactivate[27]
Summary
The human-ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunit of the voltage-gated K+ (Kv) channel that underlies the cardiac rapid delayed rectifier current, IKr. The high resolution cryo-EM structure of the open hERG channel state revealed unusual features of the inner cavity that may provide significant insight into the susceptibility of hERG to a wide range of drugs These include the presence of four hydrophobic pockets, each potentially capable of accommodating a drug molecule, which connect to the pore cavity just below the selectivity filter, converging at a region with strong negative electrostatic potential. HERG I663P channels are trapped in the open state over a wide range of voltages yet exhibit voltage-dependent inactivation that is similar to WT channels This allows us to overcome a typical challenge in studying the inactivation-dependence of drug binding, which is that the kinetics of inactivation are an order of magnitude faster than those of activation, yet drugs require the intracellular pore gate to open to access their binding site. We compared the extent of block of hERG channels by classical blockers at different voltages, which induce varying degrees of inactivation and show that the extent of block does not correlate with inactivation
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