Molecular Determinants of Dofetilide Block of HERG K ⁺ Channels

Abstract

The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K+ current (IKr). HERG/IKr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.

A structural basis for drug-induced long QT syndrome

“…2e). This finding was unexpected because previous studies concluded HERG channel inactivation was required for high-affinity drug block (11,12), suggesting that enhanced inactivation might increase drug potency.…”

“…However, the eag channel is relatively insensitive to block by dofetilide (11) and MK-499 (IC 50 Ͼ 10 M; data not shown). Unlike HERG, eag channels do not inactivate.…”

“…Previous studies have characterized single residues of HERG that when mutated altered the sensitivity of the channel to block by the methanesulfonanilide antiarrhythmic drug dofetilide. However, these residues are believed to alter drug binding by an allosteric effect related to the loss of inactivation gating (11). A more recent study found that mutation of a Phe located in the S6 domain of HERG decreased block by dofetilide and quinidine (12).…”

Rb+ Flux through hERG Channels Affects the Potency of Channel Blocking Drugs: Correlation with Data Obtained Using a High-Throughput Rb+ Efflux Assay

“…It has been reported that the removal of the rapid inactivation, which has been labeled as C-type inactivation, 17 resulted in the reduction of the sensitivity of hERG channels to E-4031 and dofetilide. 18,19 Mitcheson et al 20 showed that hERG mutations that shifted the inactivation curve toward either negative or positive directions resulted in the loss of sensitivity to drugs. These results underline the importance of the C-type inactivation in high-affinity drug binding and also suggest the involvement of other factors.…”

Do Glia Have Heart? Expression and Functional Role forEther-A-Go-GoCurrents in Hippocampal Astrocytes

“…1, compare B2, B3) and in cultured astrocytes, respectively. In addition to E4031, dofetilide has been widely used to specifically block ERG (Kiehn et al, 1996;Ficker et al, 1998) (Fig. 1 B).…”