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

Abstract

Drug block of the human ether-à -go-go-related gene Kϩ channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wildtype but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4-to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.

The Link between Inactivation and High-Affinity Block of hERG1 Channels

“…Together these findings strongly suggest that high-affinity blockers preferentially bind to the inactivated state of hERG1 channels. Contrary to this hypothesis was the observation that the IC 50 for block of wild-type and inactivation-deficient mutant channels is similar for some lowpotency compounds such as quinidine (Lees-Miller et al, 2000;Perrin et al, 2008), erythromycin, and perhexilin (Perrin et al, 2008). Moreover, drug potency of high-affinity ligands is not always proportionate to the extent of channel inactivation.…”

“…The most compelling evidence for this state-dependent block is the finding that most inactivationdeficient mutant channels exhibit dramatically reduced drug sensitivity (Suessbrich et al, 1997;Wang et al, 1997a;Ficker et al, 1998;Numaguchi et al, 2000;Perrin et al, 2008). However, there is not a simple correlation between point mutation-induced reduction or elimination of inactivation and the potency of hERG1 blockers.…”

“…However, there is not a simple correlation between point mutation-induced reduction or elimination of inactivation and the potency of hERG1 blockers. For example, the low-affinity blockers quinidine, perhexiline, and erythromycin inhibit WT and inactivation-deficient N588K hERG1 channels with equal potency (Perrin et al, 2008).…”

“…These two aromatic residues are conserved in hERG1 and ether-à-go-go (eag) channel subunits, but noninactivating eag channels are much less sensitive to most hERG1 blockers (Ficker et al, 1998). In addition, most inactivation-deficient hERG1 mutant channels (e.g., S620T, S631A, G628C/S631C) exhibit significantly reduced sensitivity to high-affinity drugs (Suessbrich et al, 1997;Wang, et al 1997a;Ficker et al, 1998;Numaguchi et al, 2000;Perrin et al, 2008). Together these findings strongly suggest that high-affinity blockers preferentially bind to the inactivated state of hERG1 channels.…”

The Pore Domain Outer Helix Contributes to Both Activation and Inactivation of the hERG K+ Channel

“…First, Chen et al (48) showed that rotation of the inner helix facilitates high affinity drug binding, which is preferentially associated with the inactivated state (51). Second, here we have shown that mutations to every third or fourth residue in the entire S5 domain perturb inactivation, suggesting that the entire S5 domain experiences a change in environment during channel inactivation.…”