Abstract
Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Here we report a novel and potent inhibitor of Kv1.5 potassium channels, N-benzyl-N-pyridin-3-yl-methyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), which exhibits features consistent with preferential block of the open state. The IC50 of S0100176 for Kv1.5 expressed in Xenopus oocytes was 0.7 microm. Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity. This approach identified Thr-479, Thr-480, Val-505, Ile-508, and Val-512 as the most important residues for block by S0100176. Mutations of these key residues to Ala or other amino acids caused marked changes in the IC50 of S0100176 (p<0.01). For example, the IC50 of S0100176 increased 362-fold for T480A, 26-fold for V505A, 150-fold for I508A, and 99-fold for V512A. We used modeling to dock S0100176 into the inner cavity of a Kv1.5 pore homology model that was generated based on the crystal structure of KcsA. The docking predicted that the five residues identified by the Ala scan were positioned less than 4.5 A from the compound. Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels.
Highlights
Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes
Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity
Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels
Summary
Voltage-gated Kϩ channel; IKur, ultrarapid delayed rectifier potassium current; QXP, Quick Explore software; S0100176, N-benzyl-N-pyridin-3-ylmethyl-2(toluene-4-sulfonylamino)-benzamide hydrochloride. Open channel blockers such as tetraethyl ammonium, other quaternary amines, and Kv1 subunits or the inactivation gate itself are thought to bind to the central cavity of voltage-gated Kϩ channels (12, 19 –22). For quaternary amines, this binding presumably involves an interaction with two different sites, including one near the selectivity filter and a second site in the S6 segment (12, 13, 23). Ala scanning of the residues in these regions in Kv1.5 combined with voltage clamp experiments of mutant channels identified four residues as putative binding sites for N-benzyl-N-pyridin-3-ylmethyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), an anthranilic acid derivative that reduced current with properties characteristic of open channel block. The positions of the residues correspond to important residues responsible for drug binding to other Kϩ channels such as HERG (human ether-a-go-gorelated gene) (18), KCNQ1 (27), and Kv1.3 (28), indicating a conservation of drug binding sites among different Kϩ channel families
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