Myokai Toshihiko, Ryu Sunghi, Shimizu Hirofumi, Oiki Shigetoshi
Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan.
Mol Pharmacol. 2008 Jun;73(6):1643-51. doi: 10.1124/mol.107.042085. Epub 2008 Mar 7.
The human ether-à-go-go related gene product (HERG) channel is essential for electrical activity of heart cells, and block of this channel by many drugs leads to lethal arrhythmias. Tyr(652) and Phe(656) of the sixth transmembrane helix are candidates for the drug binding site. In the tetrameric HERG channel, a drug with asymmetric structure should interact unevenly with multiple residues from different subunits. To elucidate the topology of the drug-binding site, we constructed tandem dimers of HERG channels and the aromatic Tyr(652) and Phe(656) residues were replaced by alanine singly or doubly. Eight types of HERG channels, including homotetrameric mutants, having different numbers and arrangements of aromatic residues at the blocking site, were studied. Effects of cisapride on channels expressed in Xenopus laevis oocytes were examined electrophysiologically. The inhibition constants (K(i)) were increased significantly as the diagonal Tyr(652) were deleted, whereas those for the diagonal Phe(656)-deleted mutant were not changed. These results suggest that Tyr(652) residues from adjacent subunits contributed to the binding. Two types of double mutants of tandem dimers showed significantly distinct affinities, suggesting that the coexistence of Tyr(652) and Phe(656) on a subunit in diagonal position is crucial to having a high affinity. Thermodynamic double-mutant cycle analyses revealed interactions between Tyr(652) and Phe(656) upon binding. The kinetics and voltage-dependence of blocking suggested transitions of the binding site from low to high affinity. These approaches using a set of mutant HERG channels gave a dynamic picture of the spatial arrangements of residues that contribute to the drug-channel interaction.
人醚 - 去 - 去相关基因产物（HERG）通道对于心脏细胞的电活动至关重要，许多药物对该通道的阻断会导致致命性心律失常。第六个跨膜螺旋的Tyr（652）和Phe（656）是药物结合位点的候选残基。在四聚体HERG通道中，具有不对称结构的药物应与来自不同亚基的多个残基发生不均匀相互作用。为了阐明药物结合位点的拓扑结构，我们构建了HERG通道的串联二聚体，并将芳香族Tyr（652）和Phe（656）残基单独或双重替换为丙氨酸。研究了八种类型的HERG通道，包括在阻断位点具有不同数量和排列的芳香族残基的同四聚体突变体。通过电生理学方法检测了西沙必利对非洲爪蟾卵母细胞中表达的通道的影响。随着对角线上的Tyr（652）缺失，抑制常数（K（i））显著增加，而对角线上Phe（656）缺失的突变体的抑制常数没有变化。这些结果表明相邻亚基的Tyr（652）残基有助于结合。串联二聚体的两种类型的双突变体表现出明显不同的亲和力，表明对角位置的亚基上Tyr（652）和Phe（656）的共存对于具有高亲和力至关重要。热力学双突变循环分析揭示了结合时Tyr（652）和Phe（656）之间的相互作用。阻断的动力学和电压依赖性表明结合位点从低亲和力向高亲和力转变。这些使用一组突变HERG通道的方法给出了有助于药物 - 通道相互作用的残基空间排列的动态图景。
