Sánchez-Chapula Jose A, Navarro-Polanco Ricardo A, Culberson Chris, Chen Jun, Sanguinetti Michael C
Unidad de Investigación Carlos Méndez del Centro Universitario de Investigaciones Biomédicas de la Universidad de Colima, 23000 Colima, México.
J Biol Chem. 2002 Jun 28;277(26):23587-95. doi: 10.1074/jbc.M200448200. Epub 2002 Apr 17.
The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K(+) channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC(50) was 8.4 +/- 0.9 microm when assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC(50) > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-pi and pi-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.
离子通道电压依赖性阻断的结构决定因素目前还知之甚少。在此，我们研究了抗疟化合物氯喹对野生型和突变型人类醚 - 去极化相关基因（HERG）钾通道的电压依赖性阻断作用。非洲爪蟾卵母细胞中表达的野生型HERG通道的阻断作用会随着膜电位逐渐去极化而增强。在4秒电压钳制脉冲至0 mV时评估，其半数抑制浓度（IC50）为8.4±0.9微摩尔。氯喹还减缓了HERG失活的表观速率，这反映了药物结合的通道无法关闭。位于HERG的S6结构域中的芳香族残基（Tyr - 652或Phe - 656）突变为丙氨酸，大大降低了氯喹对通道的阻断效力（在0 mV时IC50>1毫摩尔）。然而，Tyr - 652的突变也改变了阻断的电压依赖性。与野生型HERG相反，Y652A HERG通道的阻断作用随着膜的逐渐去极化而减弱，在+40 mV时观察到阻断作用完全解除。当将Tyr - 652的残基突变为Phe从而去除其羟基时，HERG通道阻断与电压无关。这些研究结果共同表明Tyr - 652在HERG通道的电压依赖性阻断中起关键作用。分子建模用于确定氯喹与HERG中心腔的能量最小化对接。我们的实验结果和建模表明，氯喹通过与多个亚基的Tyr - 652和Phe - 656的阳离子 - π和π堆积相互作用优先阻断开放的HERG通道。
