Department of Physiology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53706, USA.
J Gen Physiol. 2010 Nov;136(5):541-54. doi: 10.1085/jgp.201010438. Epub 2010 Oct 11.
The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.
许多细胞内孔道阻断剂（如四烷基铵化合物和局部麻醉剂）的标志是它们能够变构调节电压依赖性离子通道中电压传感器的运动。例如，当局部麻醉剂阻断钠电流时，域 III 的电压传感器特别稳定在激活状态。这种孔结合位点与电压传感器之间的长程相互作用的分子机制仍知之甚少。在这里，我们使用扫描诱变结合电压钳荧光法，系统地评估了钠通道域 III 内部门控界面的作用。我们发现 S4-S5 接头和 S5 和 S6 螺旋中的几个突变显着降低了利多卡因对域 III 电压传感器激活的稳定作用，而在饱和药物浓度下，对使用依赖性阻断没有显着影响。在野生型骨骼肌钠通道中，局部麻醉剂阻断伴随着总门控电荷减少 21%。相比之下，该关键细胞内区域的点突变会降低局部麻醉剂对这种电荷修饰的作用。我们对简单模型的分析表明，这些门控界面中的突变可能会破坏电压传感器和钠通道孔之间的各种耦合相互作用。这些发现为理解药物结合位点与电压传感器之间变构相互作用的机制提供了一个分子框架。
