Rezazadeh Saman, Hesketh J Christian, Fedida David
Department of Physiology, University of British Columbia, Vancouver, BC, Canada.
J Biomol Screen. 2004 Oct;9(7):588-97. doi: 10.1177/1087057104264798.
The nonradioactive Rb+ efflux assay has become a reliable and efficient high-throughput hERG screening method, but it is limited by its low sensitivity for potent hERG blockers. Using the patch clamp technique, the authors found that the low sensitivity is due in part to the use of Rb+ as the permeating cation in the assay. The affinities of the drugs measured by patch clamp technique in the presence of Rb+ were 3- to 10-fold lower than when measured by the same method in the presence of K+ ions. The apparent affinity of the drugs decreased even further when monitored by the Rb+ efflux assay. It was also observed that Rb+ had minimal effects on the activation properties of channels while there was a significant change in the half-inactivation potential. This voltage shift reduces hERG channel inactivation at efflux assay potentials, and will reduce the affinity of hERG-blocking drugs that bind to inactivated states of the channel. In combination with the effects of elevated extracellular ion concentrations, it is likely that Rb+ modulation of hERG channel inactivation is largely responsible for the reduced drug potencies observed in the Rb+ efflux assay.
非放射性铷离子（Rb⁺）外流测定法已成为一种可靠且高效的高通量人乙醚相关基因（hERG）筛选方法，但它对强效hERG阻滞剂的低敏感性限制了其应用。通过膜片钳技术，作者发现这种低敏感性部分归因于在该测定法中使用Rb⁺作为通透阳离子。在存在Rb⁺的情况下，通过膜片钳技术测得的药物亲和力比在存在钾离子（K⁺）的情况下用相同方法测得的低3至10倍。当通过Rb⁺外流测定法监测时，药物的表观亲和力甚至进一步降低。还观察到，Rb⁺对通道的激活特性影响极小，而半失活电位有显著变化。这种电压偏移降低了在外流测定电位下hERG通道的失活，并且会降低与通道失活状态结合的hERG阻断药物的亲和力。结合细胞外离子浓度升高的影响，Rb⁺对hERG通道失活的调节很可能是导致在Rb⁺外流测定法中观察到药物效力降低的主要原因。
