Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Dept R4PM, Bldg. AP9A, 100 Abbott Park Road, Abbott Park, IL 60064-6125, USA.
Inflamm Res. 2011 Jul;60(7):683-93. doi: 10.1007/s00011-011-0322-9. Epub 2011 Mar 11.
The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays.
The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology.
N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable.
The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.
本研究旨在使用荧光和自动膜片钳电生理学检测比较一组不同的肽类和小分子钙通道阻滞剂对天然和重组 N 型钙通道失活状态的阻断作用。
通过增加细胞外 KCl 来开放钙通道阻滞剂在 IMR-32 细胞和过表达内向整流钾通道 Kir2.1 的 HEK 细胞中的 N 型通道的药理学研究。在 Kir2.1/N 型细胞系中，通过调节细胞外 KCl 可以调节膜电位，从而确定 N 型钙通道的静息和失活状态阻断。通过自动膜片钳电生理学还确定了这些阻滞剂的效力和状态依赖性抑制程度。
为一组具有 0.001-7 μM IC50 值的阻滞剂确定了 IMR-32 细胞中的 N 型介导的钙内流，并且这与使用电生理学测量的重组通道的失活状态阻断呈正相关。与电生理学测定相比，几种化合物在基于荧光的状态依赖性测定中的效力明显较弱，尽管状态依赖性阻断程度相当。
目前的数据表明，荧光测定适用于评估阻滞剂选择性与 N 型通道失活状态相互作用的能力。
