Parihar Ashutosh S, Groebe Duncan R, Scott Victoria E, Feng Jianlin, Zhang Xu-Feng, Warrior Usha, Gopalakrishnan Murali, Shieh Char-Chang
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6125, USA.
Assay Drug Dev Technol. 2003 Oct;1(5):647-54. doi: 10.1089/154065803770381002.
Although techniques such as (86)Rb(+) flux provide a sensitive measure of K(+) channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK(Ca) alpha subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BK(Ca) channel openers, including NS1608, NS1619, and NS-8, activated rubidium efflux with EC(50) values ranging from 1 to 4 microM in both radioactive and nonradioactive efflux formats. Compounds such as iberiotoxin, paxilline, and charybdotoxin inhibited rubidium efflux responses evoked by the BK(Ca) channel opener NS1608 in both radioactive and nonradioactive efflux formats. The IC(50) values of the inhibitors in AAS format were comparable to those derived from (86)Rb(+) efflux assays. The present studies show that the pharmacological profiles of BK(Ca) channels assessed by AAS compare well with those obtained using the (86)Rb(+) efflux assay, and support the utility of nonradioactive efflux format for higher throughput screening campaigns for novel K(+) channel modulators.
尽管诸如⁸⁶Rb⁺通量等技术能够灵敏地测量钾离子通道活性，但相对较短的半衰期、高能量发射以及所产生的放射性物质数量，限制了基于通量的方法在高通量筛选中的应用。本研究详细阐述了利用火焰原子吸收光谱法（AAS）技术进行大电导钙激活钾离子通道（BK(Ca)）非放射性铷外流测定的方法。利用表达BK(Ca)α亚基的人胚肾（HEK293）细胞，建立了一种基于96孔细胞的通道开放剂和抑制剂非放射性铷外流筛选方法。已知的BK(Ca)通道开放剂，包括NS1608、NS1619和NS - 8，在放射性和非放射性外流形式中均能激活铷外流，其半数有效浓度（EC₅₀）值范围为1至4 μM。诸如iberiotoxin、paxilline和charybdotoxin等化合物在放射性和非放射性外流形式中均抑制BK(Ca)通道开放剂NS1608诱发的铷外流反应。AAS形式中抑制剂的半数抑制浓度（IC₅₀）值与⁸⁶Rb⁺外流测定所得值相当。本研究表明，通过AAS评估的BK(Ca)通道药理学特征与使用⁸⁶Rb⁺外流测定获得的特征具有良好的可比性，并支持非放射性外流形式在新型钾离子通道调节剂高通量筛选活动中的实用性。
