Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators.
Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators.
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DOI：
10.1021/acsptsci.1c00233
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发表时间：
2022-03-11
影响因子：
--
通讯作者：
Pan JQ
中科院分区：
文献类型：
--
作者：
Zhang YL;Moran SP;Allen A;Baez-Nieto D;Xu Q;Wang LA;Martenis WE;Sacher JR;Gale JP;Weïwer M;Wagner FF;Pan JQ
T-型电压门控Ca 2+通道与许多人类疾病有关，并且开发具有潜在临床应用价值的高选择性和有效的T-型Ca 2+通道调节剂的兴趣日益增加。然而，T型钙离子通道独特的生物物理特性不利于开发高通量筛选（HTS）测定来鉴定调节剂，特别是增效剂。为了说明，T型Ca 2+通道在很大程度上失活，并且不能在−25 mV（细胞筛选试验中常用的细胞系的典型静息膜电位）下打开以允许Ca 2+流入。为了解决这个问题，我们开发了表达Kir2.3通道的细胞系，使膜电位升高至-70 mV，从而使T型通道恢复到其静息状态，随后在细胞外KCl存在下通过膜去极化激活。此外，为了简化HTS测定并降低试剂成本，我们稳定地表达了膜系留的遗传钙传感器GCaMP 6s-CAAX，与未系留的GCaMP 6或合成的Ca 2+传感器Fluo-4AM相比，其显示出比背景上级的信号。在这里，我们描述了一种新的基于GCaMP 6s-CAAX的钙测定，其利用高通量荧光成像板读数器（Molecular Devices，桑尼维尔，CA）格式，其可以识别T型Ca 2+通道的激活剂和抑制剂。最后，我们证明了这种新的基于荧光的测定的实用性，以评估两种不同的G蛋白偶联受体的活性，从而将GCaMP 6s-CAAX的使用扩展到与开发药物发现中的细胞测定相关的广泛应用。
T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at −25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to −70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.
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影响因子：
3.5
作者：
Holdsworth, G;Slocombe, P;Milligan, G
通讯作者：
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影响因子：
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作者：
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作者：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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