Immunofluorescence-based assay to identify modulators of the number of plasma membrane KCa3.1 channels
Immunofluorescence-based assay to identify modulators of the number of plasma membrane KCa3.1 channels
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DOI：
10.4155/fmc.10.182
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发表时间：
2010-05-01
影响因子：
4.2
通讯作者：
Devor, Daniel C.
中科院分区：
文献类型：
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作者：
Balut, Corina M.;Gao, Yajuan;Devor, Daniel C.
背景：中电导钙依赖性钾通道（KCa3.1）已被提出作为许多疾病的治疗靶点。我们最近的特点，这些通道的内吞命运，导致的可能性，这可以被操纵，从而改变在质膜通道的数量（N）。结果与讨论：我们证明，质膜定位的KCa3.1可以快速（10分钟）标记的荧光团使用的生物素连接酶（BirA）受体肽标记的通道和ER定位的BirA的组合。使用96孔板形式定量KCa 3.1的内吞作用，证明泛素活化酶El抑制剂UBEI-41阻断KCa 3.1的内吞作用。结论：我们描述了一种新的方法，用于识别KCa内吞作用的调节剂，并证明这可用于调节N在质膜。预计改变N将为靶向疾病中的这些通道提供新的治疗策略。
Background: Intermediate conductance Ca(2+)-dependent K(+) channels (KCa3.1) have been proposed as therapeutic targets for numerous diseases. We recently characterized the endocytic fate of these channels; leading to the possibility that this can be pharmacologically manipulated, thereby altering the number of channels (N) at the plasma membrane. Results & discussion: We demonstrate that plasma membrane-localized KCa3.1 can be rapidly (10 min) tagged with a fluorophore using a combination of a biotin ligase (BirA) acceptor peptide-tagged channel and an ER-localized BirA. Endocytosis of KCa3.1 was quantified using a 96-well plate format, demonstrating that the ubiquitin-activating enzyme El inhibitor UBEI-41, blocks the endocytosis of KCa3.1. Conclusion: We describe a novel method for identifying modulators of KCa endocytosis and demonstrate this can be used to modulate N at the plasma membrane. It is anticipated that altering N will provide novel therapeutic strategies for targeting these channels in disease.