Institut für Integrative Neuroanatomie, Centrum für Anatomie, Charité-Universitätsmedizin Berlin, Germany.
Cell Mol Neurobiol. 2011 Mar;31(2):323-35. doi: 10.1007/s10571-010-9625-6. Epub 2010 Nov 17.
Monoaminergic and cholinergic systems are important regulators of cortical and subcortical systems, and a variety of vegetative functions are controlled by the respective neurotransmitters. Neuronal excitability and transmitter release of these neurons are strongly regulated by their potassium conductances carried by Kir and K(2P) channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K(2P) channel. After removal of cross-reactivities and affinity purification the antibody was characterized by ELISA, immunocytochemistry of TASK-3 transfected cells, and Western blots indicating that the antibody only detects TASK-3 protein, but not its paralogs TASK-1 and TASK-5. Western blot analysis of brain membrane fractions showed a single band around 45 kD, close to the predicted molecular weight of the TASK-3 protein. In addition, specific immunolabeling using the anti-TASK-3 antibody in Western blot analysis and immunocytochemistry was blocked in a concentration dependent manner by its cognate antigen only. Immunocytochemical analysis of rat brain revealed strong expression of TASK-3 channels in serotoninergic neurons of the dorsal and median raphe, noradrenergic neurons of the locus coeruleus, histaminergic neurons of the tuberomammillary nucleus and in the cholinergic neurons of the basal nucleus of Meynert. Immunofluorescence double-labeling experiments with appropriate marker enzymes confirmed the expression of TASK-3 in cholinergic, serotoninergic, and noradrenergic neurons. In the dopaminergic system strong TASK-3 expression was found in the ventral tegmental area, whereas TASK-3 immunoreactivity in the substantia nigra compacta was only weak. All immunocytochemical results were supported by in situ hybridization using TASK-3 specific riboprobes.
单胺能和胆碱能系统是皮质和皮质下系统的重要调节者，各种植物性功能受各自的神经递质控制。这些神经元的兴奋性和递质释放受到 Kir 和 K(2P)通道所携带的钾电导的强烈调节。本文描述了一种针对大鼠 TASK-3 的多克隆单特异性抗体的产生和特性，TASK-3 是主要的脑 K(2P)通道。在去除交叉反应性和亲和纯化后，该抗体通过 ELISA、转染 TASK-3 细胞的免疫细胞化学和 Western blot 进行了表征，表明该抗体仅检测 TASK-3 蛋白，而不检测其同源物 TASK-1 和 TASK-5。脑膜部分的 Western blot 分析显示，约 45 kD 处有一个单带，接近 TASK-3 蛋白的预测分子量。此外，在 Western blot 分析和免疫细胞化学中使用抗 TASK-3 抗体进行的特异性免疫标记，仅在其同源抗原的浓度依赖性方式下被阻断。大鼠脑的免疫细胞化学分析显示，TASK-3 通道在背侧和中缝核的 5-羟色胺能神经元、蓝斑核的去甲肾上腺素能神经元、乳头体核的组胺能神经元以及梅内尔特基底核的胆碱能神经元中表达强烈。用适当的标记酶进行免疫荧光双标记实验证实了 TASK-3 在胆碱能、5-羟色胺能和去甲肾上腺素能神经元中的表达。在多巴胺能系统中，在腹侧被盖区发现了强烈的 TASK-3 表达，而在黑质致密部的 TASK-3 免疫反应性则较弱。所有免疫细胞化学结果都得到了使用 TASK-3 特异性核糖探针的原位杂交的支持。
