Plotkin M D, Kaplan M R, Peterson L N, Gullans S R, Hebert S C, Delpire E
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Am J Physiol. 1997 Jan;272(1 Pt 1):C173-83. doi: 10.1152/ajpcell.1997.272.1.C173.
We used in situ hybridization and immunocytochemistry with polyclonal antibodies against the mouse bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (mBSC2) to determine the location of this cotransporter in rat brain. Northern blots and in situ hybridization showed the presence of cotransporter mRNA in the brain, with an especially high level of expression in the choroid plexus (CP). Affinity-purified anti-BSC2 antibody identified proteins of 145-155 kDa on Western blot analysis and immunoprecipitation of brain and CP membrane protein. Indirect immunofluorescence demonstrated that BSC2 protein is located on the apical surface of the CP and is heterogeneously distributed in cell bodies and dendrites of neurons in the central and peripheral nervous system. The apical localization of BSC2 in the CP was confirmed by 86Rb+ uptakes in primary cultures of CP cells grown on permeable filters and confocal immunofluorescence microscopy. The apical localization of the cotransporter in CP epithelium suggests a role for the cotransporter in cerebrospinal fluid K+ homeostasis. In neurons, the cotransporter may help regulate intracellular Cl- concentration and thereby affect neuronal response to gamma-aminobutyric acid.
我们运用原位杂交技术以及针对小鼠布美他尼敏感的钠-钾-2-氯协同转运蛋白（mBSC2）的多克隆抗体进行免疫细胞化学实验，以确定该协同转运蛋白在大鼠脑中的位置。Northern印迹法和原位杂交显示脑中存在协同转运蛋白的信使核糖核酸（mRNA），脉络丛（CP）中的表达水平尤其高。亲和纯化的抗BSC2抗体在蛋白质印迹分析以及脑和CP膜蛋白的免疫沉淀实验中鉴定出了145 - 155千道尔顿的蛋白质。间接免疫荧光表明，BSC2蛋白位于CP的顶端表面，并且在中枢和外周神经系统的神经元细胞体和树突中呈异质性分布。通过在可渗透滤膜上生长的CP细胞原代培养物中的86Rb +摄取以及共聚焦免疫荧光显微镜观察，证实了BSC2在CP中的顶端定位。协同转运蛋白在CP上皮细胞中的顶端定位表明其在脑脊液钾离子稳态中发挥作用。在神经元中，该协同转运蛋白可能有助于调节细胞内氯离子浓度，从而影响神经元对γ-氨基丁酸的反应。
