MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain.
MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain.
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DOI：
10.1016/j.neuroscience.2015.12.058
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发表时间：
2016-03-11
期刊：
影响因子：
3.3
通讯作者：
Toro L
中科院分区：
文献类型：
--
作者：
Singh H;Li M;Hall L;Chen S;Sukur S;Lu R;Caputo A;Meredith AL;Stefani E;Toro L
大电导电压和钙激活钾 (MaxiK) 通道通过膜去极化和胞质 Ca2+ 升高而激活。在大脑中，它们定位于神经元和星形胶质细胞，发挥作用，例如在动作电位期间重置膜电位、神经递质释放和神经血管耦合。已知 MaxiK 通道与多种调节蛋白和辅助亚基相关，并且每种相互作用都可能产生不同的生理后果。为了发现 MaxiK 通道脑生理学的新参与者，我们应用定向蛋白质组学方法并使用特定抗体获得了 MaxiK 通道成孔 α 亚基脑相互作用组。对照包括分别用兔 IgG 和野生型和 MaxiK 通道敲除小鼠 (Kcnma1−/−) 中的抗 MaxiK 抗体进行免疫沉淀。我们发现了已知和未报道的相互作用伙伴，它们定位于质膜、细胞外空间、细胞质和细胞内细胞器，包括线粒体、细胞核、内质网和高尔基体。使用与 MitoTracker 共标记的纯化脑线粒体进一步证实了 MaxiK 通道对线粒体的定位。针对 GABA 转运蛋白 3 (GAT3) 和热休克蛋白 60 (HSP60)，给出了 MaxiK 通道与先前未识别的伙伴相互作用的独立证据。在 HEK293T 细胞中，GAT3 和 HSP60 均与 MaxiK 通道共免疫沉淀并共定位；主要在细胞外周用 GAT3 观察到共标记，在细胞内用 HSP60 观察到共标记，蛋白质邻近指数分别为 ~0.6 和 ~0.4。在大鼠原代海马神经元中，GAT3 的共定位指数相同 (~0.6)，HSP60 与 MaxiK 通道相关的共定位指数略高 (~0.5)。本研究结果提供了MaxiK通道在小鼠大脑中的完整相互作用组，进一步确立了MaxiK通道在小鼠大脑线粒体中的定位，并证明了MaxiK通道与神经元中GAT3和HSP60的相互作用。 MaxiK 通道与 GAT3 的相互作用开启了 MaxiK 通道在 GABA 稳态和信号传导中发挥作用的可能性。
Large conductance voltage and calcium-activated potassium (MaxiK) channels are activated by membrane depolarization and elevated cytosolic Ca2+. In the brain, they localize to neurons and astrocytes, where they play roles such as resetting the membrane potential during an action potential, neurotransmitter release, and neurovascular coupling. MaxiK channels are known to associate with several modulatory proteins and accessory subunits, and each of these interactions can have distinct physiological consequences. To uncover new players in MaxiK channel brain physiology, we applied a directed proteomic approach and obtained MaxiK channel pore-forming α subunit brain interactome using specific antibodies. Controls included immunoprecipitations with rabbit IgG and with anti-MaxiK antibodies in wild type and MaxiK channel knockout mice (Kcnma1−/−), respectively. We have found known and unreported interactive partners that localize to the plasma membrane, extracellular space, cytosol and intracellular organelles including mitochondria, nucleus, endoplasmic reticulum and Golgi apparatus. Localization of MaxiK channel to mitochondria was further confirmed using purified brain mitochondria colabeled with MitoTracker. Independent proof of MaxiK channel interaction with previously unidentified partners is given for GABA transporter 3 (GAT3) and heat shock protein 60 (HSP60). In HEK293T cells, both GAT3 and HSP60 coimmunoprecipitated and colocalized with MaxiK channel; colabeling was observed mainly at the cell periphery with GAT3 and intracellularly with HSP60 with protein proximity indices of ~0.6 and ~0.4, respectively. In rat primary hippocampal neurons, colocalization index was identical for GAT3 (~0.6) and slightly higher for HSP60 (~0.5) association with MaxiK channel. The results of this study provide a complete interactome of MaxiK channel the mouse brain, further establish the localization of MaxiK channel in the mouse brain mitochondria and demonstrate the interaction of MaxiK channel with GAT3 and HSP60 in neurons. The interaction of MaxiK channel with GAT3 opens the possibility of a role of MaxiK channel in GABA homeostasis and signaling.