Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.
Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.
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作者：
Ou, J. W.;Kumar, Y.;Alioua, A.;Sailer, C.;Stefani, E.;Toro, L.
大电导、电压和Ca 2+激活的K+通道（MaxiK）是广泛表达的离子通道，由4个成孔α亚基（MaxiKα）组成，通常作为质膜蛋白存在于各种细胞类型中。在小鼠星形胶质细胞原代培养中，我们发现MaxiKα主要局限于微管网络。通过三种独立的标记方法观察MaxiKα的独特微管分布：1）MaxiKα特异性抗体，2）表达的EGFP标记的MaxiKα，和3）荧光团缀合的伊比利亚毒素，一种特异性MaxiK孔阻断剂。通过体外His-标签下拉、脑裂解物的免疫共沉淀和微管解聚实验进一步证实了MaxiKα与微管的结合。一般药理学药物咖啡因或毒胡萝卜素引起的细胞内Ca 2+变化导致质膜MaxiKα标记增加。更值得注意的是，U46619是血栓烷A2（TXA 2）的类似物，可触发Ca 2+释放途径，其水平在脑出血/创伤期间增加，也可引起MaxiKα表面标记的类似增加。U46619刺激细胞的全细胞膜片钳记录显示电流振幅增加约3倍，表明TXA 2刺激导致额外的功能性MaxiK通道募集至表面膜。虽然成熟星形胶质细胞中基本不存在微管，但脑切片中的免疫组织化学结果显示，新生小鼠（P1）的皮质星形胶质细胞显示出与MaxiKα显著共定位的微管的稳健表达。本研究的结果提供了新的见解，表明从细胞内储存释放的Ca 2+可能在调节细胞内微管相关MaxiKα储存向发育中的小鼠星形胶质细胞质膜的运输中发挥关键作用。
Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming α-subunits (MaxiKα) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiKα is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKα was visualized by three independent labeling approaches: 1) MaxiKα-specific antibodies, 2) expressed EGFP-labeled MaxiKα, and 3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiKα association with microtubules was further confirmed by in-vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiKα labeling at the plasma membrane. More notably U46619, an analog of thromboxane A2 (TXA2) which triggers Ca2+-release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKα surface labeling. Whole-cell patch clamp recordings of U46619 stimulated cells develop a ~3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiKα. The results of this study provide the novel insight that suggests Ca2+ released from intracellular stores, may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiKα stores to the plasma membrane of developing murine astrocytes.
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DOI：
10.1083/jcb.85.3.890
发表时间：
1980-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
McCarthy KD;de Vellis J
通讯作者：
de Vellis J
影响因子：
3.8
作者：
Anderson, C S;MacKinnon, R;Smith, C;Miller, C
通讯作者：
Miller, C
DOI：
10.1007/bf00495643
发表时间：
1984-01-01
期刊：
HISTOCHEMISTRY
影响因子：
--
作者：
HAJOS, F;ROSTOMIAN, MA
通讯作者：
ROSTOMIAN, MA
影响因子：
5
作者：
Honma, Shigeyoshi;Saika, Manami;Nakahata, Norimichi
通讯作者：
Nakahata, Norimichi
影响因子：
5.5
作者：
Lu, R;Alioua, A;Toro, L
通讯作者：
Toro, L