Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain
Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain
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DOI：
10.1074/jbc.m109.086553
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发表时间：
2010-06-04
影响因子：
4.8
通讯作者：
Devor, Daniel C.
中科院分区：
文献类型：
--
作者：
Gao, Yajuan;Balut, Corina M.;Devor, Daniel C.
调节内皮细胞表面 Ca2+ 激活的 K+ 通道数量有助于控制内皮源性超极化因子反应，尽管这一过程尚不清楚。为了解决质膜定位 KCa2.3 的命运，我们在人胚胎肾细胞和人微血管内皮细胞系 HMEC-1 中结合使用细胞外表位标记通道以及荧光和生物素化技术。 KCa2.3 从质膜内化并以 18 小时的时间常数降解。细胞表面生物素化表明 KCa2.3 被快速内吞并再循环回质膜。与回收一致，显性失活 (DN) RME-1 或 Rab35 以及野生型 EPI64C（Rab35 GTP 酶激活蛋白）的表达导致 KCa2.3 在细胞内积聚。 DN RME-1、DN Rab35 或野生型 EPI64C 的表达导致稳态质膜表达减少。 EPI64C 的敲低增加了 KCa2.3 的细胞表面表达。此外，EPI64C 的作用取决于其 GTP 酶激活蛋白活性。免疫共沉淀研究证实 KCa2.3 与 Rab35 和 RME-1 之间存在关联。与 KCa2.3 相比，KCa3.1 被快速内吞并以不依赖 RME-1 和 Rab35 的方式降解。一系列 N 端缺失确定了一个 12 个氨基酸区域，Gly(206)-Pro(217)，这是 KCa2.3 快速回收所必需的。 Gly(206)-Pro(217)的删除对KCa2.3与Rab35的关联没有影响，但显着降低与RME-1的关联。这些代表了首次研究阐明 KCa2.3 在质膜上的维持机制。
Regulation of the number of Ca2+-activated K+ channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly(206)-Pro(217), as being required for the rapid recycling of KCa2.3. Deletion of Gly(206)-Pro(217) had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.