KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells
KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells
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DOI：
10.1007/s11103-007-9252-x
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发表时间：
2008-01-01
影响因子：
5.1
通讯作者：
Costa, Alex
中科院分区：
文献类型：
--
作者：
Bregante, Monica;Yang, Yingzhen;Costa, Alex
Shaker钾通道是由四个α-亚基组装而成的四聚体蛋白。同种和异种α亚基之间均可发生齐聚反应。KDC1是一种胡萝卜振荡型钾通道，在植株根的表皮和体细胞胚胎的原真皮中表达。KDC1以前在CHO和非洲爪哇卵母细胞中具有电生理学特征，但在这些系统中进行的实验并没有提供确凿的证据表明KDC1在植物细胞中形成了一个功能上的同源通道。在这篇报道中，我们发现KDC1定位于转化KDC1：：GFP融合基因烟草植株的根细胞质膜上。膜片钳实验表明，在瞬时转化KDC1：：GFP的烟草叶肉原生质体中，KDC1存在于细胞膜上，并且没有表现出任何内向钾电流。KDC1：：GFP与拟南芥钾通道AKT1在烟草叶肉原生质体中的共表达具有将KDC1的定位从内膜转移到质膜的作用。对单独表达AKT1和与KDC1：：GFP联合表达AKT1的烟草叶肉原生质体进行了膜片钳实验，发现电压激活的内向钾电流具有不同的特性。在镀液中加入锌离子后，AKT1单独转化的原生质体的钾电流明显降低，而AKT1+KDC1：：GFP共转化的原生质体的钾电流明显增加。在酵母细胞中进行的分裂泛素实验证实了AKT1和KDC1之间的相互作用。
The Shaker potassium channels are tetrameric proteins formed by the assembly of four alpha-subunits. The oligomerization can occur among both homo- and hetero-alpha-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1::GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1::GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1::GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1::GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn2+ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1::GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.