The phosphoinositide PI(3,5)P2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells
The phosphoinositide PI(3,5)P2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells
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DOI：
10.1007/s00018-014-1623-2
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发表时间：
2014-11-01
影响因子：
8
通讯作者：
Carpaneto, Armando
中科院分区：
文献类型：
--
作者：
Boccaccio, Anna;Scholz-Starke, Joachim;Carpaneto, Armando
双孔通道蛋白（TPC）编码动物和植物细胞内的离子通道。在哺乳动物细胞中，两种同种型（TPC 1和TPC 2）定位于内溶酶体区室，而植物TPC 1蛋白靶向于大的裂解空泡周围的膜。虽然它是公认的，植物TPC 1通道激活在体外的电压和钙依赖性的方式，仍然存在争议，其在生理条件下的激活。同样，动物TPC激活的模式在两个阵营之间存在很大争议，这两个阵营支持烟酸腺嘌呤二核苷酸磷酸（NAADP）或磷酸肌醇PI（3，5）P-2作为激活剂。在这里，我们研究TPC电流响应这些第二信使的全液泡膜片钳实验上分离的拟南芥液泡。在拟南芥tpc 1基因敲除植株的叶肉原生质体中表达后，我们在中央大液泡的膜上检测到拟南芥TPC 1-EGFP和人TPC 2-EGFP融合蛋白。无论是NAADP或PI（3，5）P-2浴（胞质）应用不影响AtTPC 1-EGFP的电压和钙依赖性的特性。PI（3，5）P-2在含hTPC 2-EGFP的空泡中引起大的钠电流，而NAADP没有这种作用。当PI（3，5）P-2应用于在面包酵母巨空泡中表达的hTPC 2时，获得了类似的结果。我们的研究结果强调了动物和植物TPC蛋白之间的电流激活和离子选择性模式的根本差异，并证实了PI（3，5）P-2介导的激活和哺乳动物TPC 2的Na+选择性。
Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P-2. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P-2 did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P-2 elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P-2 was applied to hTPC2 expressed in baker's yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P-2-mediated activation and Na+ selectivity of mammalian TPC2.