Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts
Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts
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DOI：
10.1074/jbc.m708213200
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发表时间：
2008-01-25
影响因子：
4.8
通讯作者：
Uozumi, Nobuyuki
中科院分区：
文献类型：
--
作者：
Hamamoto, Shin;Marui, Junichiro;Uozumi, Nobuyuki
液泡膜 K+ 膜转运系统在维持植物细胞 K+ 稳态方面发挥着至关重要的作用。在这里，我们从烟草品种中分离出编码双孔 K+ 通道 (NtTPK1) 的 cDNA。 SR1 和培养的 BY-2 烟草细胞。 NtTPK1 的四个变体中的两个包含 VHG 和 GHG，而不是第二个孔区域中的 GYG 特征序列。当在大肠杆菌细胞膜中表达时，所有四种产物均具有功能，并且 NtTPK1 靶向烟草细胞中的液泡膜。从烟草品种中分离出的三个启动子序列中的两个。 SR1 是活跃的，盐胁迫或高渗透压休克使这些表达增加了近 2 倍。为了确定 NtTPK1 的特性，我们扩大了具有失活的内源性液泡膜通道的突变酵母细胞，并制备了适合膜片钳记录的液泡膜，从而可以将 NtTPK1 相关通道电导与小的内源电流区分开来。 NtTPK1 对 K+ 表现出比 Na+ 更强的选择性。 NtTPK1 活性对亚精胺和精胺敏感，这两种物质存在于烟草细胞中。 NtTPK1 在没有 Ca2+ 的情况下具有活性，但 45 μM Ca2+ 的胞质浓度导致 K+ 电流幅度增加 2 倍。细胞质 toppH5.5 的酸化也显着增加了 NtTPK1 介导的 K+ 电流。这些结果表明，NtTPK1 是一种新型液泡膜 K+ 通道，与之前表征的液泡通道 SV、FV 和 VK 属于不同的组。
The tonoplast K+ membrane transport system plays a crucial role in maintaining K+ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K+ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased similar to 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K+ over Na+. NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca2+, but a cytosolic concentration of 45 mu M Ca2+ resulted in a 2-fold increase in the amplitude of the K+ current. Acidification of the cytosol topH5.5 also markedly increased NtTPK1-mediated K+ currents. These results show that NtTPK1 is a novel tonoplast K+ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.