Uozumi N, Nakamura T, Schroeder J I, Muto S
Bioscience Center, Nagoya University, Nagoya, 464-8601, Japan.
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9773-8. doi: 10.1073/pnas.95.17.9773.
We report here that the inward-rectifying potassium channels KAT1 and AKT2 were functionally expressed in K+ uptake-deficient Escherichia coli. Immunological assays showed that KAT1 was translocated into the cell membrane of E. coli. Functional assays suggested that KAT1 was inserted topologically correctly into the cell membrane. In control experiments, the inactive point mutation in KAT1, T256R, did not complement for K+ uptake in E. coli. The inward-rectifying K+ channels of plants share a common hydrophobic domain comprising at least six membrane-spanning segments (S1-S6). The finding that a K+ channel can be expressed in bacteria was further exploited to determine the KAT1 membrane topology by a gene fusion approach using the bacterial reporter enzymes, alkaline phosphatase, which is active only in the periplasm, and beta-galactosidase. The enzyme activity from the alkaline phosphatase and beta-galactosidase fusion plasmid showed that the widely predicted S1, S2, S5, and S6 segments were inserted into the membrane. Although the S3 segment in the alkaline phosphatase fusion protein could not function as an export signal, the replacement of a negatively charged residue inside S3 with a neutral amino acid resulted in an increase in alkaline phosphatase activity, which indicates that the alkaline phosphatase was translocated into the periplasm. For membrane translocation of S3, the neutralization of a negatively charged residue in S3 may be required presumably because of pairing with a positively charged residue of S4. These results revealed that KAT1 has the common six transmembrane-spanning membrane topology that has been predicted for the Shaker superfamily of voltage-dependent K+ channels. Furthermore, the functional complementation of a bacterial K+ uptake mutant in this study is shown to be an alternative expression system for plant K+ channel proteins and a potent tool for their topological analysis.
我们在此报告，内向整流钾通道KAT1和AKT2在钾摄取缺陷型大肠杆菌中实现了功能表达。免疫分析表明，KAT1转位至大肠杆菌细胞膜。功能分析表明，KAT1在拓扑结构上正确插入细胞膜。在对照实验中，KAT1中的无活性点突变T256R不能补充大肠杆菌中的钾摄取。植物的内向整流钾通道具有一个共同的疏水结构域，该结构域包含至少六个跨膜片段（S1 - S6）。钾通道能够在细菌中表达这一发现，被进一步用于通过基因融合方法确定KAT1的膜拓扑结构，该方法使用仅在周质中具有活性的细菌报告酶碱性磷酸酶和β - 半乳糖苷酶。碱性磷酸酶和β - 半乳糖苷酶融合质粒的酶活性表明，广泛预测的S1、S2、S5和S6片段插入了膜中。尽管碱性磷酸酶融合蛋白中的S3片段不能作为输出信号，但用中性氨基酸取代S3内部的带负电荷残基会导致碱性磷酸酶活性增加，这表明碱性磷酸酶转位至周质。对于S3的膜转位，可能需要中和S3中的带负电荷残基，大概是因为它与S4的带正电荷残基配对。这些结果表明，KAT1具有电压依赖性钾通道的Shaker超家族所预测的常见六跨膜膜拓扑结构。此外，本研究中细菌钾摄取突变体的功能互补被证明是植物钾通道蛋白的另一种表达系统，也是对其进行拓扑分析的有力工具。
