Determination of transmembrane topology of an inward-rectifying potassium channel from Arabidopsis thaliana based on functional expression in Escherichia coli

Nobuyuki Uozumi, Tatsunosuke Nakamura, Julian I. Schroeder, Shoshi Muto

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Abstract

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 β-galactosidase. The enzyme activity from the alkaline phosphatase and β-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.

Original languageEnglish
Pages (from-to)9773-9778
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number17
DOIs
Publication statusPublished - 1998 Aug 18
Externally publishedYes

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