DETERMINATION OF TRANSMEMBRANE TOPOLOGY OF AN INWARD-RECTIFYING POTASSIUM CHANNEL FROM ARABIDOPSIS-THALIANA BASED ON FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI

We report here that the inward-rectifying potassium channels KAT1 and AKT2 were functionally expressed in K+ uptake-deficient Escherichia co li. Immunological assays showed that KAT1 was translocated into the ce ll membrane of E. coli. Functional assays suggested that KAT1 was inse rted topologically correctly into the cell membrane. In control experi ments, 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-spa nning segments (S1-S6), The finding that a K+ channel can be expressed in bacteria was further exploited to determine the KAT1 membrane topo logy by a gene fusion approach using the bacterial reporter enzymes, a lkaline phosphatase, which is active only in the periplasm, and beta-g alactosidase. The enzyme activity from the alkaline phosphatase and be ta-galactosidase fusion plasmid showed that the widely predicted S1, S 2, S5, and S6 segments were inserted into the membrane. Although the S 3 segment in the alkaline phosphatase fusion protein could not functio n as an export signal, the replacement of a negatively charged residue inside S3 with a neutral amino acid resulted in an increase in alkali ne 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 requ ired presumably because of pairing with a positively charged residue o f S4, These results revealed that KAT1 has the common six transmembran e-spanning membrane topology that has been predicted for the Shaker su perfamily of voltage-dependent K+ channels. Furthermore, the functiona l complementation of a bacterial K+ uptake mutant in this study is sho wn to be an alternative expression system for plant K+ channel protein s and a potent tool for their topological analysis.