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

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 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.

Figure 1

Effect of K⁺ concentrations on the growth of the K⁺ uptake-deficient E. coli mutant strain LB2003 with pPAB-KAT1 (•), pBS-AKT2 (○), pPAB-D141V-KAT1 (▴), pPAB-T256R-KAT1 (▾), and vector plasmids, pPAB404 (■) and pBluescript KSII (□) in liquid culture. The initial K⁺ concentrations were measured by flame photometry. •, 8.5 mM; ○, 8.5 mM; 8.8 mM; ▴, 7.6 mM; ▾, 10.4 mM; □, 10.4 mM

Figure 2

Hydropathy plot and deduced transmembrane domains of KAT1 (A) and deduced amino acid sequence (B). Hydrophobicity plot was generated by the method of Hopp and Woods with a window of 13 amino acids (6, 52). Negative values show hydrophobic regions. The positions of fusion sites selected for PhoA fusion are indicated by the bars (A) and arrowheads (B). ■ Shows the amino acids that were replaced with valines (Materials and Methods). • Represents the positively charged amino acids in the putative S4 segment. The putative transmembrane segments (S1, S2, S3, S4, S5, H5, S6, and S7) are indicated by horizontal bars. The horizontal dotted line represents the putative nucleotide-binding domain (NBD).

Figure 3

Immunoblot analysis for KAT1-PhoA fusion proteins expressed in E. coli UT5600 (A) and KAT1-LacZ fusion proteins expressed in E. coli MC4100 (B). Transformed E. coli were grown on solid medium for 1 day, and membranes were prepared as described in the text. Membrane proteins (10 μg) were subjected to SDS/10% (A) or SDS/8% (B) polyacrylamide gel electrophoresis followed by Western blotting.

Figure 4

Proposed membrane topology of KAT1 as derived from PhoA and LacZ fusions. Transmembrane segments (S1–S6) are shown as open rectangles. The position of charged amino acids in loops are indicated by + (Arg and Lys) and − (Asp and Glu). The phoA and lacZ gene fusion positions are indicated by arrows.