Gain-of-function mutations indicate that Escherichia coli Kch forms a functional K+ conduit in vivo

Abstract

Although Kch of Escherichia coli is thought to be a K(+) channel by sequence homology, there is little evidence that it actually conducts K(+) ions in vitro or in vivo. We isolated gain-of-function (GOF) Kch mutations that render bacteria specifically sensitive to K(+) ions. Millimolar added K(+), but not Na(+) or sorbitol, blocks the initiation or continuation of mutant growth in liquid media. The mutations are mapped at the RCK (or KTN) domain, which is considered to be the cytoplasmic sensor controlling the gate. Additional mutations directed to the K(+)-filter sequence rescue the GOF mutant. The apparent K(+)-specific conduction through the 'loose-cannon' mutant channel suggests that the wild-type Kch channel also conducts, albeit in a regulated manner. Changing the internal ATG does not erase the GOF toxicity, but removes kch's short second product, suggesting that it is not required for channel function in vivo. The mutant phenotypes are better explained by a perturbation of membrane potential instead of internal K(+) concentration. Possible implications on the normal function of Kch are discussed.

Fig. 1. The structure of Kch, a subunit of the putative K⁺ channel in E.coli. (A) Membrane topology of a Kch subunit is composed of a channel body (S1–S6, cylinder) with a pore region between S5 and S6, and a RCK domain (M240–N393) (Jiang et al., 2001) after S6. M240 also initiates the production of RCK protein (see Figure 6A). (B) Sequence alignment of the pore region from selected K⁺ channels with the conserved K⁺ signature sequence (GYG) highlighted in gray. Kch, E. coli (NCBI protein database code GI: 11354242); Shaker, Drosophila melanogaster (GI: 13432103); KcsA, Streptomyces lividans (GI: 8134524); MthK, Methanothermobacter thermautotrophicus (GI: 21542150); Kat1, Arabidopsis thaliana (GI: 421842); eag, Drosophila melanogaster (GI: 399253); Herg, Homo sapiens (GI: 7531135); Irk1, Mus musculus (GI: 6680530); mSlo1, Mus musculus (GI: 6754436).

Fig. 2. Isolation of the GOF Kch mutants. (A) The experimental scheme. Kch coding region together with pBluescript vector were mutagenized in vivo using XL1-Red Cells. The mutated kchs were further cloned in pB11d behind IPTG-inducible LacUV5 promoter and used to transform kch-null FRAG1 E.coli. Transformed colonies whose replicas failed to grow on any of the restrictive plates were picked, DNA sequenced and phenotyped. (B) Growth phenotype on the permissive (panel 1) and various restrictive plates (panels 2–8) with (1–4) or without (5–8) IPTG. Each row shows the growth pattern of five 5-µl drops of inoculate from cultures of stationary cells (OD₆₀₀ ∼ 3.5–4.5) diluted 10²-, 10³-, 10⁴-, 10⁵- and 10⁶-fold.

Fig. 3. Expression of Kch from the native promoter. (A) A 2 kb E.coli genomic fragment containing the C-terminus of the neighboring gene yciI, kch’s native promoter and kch coding region was cloned in a multi-copy vector, pGEM, between BamHI and PstI. (B) Plate phenotype of the GOF Kch mutants expressed from native promoter on the LB-based plates. The bacteria with the naturally promoted C312F, N325S or S331P Kch mutants were sensitive to only K⁺ and not to Na⁺ or sorbitol. (C) On tryptone–agarose plates, the colony-forming units of these mutants decrease dramatically with the concentration of additional K⁺ between 1 and 10 mM.

Fig. 4. Growth in tryptone-based liquid media. Growth curves of cultures in various test media (see Materials and methods) inoculated with stationary-phase bacteria bearing pGEM-empty (left panels), pGEM-WT (middle) or pGEM-S331P (right). Mean ± SD given (n ≥ 3, SD smaller than symbol when not visible). (A) Growth of cells in the T medium (open squares, broken line), TK5 (open triangles), TNa5 (open circles) and TS10 (open diamonds). (B) Growth of cells in T medium (pH 7.3, open squares, broken line), TH5 (pH 5.8, gray squares, only pGEM-S331P was measured), TK5 (pH 7.3, open triangles) and TK5H5 (pH 5.8, gray triangles). The pH variations of the cultures remained within ±0.1 U in the first 4 h. (C) Growth of cells before and after the addition of 10 mM KCl (black triangles), NaCl (gray circles) or none (open squares, broken line), at the logarithmic-phase of growth (arrows).

Fig. 5. Effect of representative filter mutations on the S331P GOF mutant. (A) Additional G187Q (S331P-G187Q) mutation on the K⁺ filter rescues the S331P mutant on K200 plates (bottom row). The Y188F (S331P-Y188F) mutation is the only K⁺ filter mutation we could isolate that did not rescue the S331P mutant on K200 plates (row 3). (B) Western blot analysis of the membrane fractions prepared from the cells carrying vectors: pGEM-empty (lane 1), pGEM-WT (lane 2), pGEM-S331P (lane 3) and pGEM-S331-G187Q (lane 4). All strains produce a comparable amount of Kch and RCK proteins in the bacterial membrane. RCK: RCK protein (see Figure 6A). M: MagicMark (Invitrogen, CA).

Fig. 6. Removing the internal start codon eliminates the RCK protein but does not suppress the toxicity of the GOF mutations. (A) Diagram of the relative position of the internal start and the two translation products, Kch and RCK protein. The protein secondary structures are indicated as gray rectangles (α-helix) and arrows (β-sheet). (B) Western blot analysis of the Kch proteins. The samples were prepared by directly boiling the stationary cells in SDS–PAGE sample buffer. As expected, both the larger (Kch) and the smaller (RCK) proteins are found in the wild-type (lane 2) and the GOF mutants (lanes 4, 6 and 8). Genetically engineered removal of the internal translational initiation removes the smaller peptide in both the wild-type (lane 3) and the GOF mutants (lanes 5, 7 and 9). M: MagicMark (Invitrogen, CA). (C) Growth analysis. Removal of the RCK proteins does not alter the growth phenotype of the wild-type (rows 1 and 2). It also does not affect the toxic phenotype of the GOF mutants (rows 3–8).

Fig. 7. Mutation on the K⁺ filter does not rescue bacteria from the toxicity of Kch overproduction. When wild-type Kch is overproduced it is toxic to bacteria. A mutation directed at its K⁺ filter (G189Q) to block the K⁺ flux is unable to erase this toxicity.

Fig. 8. A model of the functional parts of Kch and the locations of the GOF mutations in RCK. (A) Diagram of the wild-type, the GOF mutant and the double mutant Kch channels. Top: the wild-type Kch only opens briefly when ligands (black circles) bind to its cytoplasmic sensor domain (RCK). Middle: the GOF mutations (black triangles) in the RCK domain bias the sensor towards its open configuration even without the ligand. Bottom: a mutation (black diamonds) in the K⁺ filter disrupts the filter and blocks the K⁺ permeation. (B) A map of the GOF mutations in the RCK dimer of Kch (Jiang et al., 2001; PDB code 1ID1). I247, A307 and C312 are toward the core of the Rossmann fold. N325 and S331 are on the hydrophobic interface. I361 and S364 are at the hinge/flexible interface.