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.

Structures of the MthK RCK Domain and the Effect of Ca2+ on Gating Ring Stability

“…This co-expression phenomenon was also observed in several other prokaryotic RCK-regulated K ϩ channels such as the E. coli K ϩ channel (21), MjK1, and MjK2 from M. jannaschii (22,23). K ϩ uptake complementation assays of MjK2 (23) and gain-of-function assays of the E. coli K ϩ channel (21) show that these two channels can still conduct K ϩ after the expression of the extra RCK domain is abolished by mutating the internal start methionines. However, several observations lead us to believe that the co-expression of the extra RCK domain together with the channel is a functionally relevant phenomenon that plays an important role in the regulation of channel gating.…”

Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity

“…Despite the elegant simplicity of the model, there are experimental data suggesting that the gating machinery may not require free RCK domains (15,16). Moreover, the isolated KTN domain forms a variety of assembly modes of the canonical dimers, including tetramer in solution (14) and also octamers in the crystals (17), indicating that the flexibility of the RCK/KTN domain assembly can result in different modes of oligomers.…”

Patch Clamp and Phenotypic Analyses of a Prokaryotic Cyclic Nucleotide-gated K+ Channel Using Escherichia coli as a Host

“…2, MmaK renders E. coli sensitive to externally added K ϩ , but not Na ϩ . This K ϩ -sensitive phenotype recapitulates the gain-of-function phenotype discovered after mutagenizing Kch, the gene that encodes the K ϩ channel native to E. coli (18). Externally enriched K ϩ apparently causes membrane depolarization through the gain-of-function Kch channels, resulting in the K ϩ -specific growth phenotype (31).…”

“…3B, panel 2). This K ϩ -sensitive phenotype is very similar to that caused by the gain-of-function mutants of the native Kch channel of E. coli (18), indicating that the E. coli cells are able to express MmaK and to form a functional K ϩ conduit in its membrane. Note that the K ϩ -sensitive phenotype observed in both cases was carried out in the absence of the corresponding inducers, reiterating that the leakage levels of the inducible promoters are enough to produce sufficient number of channels to give the observable phenotypes.…”

“…For plasmid retention, 100 g/ml ampicillin was added in all subsequent culture media. The phenotype assay was carried out following the protocol as previously described (18). Tryptone medium contain 10 g/liter of tryptone (211705; Bacto Tryptone), which has ϳ0.5 mM contaminating K ϩ (measured with a flame photometer).…”