Identification of mutations that alter the gating of the Escherichia coli mechanosensitive channel protein, MscK.
Mechanosensitive channels allow bacteria to survive rapid increases in turgor pressure. Substantial questions remain as to how these channels sense and respond to mechanical stress. Here we describe a set of mutants with alterations in their MscK channel protein. The mutants were detected fortuitously by their enhanced ability to modify the accumulation of quinolinic acid. Some amino acid changes lie in the putative pore region of MscK, but others affect sequences that lie amino-terminal to the domain aligning with MscS. We demonstrate that the alterations in MscK cause the channel to open more frequently in the absence of excessive mechanical stress. This is manifested in changes in sensitivity to external K(+) by cells expressing the mutant proteins. Single-channel analysis highlighted a range of gating behaviours: activation at lower pressures than the wild type, inability to achieve the fully open state or a modified requirement for K(+). Thus, the dominant uptake phenotype of these mutants may result from a defect in their ability to regulate the gating of MscK. The locations of the substituted residues suggest that the overall gating mechanism of MscK is comparable to that of MscS, but with subtleties introduced by the additional protein sequences in MscK.
Li
Chan
C
School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, UK.
Edwards
Michelle D
MD
Jeong
Hotcherl
H
Jeong
Hochterl
H
Roth
John
J
Booth
Ian R
IR
D000483:Alleles; D000595:Amino Acid Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000166:cytology / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005816:Genetic Complementation Test; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism; D040542:Mechanotransduction, Cellular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli; D015221:Potassium Channels; C461459:kefA2 protein, E coli