Use of small fluorescent molecules to monitor channel activity.
The Mechanosensitive channel of Large conductance (MscL) allows bacteria to rapidly adapt to changing environmental conditions such as osmolarity. The MscL channel opens in response to increases in membrane tension, which allows for the efflux of cytoplasmic constituents. Here we describe the cloning and expression of Salmonella typhimurium MscL (St-MscL). The amino acid sequence encoding for this MscL exhibits a high degree of similarity to Escherichia coli MscL (Eco-MscL). Using a fluorescence efflux assay, we demonstrate that efflux through the MscL channel during hypoosmotic shock can be monitored using endogenously produced fluorophores. These fluorophores are synthesized by a cotransformed gene, cobA. In addition, we observe that thermal stimulation, i.e., heat shock, can induce efflux through MscL.
Jones
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SE
Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright Patterson AFB, Ohio 45433, USA.
Naik
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RR
Stone
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MO
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D005453:Fluorescence; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D012486:Salmonella typhimurium / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli