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 all ows for the efflux of cytoplasmic constituents. Here we describe the clonin g and expression of Salmonella typhimurium MscL (St-MscL). The amino acid s equence encoding for this MscL exhibits a high degree of similarity to Esch erichia coli MscL (Eco-MscL). Using a fluorescence efflux assay, we demonst rate that efflux through the MscL channel during hypoosmotic shock can be m onitored using endogenously produced fluorophores. These fluorophores are s ynthesized by a cotransformed gene, cobA. In addition, we observe that ther mal stimulation, i.e., heat shock, can induce efflux through MscL. (C) 2000 Academic Press.