Developing an in vivo assay for bacterial cyclic nucleotide gated channels

Jonathan E. King, Joshua A. Maurer, Chemistry Department, Washington University, St. Louis, MO

Our group has recently cloned a bacterial cyclic nucleotide gated ion channel from Chromobacterium violaceum (Cv-bCNG).  By transfection of a vector encoding the gene from this protein, we were able to overexpress Cv-bCNG in E. coli.  Once the protein was overexpressed in the cell membranes of the E. coli, we sought to develop an assay for studying in vivo channel function.  Thus far, our data suggests that the level of cAMP within glucose-limited E. coli cultures is sufficient to trigger channel opening.  However, it seems that when bacteria are grown in media with increased concentrations of glucose, cAMP levels are below the critical concentration for channel opening.  In the future, we plan to use protein engineer to design channels that open in response to other molecules, such as TNT.  This assay is appropriate for and could be used as a selection tool to identify these biosensors.  Additionally, we would like to understand the protein movements required to transition the channel between the open and closed states; this assay could be used to determine how specific mutations of Cv-bCNG affect the functional components of the binding domain and how they interact in the opening and closing processes