The ability to detect mechanical and thermal stimuli is at the foundation of many life-sustaining systems including renal function as well as cardiovascular and thermal regulation. In addition, mechanosensation is the necessary primary event for the senses of hearing, balance, touch and pain. However, little is known about the molecular mechanisms underlying these senses. The best candidates for sensors at the base of these phenomena in mammalian systems are ion channels that have only recently been discovered. Previously, researchers have demonstrated heterologous functional expression of many eukaryotic transporters and channels in bacteria and yeast. Given these numerous examples, we anticipate that some of the newly discovered candidate mechano- and thermo-sensitive channels may be functionally expressed in microbial systems. Microbial heterologous expression has the unique advantage that the expressed gene can be randomly mutated and rare mutational events rapidly screened or selected. In this way, structural changes can be correlated with functional differences, thus giving insight into the molecular mechanisms of the protein. Here we propose to utilize the power of microbial genetics to study the structure-function relationships of eukaryotic channels which are thought to gate in response to mechanical and/or thermal stimuli. Previously, it was demonstrated that E. coli strains deficient in mechanosensitive channels have an osmotic-dependent cell death phenotype. Candidate mammalian sensors will be heterologously expressed in one of these strains and their ability to suppress this phenotype assayed. Several candidates will be examined and the one(s) that give the most promising results will be aggressively pursued. Mutations that evoke improved suppression, slowed growth or flux-dependent phenotypes will be determined. All resulting mutants will be assayed for osmotic-dependent ion fluxes and channel activity to allow for the correlation of structural with functional changes. Finally, we will pursue the development of a system that will allow for a similar study of homologously and heterologously expressed mechano- and thermo-sensitive eukaryotic channels in yeast (S. pombe).
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