Molecular Mechanisms of Mechanosensitive Channel Gating

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The study of the bacterial mechanosensitive MscL channel has biomedical significance for several reasons. First, the channel serves a vital function in maintaining osmotic homeostasis of microbes; when the channel misfunctions it can lead to the death of the microbial cell. Hence, it appears to be a viable pharmacological target. Second, as nanotechnology progresses, the potential for biological sensors, especially MscL, to be used in biomedical nanomachines or drug delivery devices is being realized. Third, MscL has, and will continue to serve as a molecular paradigm for the investigation of mechanosensory transduction. With a crystal structure of what appears to be a 'nearly-closed' state of MscL, the channel has advanced the field considerably by opening the avenues of structural, genetic and molecular analyses coupled with electrophysiology and flux assays, all allied to a well-defined physiological role. MscL continues to serve as a tractable model for determining the molecular mechanisms of channel gating as well as general principles for how a protein detects and responds to membrane tension. To truly exploit this system, however, a better understanding of the molecular mechanisms of how MscL senses and responds to membrane tension must be obtained; this is the objective of this proposal. While models for structural transitions during gating have been proposed, they are not consistent and even many of the fundamental features are not yet resolved. The experiments within this proposal are designed to ally the solved structure with molecular, biochemical, genetic and electrophysiological analyses to determine the functional role that regions of the protein play in sensing and responding to membrane stretch and to define transitions that occur upon gating. The approaches used include: the generation of chimeras to determine the structural elements associated with functional differences of homologues, reconstitution of orthologues into native and defined membranes, utilizing the "Substituted Cysteine Accessibility Method" (SCAM) to determine at what point in the gating process pore residues are exposed to the aqueous environment, disulfide trapping to define transition and open states of the channel, and a genetic approach to test if residues approach each other upon gating transition. PUBLIC HEALTH: Studying how a bacterial sensor detects forces will allow insight into the mechanisms of how human mechano-sensors, e.g. those used in blood pressure and kidney regulation, may function; thus, we may eventually speculate how such sensors can be modulated by drugs. This work could also have implications in anti-bacterial drug design and the utilization of biological sensors for future technological feats, such as nanodevices for drug delivery.