Activation and regulation of mechanosensitive ion channels

Much of our understanding of mechanotransduction and adaptation is based on two model systems. Genetic information from the nematode Caenorhabditis elegans implicates numerous proteins involved in touch sensation but provides little functional data. In contrast, experiments on saccular hair cells of the bullfrog Rana catesbeiana describe mechanotransduction and adaptation involved in hearing and balance but provide no identified proteins. Both models propose a mechanically gated ion channel attached to intracellular and extracellular structures. This thesis tests key predictions of both models.; In nematodes, MEC-4 and MEC-10 encode proteins required for touch sensitivity. Based on primary sequence, these and other degenerins were predicted to form mechanosensitive channels that open to detect membrane stretch or touch. Similarly, the degenerin UNC-105 is thought to form a mechanosensitive channel in muscle cells. In this thesis, I studied wild type and mutant UNC-105 by heterologous expression. Mutant UNC-105 alone produces constitutively active channels blocked by amiloride, explaining the hypercontraction phenotype seen in the mutant nematode muscle. Wild type UNC-105 produces no detectable currents, suggesting that additional proteins are required for mechanical gating of UNC-105. Thus, UNC-105 is shown to be a channel, and by extension, MEC-4 and Mec-10 are as well.; In hair cells, a molecular motor regulates tension and adaptation on transduction channels. Earlier cloning efforts produced a motor candidate, myosin-Iβ. In this thesis, confocal and electron microscopy were used to identify myosin-Iβ at the tips of hair cell stereocilia. A quantitative immuno-gold approach found enrichment of myosin-Iβ at and near the upper insertions of tip links, the proposed sites of adaptation. This labeling was absent in control bundles. Thus, myosin-Iβ may function as a linker between the transduction channel and cytoskeleton and as the adaptation motor.; Finally, I used myosin-Iβ as a yeast two-hybrid bait protein to search for interacting proteins involved in mechanotransduction and adaptation. While initial studies were unsuccessful, I found that three calmodulin-binding IQ motifs upstream of the myosin-Iβ tail region enable soluble protein expression and the nuclear localization necessary for yeast two-hybrid screens. Thus, the yeast two-hybrid system is now a promising method of identifying proteins that bind myosin-Iβ.