Title: The role of TRP channels in mechanosensation
Description: Members of the TRP ion channel superfamily have been implicated in a wide variety of mechanically-sensitive processes. We have developed a set of criteria to help determine not only if a candidate channel protein mediates a mechanosensory process, but also to determine if the candidate is directly gated by mechanical force. These became the basis for three related projects: For the ion channel TRPA1, which is thought to play a role in noxious touch, I created a set of pore mutants to better understand ion permeation through the channel and also for future use as a tool to understand its physiological role. I determined that the pore of TRPA1, at 8.3 Å diameter, is large relative to other cation channels, and that residue D915 is the constriction site of the pore. Thus D915 plays an important role in ion selectivity and in allowing ions to pass through the TRPA1 channel. A second site, E920, was found to be an important surface charge residue that collects ions into the mouth of the pore to increase channel currents. A second project was an attempt to identify a candidate for the hair cell transduction channel, using a functional screen in hair cells of the inner ear for all TRP ion channel family members. Although the screen did not give unambiguous results, a functional screen remains the best chance of determining the identity of the hair cell transduction channel. A third project, on mechanosensation mediated by the primary cilium of epithelial kidney cells, involved attempting to record currents from the putative mechanosensory channel in these cells. These attempts proved fruitless; hypotheses to explain why are discussed. Finally, the appendices describe previously published work into the physiological role of TRPA1 as investigated by using a knockout mouse model, as well as a therapeutic technique to treat brain tumors using a mutated channel.