Date of Award
Doctor of Philosophy (PhD)
Mechanosensation is arguably the least understood of all senses. For most physiological processes, the first response to membrane stress is thought to be the opening or closing of mechanosensitive channels1, but the clonal nature of the first mechanotransducers is still largely unknown. The objective of my research was to identify molecules involved in mechanosensory transduction by both studying known channels as well as performing screens to identify previously uncharacterized channels. Shortly before my research began, Daniel Schmidt from the MacKinnon Lab showed that certain voltage gated potassium channels, not previously associated with mechanosensation, are in fact remarkably sensitive to membrane tension in isolated membrane patches2. I therefore began investigating the possibility of voltage gated potassium channels being mechanosensitive in physiological contexts. Results using hypo-osmotic swelling provided additional support that Paddle Chimaera and Kv2.1 are indeed mechanosensitive in cellular contexts3. Given the close structural similarity between voltage gated potassium channels and other ion channel families, I extended these studies to include sodium and calcium selective voltage gated ion channels using patch inflation, swelling, poking, and stretching. However the sodium selective voltage gated channel, Nav1.7, and calcium selective voltage gated channels, Cav1.2 and Cav1.3, were not found to display major mechanosensitive properties. In a complimentary approach, I performed a screen of 10 different cell lines using the poking assay to identify novel molecules involved in mechanical transduction. My results identified multiple undescribed slow-inactivating mechanosensitive currents in cell lines from a variety of sources including numerous cancer cell lines, human stem cells and mouse stem cells. Further work using transcriptome analysis, bioinformatic techniques, and electrophysiological recordings identified that the pore forming subunit responsible for the slowinactivating mechanosensitive conductances in mouse embryonic stem cells is Piezo1, a mechanosensitive channel canonically known for displaying fastinactivating kinetics. With very few modulators known to date45, the mechanism by which Piezo1 could produce slow inactivating currents was not known. To address possible novel sources of modulation of Piezo1 currents, I performed transcriptome analyses that identified 2 potential candidates including one novel protein subsequently confirmed to modify the behavior of Piezo1 in vitro. This protein, Plp2, is a small transmembrane protein of undescribed function that amplifies the magnitude and slows down the kinetics of Piezo1 in heterologous expression. The other protein, Cd63, is also a transmembrane protein that only amplifies the magnitude of Piezo1 currents, with no modification of its kinetics, in heterologous expression. Given the remarkably large set of functions that have been attributed to Piezo channels6,7,8,9 in the very few years since its discovery, and how little we still know of its functional mechanisms, the identification of novel modulators provides a crucial next step in elucidating the molecular basis of mechanosensation.
del Marmol, Josefina Ines, "Molecular Basis of Mechanosensitivity" (2016). Student Theses and Dissertations. 418.