Structure and Function of Mechanosensitive Channels in Inner Ear Hair Cells - Project Summary The goal of this project is to investigate the structure and function of a poorly understood family of ion channels known as TMEM63s. There are three members of the family in animals, known as TMEM63A, B and C. TMEM63s form mechanosensitive ion channels and are thought to be the animal orthologues of the plant OSCA channels. TMEM63A mutations have been identified in young patients with hypo-myelinating leukodystrophies characterized by myelin deficits suggesting a functional role of TMEM63A in myelination and neuronal development. TMEM63B deficiency in mice leads to deafness and it is hypothesized to act as an osmosensor in auditory hair cells. TMEM63B mutations in humans lead to a range of disorders including severe neurodevelopmental disorders, epileptic encephalopathy, hematological abnormalities, and hearing loss. A genomic analysis in hypertensive rats implied a role for TMEM63C in kidney damage. When expressed in heterologous cells, TMEM63A and TMEM63B can be activated by mechanical stimulation, revealing small currents of 10s to 100s of pA. These data, while intriguing, raise fundamental questions regarding the structure and function TMEM63 mechanosensitive ion channels and whether TMEM63 channels share common molecular, structural, and gating mechanisms with other mechanosensitive ion channels. The project is organized around three specific aims. For aim one, we will determine the structures of human p.V44M variant of TMEM63B using single-particle cryo-EM. We hypothesize this variant biases the channel toward the open state. For aim two, we will investigate the function of TMEM63B in proteoliposomes and in heterologous cell lines. We will evoke mechanically activated currents using pressure steps and perfusion of hypoosmotic bath solutions. We will investigate structural and functional aspects of WT TMEM63B and will use site-directed mutagenesis to investigate the function of various structural features, including the putative pore region, the intracellular loop, IL2, and the hypothesized gating helices. We will also investigate the functional consequences of human TMEM63B mutations including the p.V44M variant. For the third aim, we will investigate the physiological contributions of TMEM63B expressed in inner ear hair cells. We hypothesize that intense auditory stimulation promotes massive influx of potassium and calcium in hair cells, raising intracellular osmolarity and causing ionic imbalance. With the inside of the cell being hypertonic relative to external hypotonic bath solution, cell swelling may ensue which may, in turn, activate TMEM63B channels. Calcium influx via TMEM63B is postulated to activate neighboring calcium-activated potassium channels promoting potassium efflux and ionic equilibration. Disruption of this pathway is thought to lead to hair cell death and hearing loss. The experiments proposed herein will shed light on the role of TMEM63B in hair cells and hearing and will illuminate structural and functional features of this newly discovered, poorly understood family of mechanosensitive ion channels.
