David Paul Corey

We are interested in the gating of mechanically sensitive ion channels, which open in response to force on the channel proteins. We study these channels in vertebrate hair cells—the receptor cells of the inner ear, which are sensitive to sounds or accelerations. Hair cells are epithelial cells, with a bundle of stereocilia rising from their apical surfaces. Mechanical deflections of the bundles of just nanometers change the tension in fine "tip links" that stretch between the stereocilia; these filaments are thought to pull directly on the mechanically gated transduction channels to regulate their opening.

In recent years, many protein components of the transduction channel complex have been identified through discovery of deafness genes. These include the tip link proteins (CDH23 and PCDH15), three small accessory proteins (TMIE, LHFPL5 and CIB2), and two membrane proteins that form the transduction channels through which ions flow (TMC1 and TMC2). But we have no firm idea how the proteins are arranged in a complex, how they bind to each other, how force causes a conformational change to open channels, or where Ca²⁺ binds to the proteins to mediate a fast adaption process. Human genetics has handed us, in a sense, a box of watch parts, and it is our job to fit them together.  We are doing this with a combination of electrophysiology, electron microscopy, cell biology, protein chemistry, structural biology, and single-molecule biophysics:

For the tip-link proteins, we determined the X-ray crystal structure of the N-termini of PCDH15 bound to CDH23, and used steered molecular dynamics to determine the elastic properties and unbinding force of the cadherins. The crystal structures and molecular dynamics together have helped explain how deafness-producing mutations in the tip link disrupt its structure (Sotomayor et al., 2010; 2012). We then used single-molecule force spectroscopy to pull directly on single pairs of PCDH15 and CDH23 proteins to measure the force needed for unbinding. These experiments suggest that each tip link has a lifetime of ~10 s, which has implications for understanding noise-induced hearing loss. New experiments are testing this in intact hair cells.

Models developed through these methods are tested with site-directed mutagenesis of the different proteins. The mechanotransduction complex cannot be reconstituted in heterologous cells—there are two many known proteins, there are probably others still unknown, and there is no easy way to pull on them—so we express mutated proteins in the inner ears of mice lacking wild-type genes, usually with AAV vectors.  The function of modified transduction complexes is tested with single-cell electrophysiology.

We have translational interests as well: The viral vectors developed to study protein function are efficient at gene delivery to hair cells, and we have used them to rescue hearing and balance deficits in mice lacking LHFPL5, CLRN1, PCDH15, TMC1 or TMC2.  A special interest is PCDH15, the tip-link protein, in which mutations cause Usher syndrome type 1F, which is characterized by congenital deafness and progressive blindness.  The PCDH15 coding sequence is too large to fit in a single AAV capsid, so we have developed a variety of methods to deliver a functional coding sequence to hair cells and photoreceptors, of mice, zebrafish, human organoids, and nonhuman primates.  We are optimistic about beginning clinical trials in the near future.