PiN Faculty Member - David Corey, PhD

David Corey, PhD

Bertarelli Professor of Translational Medical Science
Investigator, Howard Hughes Medical Institute

Harvard Medical School
Department of Neurobiology
Goldenson Building, Room 443
220 Longwood Avenue
Boston, MA 2115
Tel: 617-432-2506
Fax: 617-432-2508
Visit my lab page here.

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 primarily 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, mostly by positional cloning of genes mutated in hereditary deafness. These include the tip link proteins (CDH23 and PCDH15), three small accessory proteins (TMIE, LHFPL5 and CIB2), and two membrane proteins thought to form the transduction channels through which ions flow (TMC1 and TMC2). But we have no idea how the proteins are arranged in a complex, how they bind to each other, where the ion conduction pathway is within the channels, 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 to understand the detailed mechanism of a timepiece We are doing this with a combination of electrophysiology, electron microscopy, cell biology, protein chemistry, structural biology, and single-molecule biophysics:

Candidate components of the transduction complex are localized with immunogold electron microscopy, using transmission EM, scanning EM with backscatter detection, or—for 3D reconstructions—focused ion beam scanning electron microscopy.

For the tip-link proteins, we have determined the X-ray crystal structure of the N-termini of PCDH15 bound to CDH23, and have 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 are now using single-molecule force spectroscopy to pull directly on single pairs of PCDH15 and CDH23 N-termini, to measure the force needed for unbinding. This has implications for understanding noise-induced hearing loss. For the ion channel, we use cryo-electron microscopy to determine the atomic structures of TMC proteins. We study binding between proteins with microscale thermophoresis, multi-angle light scattering, and biolayer interferometry.

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 in any case—so we express mutated proteins in the inner ears of mice lacking wild-type genes. We have developed a variety of methods to express exogenous proteins in hair cells, ranging from electroporation in vitro to viral delivery in vivo. The function of modified transduction complexes is tested with dye-accumulation assays and 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. We are working to bring these vectors and others to the clinic for treating hereditary deafness.

Last Update: 6/21/2017


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