Neurobiology

David P. Corey, Ph.D.

Professor of Neurobiology

Investigator, Howard Hughes Medical Institute

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 deflection of the bundles changes the tension in fine filaments ("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 earlier work, we found that cutting these links immediately and irreversibly abolished the mechanical sensitivity, consistent with the model. We also used calcium imaging to locate the transduction channels (which pass calcium as well as other cations) and found that they can be at both ends of the tip links.

Hair cells have a "slow" adaptation mechanism, which acts to maintain a constant tension in each tip link. Manipulations that change the rates of adaptation cause the bundle to move, by up to 100 nanometers, supporting the idea of an active motor mechanism. This is thought to be a complex of 10-30 myosin molecules just under the membrane at each end of each tip link. In order to identify a candidate for the motor myosin, we cloned fragments of most of the myosins expressed in hair cells. We found one of these, myosin-1c, to be located at the tips of stereocilia, specifically at the ends of tip links. Inhibiting the function of myosin-1c with engineered inhibitors blocks adaptation. A second, faster form of adaptation results from Ca2+ entering through transduction channels, binding to an intracellular site on the channel, and closing it. With optical trap measurements of bundle mechanics, we found that a Ca2+-bound channel requires about 2.5 piconewtons more force to open than an unbound channel.

To identify the transduction channel itself, we carried out a screen of the TRP channel superfamily, by finding all TRP channels in the mouse genome and looking for inner ear expression with in situ hybridization. Several TRPs are expressed in the inner ear, and RT-PCR showed that expression of one of them, TRPA1, rises just before hair cells become mechanically sensitive. Antibodies to TRPA1 label the tips of stereocilia. Inhibiting expression of TRPA1—with  morpholino oligonucleotides in zebrafish, or with siRNA-encoding adenovirus in mouse—inhibited hair cell function. We are now focusing on this channel and its linkage to other components of the transduction apparatus.

Three hair cells from the sensory epithelium of a bullfrog saccule. The ciliary bundle of each cell is composed of about 60 stereocilia, which contain the mechanically gated channels at their tips, and one kinocilium (with bulb), which links the bundle to an overlying structural matrix.