Research Interests:

Work in my laboratory is focused on understanding the gating and regulation of mechanically sensitive ion channels. Much of this work involves the hair cells of the inner ear, which convert the mechanical stimulus of a sound wave into an electrical signal that is sent to the brain. The mechanosensitive organelle of the hair cell is a bundle of stereocilia that protrudes from the top surface of the cell. Stereocilia are connected at their tips by fine filaments called tip links, which are stretched each time the bundle is deflected in one direction by a sound vibration. Tip links are thought to pull directly on ion channels in the tips of the stereocilia, which open in response to the tension, allowing electric current in the form of potassium ions to flow into the hair cell to change its internal voltage.

The hair cell can adapt to static deflections of its hair bundle, and acts within milliseconds to bring the fraction of open channels back to a resting value of ~15 percent. Two mechanisms have been proposed to mediate adaptation: an adjustment of tip link tension caused by movement of a tiny molecular motor complex inside the tips of a stereocilium, and a direct modulation of the channel by calcium ions that enter through the channel and then bind to a site on or near it.

Mechanics of Adaptation
We wish to determine whether either or both of the proposed adaptation mechanisms occur in hair cells. The two models make similar predictions about the electrophysiology of adaptation but are quite different in the expected mechanical correlates. In brief, the motor model predicts a mechanical relaxation as channels close, and the calcium-binding model predicts a tightening. A gradient-force optical trap ("laser tweezers") is used to apply minute forces to hair bundles within microseconds and simultaneously measure the resulting movement. These measurements, in combination with manipulations that alter adaptation, will help to determine which mechanisms are active and to measure the corresponding forces and movements.

Molecules of Adaptation
The stereocilia have dense cores of the filamentous protein actin, suggesting that a motor complex would include some type of myosin, a protein that moves on actin. We and others cloned a myosin type 1c (also known as myosin Ib) from hair cells and have asked whether it is properly located near the tip links. We made antibodies to the unique tail portion of myosin-1c and tagged them with gold beads to make them visible in the electron microscope. Beads marking myosin-1c were found at highest density within a few hundred nanometers of the ends of tip links, where we would expect the myosin to be if it is part of the motor complex.

As a more definitive test of myosin-1c's involvement, we have sought a way to use myosin-1c specifically to interfere with adaptation. In collaboration with Peter Gillespie (Oregon Health Sciences University) and John Mercer (McLaughlin Institute), we have studied a transgenic mouse whose myosin-1c has been engineered to be inhibitable by a bulky analog of ADP. These mice develop normally and have normal transduction and adaptation. The analog can be delivered to the cytoplasm of the stereocilia by putting it in the recording pipette. It blocks adaptation within a few minutes in hair cells from transgenics but does not affect adaptation in hair cells from wild-type littermates. This is the first positive identification of a protein of the transduction apparatus.

For further tests of myosin-1c and other proteins in hair cell function, we sought a simple method to express exogenous proteins in hair cells, and explored a variety of viruses as vectors for gene delivery. Only one virus—an adenovirus—worked, but this efficiently and specifically infected hair cells that were removed from a mouse and maintained in culture. We are testing the functions of ion channels normally made by hair cells, by using adenovirus to deliver dominant-negative mutants of these channels.

A Fluorescent Marker for Functional Hair Cells
FM1-43 is a small organic dye that becomes fluorescent when it partitions into membranes. Because it cannot cross membranes, FM1-43 has been used as a marker of synaptic vesicles, which trap dye when vesicle membrane is brought into the cell and release dye when vesicles are released. A very few types of cells, however, take in dye and never release it. These include hair cells, both in the inner ear and in the lateral line organs found in aquatic vertebrates. It has been suggested that transduction channels of hair cells have a pore large enough to allow dye to pass through; once in the cytoplasm, this dye would be trapped by its charge. We tested this idea and found that closing transduction channels with mechanical stimuli or drugs, or by breaking the transduction apparatus, blocked dye entry. The dye thus provides a simple and fast assay for functional hair cells, which could be used in genetic screens. Some other ion channels, including the heat-activated TRPV1 and ATP-activated P2X2 channels of other sensory cells, also allow dye entry. Injected subcutaneously in mice, FM1-43 circulates systemically until becoming trapped in some sensory cells that apparently have such channels. Hair cells, sensory cells of the trigeminal and dorsal root ganglia, and certain taste receptor cells can be fluorescently labeled and retain dye for weeks.

Genomics of Hearing and Deafness
A hair cell probably expresses between 10 and 15 thousand genes, a significant fraction of the entire genome. Many of these are "housekeeping" genes, necessary for such basic functions as protein synthesis, energy production, and cytoplasmic transport. Hundreds or thousands of other genes play roles in the unique mechanosensory function of the hair cell. To understand hair cell function fully, we must start by identifying these genes and the timing and determinants of their expression. Conventional molecular biology methods that focus on one gene at a time are too slow for a task of this size, so my lab is mapping gene expression in hair cells with oligonucleotide arrays.

We first looked at changes in gene expression during development. Although the hair cell sensory epithelium is a complex structure with half a dozen cell types, we can identify the hair cell genes by chemically dissecting away the nerve layer to assay just hair cells and supporting cells, and then killing hair cells with aminoglycoside antibiotics. To group genes into functional pathways, we use a self-organizing-map algorithm to cluster genes with similar temporal patterns of expression. For instance, this analysis clustered a large number of genes that are consistently down-regulated from E14 through P12. Dozens of these are related to the cell cycle and include genes involved in replication of DNA, modulation of chromatin formation, and control of the cell cycle. Genes in this group may be important in understanding what blocks division of mature hair cells and may offer tools for promoting the regeneration of hair cells to restore hearing.

We then looked for genes that function in sensory transduction, by looking for targets of a transcription factor needed for development of the sensory hair bundle. These studies are still under way, but we have identified genes that encode additional structural proteins or that regulate their expression. This approach may reveal some proteins of the transduction complex.

Selected Publications:

Duggan, A., García-Añoveros, J. and Corey D.P. (2001) The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of DRG neurons and dendrites of central neurons. J. Biol. Chem. 277: 5203

Holt, J. R., Gillespie, S. K. H., Provance, D. W., Shah, K., Shokat, K. M., Corey, D. P., Mercer, J. A. and Gillespie, P. G. (2002) A chemical-genetic strategy demonstrates myosin 1c mediates sensory adaptation in hair cells. Cell 108:371-381

Rehm, H. L., Zhang, D-S., Brown, M. C., Burgess, B., Halpin, C., Berger, W., Morton, C. C., Corey, D.P. and Chen, Z-Y. (2002) Vascular system defects and sensorineural deafness in a mouse model of Norrie disease. J. Neurosci. 22:4286-4292