Work in my laboratory is focused on understanding the gating and regulation of mechanically sensitive (force-gated) ion channels. Much of this work involves the hair cells of the inner ear, which convert the mechanical stimulus of a sound wave into electrical signals that are sent to the brain. The mechanosensitive organelle of the hair cell is a bundle of stereocilia that protrude from the top surface of the cell. Stereocilia are connected at their tips by fine filaments called tip links, which are pulled each time the bundle is deflected in one direction by 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 and allow electric current in the form of potassium ions to flow into the hair cell to change its internal voltage.
Mechanics of the Hair Bundle
Each hair cell has a bundle of 30–300 stereocilia that are connected by various links and that tend to move as a cohesive unit. The exact mechanism of their connection has important implications for the biophysical interaction between the transduction channels of a bundle. If the tip links hold a bundle together, then channels are largely in series and the opening of one relaxes the tension on channels in the adjacent shorter stereocilium. If other links hold the bundle together, then the channels are mechanically in parallel, and a given deflection of the bundle stimulates all channels equally and independently.
We have used high-resolution and high-speed optical imaging to observe the movement of stereocilia upon stimulation. At stimulus frequencies of almost 1 kilohertz, all the stereocilia of a bundle move by about the same angular amount and separate by no more than 10 nanometers. Chemically cutting the tip links does not change this motion, suggesting that something other than tip links holds the bundle together. These results suggest a "sliding adhesion" mechanism that allows adjacent stereocilia membranes to slide rapidly past each other by 100 nm or more, without separating by more than a few nanometers. They also indicate that transduction channels are mechanically in parallel.
Structure of the Tip Link
The major proteins of the tip link are two cadherins that are mutated in hereditary deafness. It seems likely that a parallel dimer of protocadherin 15 extending from the tip of one stereocilium joins a parallel dimer of cadherin 23 coming from the side of the adjacent taller stereocilium. To understand the junction between these two dimers, we have synthesized and crystallized the first 200 amino acids of cadherin 23. The distal end of cadherin 23 is unlike that of conventional cadherins; specifically, the tryptophan residues that participate in conventional cadherin binding are absent, and the first ? strand extends to the very tip, where it helps form a novel Ca2+-binding site. Molecular dynamics simulations have highlighted the importance of three Ca2+ ions situated between each of the 27 individual segments that make up the extracellular domain. Many mutations that cause inherited deafness occur in the acidic residues that bind these Ca2+ ions.
Mechanics of Adaptation
We wish to understand the action of Ca2+ in mediating a fast adaptation in hair cells. Because fast adaptation apparently produces a fast movement of the hair bundles, we used a gradient-force optical trap ("laser tweezers") to apply minute forces to hair bundles within microseconds and to measure the resulting movement. We simultaneously measured the channel opening and closing with electrical recording.
Ca2+ enters through transduction channels and acts at an intracellular site to close them. We asked where in the transduction apparatus Ca2+ acts, by measuring the movement that occurs when Ca2+ is allowed into stereocilia. Ca2+ could bind to an internal elastic protein to change its stiffness, it could bind to a protein that then relaxes by a fixed distance, or it could bind to the transduction channel itself to change the relationship between force and open probability. Each model predicts a different dependence of movement on the static bias applied to the bundle. Only one model fits the data: Ca2+ binding to the channel (or a closely associated subunit) tends to close the channel, so that it takes 23 piconewtons more force to open when Ca2+ is bound.
Turnover of Hair Cell Proteins
Hair cells in humans do not divide and are thought to exist for the life of the individual. At the same time, each cell must have a mechanism to degrade and replace its proteins, even as it maintains the shape of its hair bundle and the function of its mechanosensory apparatus.
In collaboration with Claude Lechene (Harvard Medical School), we have used multi-isotope imaging mass spectroscopy (MIMS) to study the synthesis and turnover of proteins in hair cells. Animals are given food that contains the stable but rare isotope 15N, which is incorporated into newly synthesized protein. After a period of days to months, the inner ear is fixed and sectioned. A cesium ion beam sputters ions out of the surface, which are then separated by mass to measure the ratio between the naturally occurring 14N and the new 15N. We can form an image representing the proportion of newly synthesized protein at each location in a sample, with resolution approaching that of electron microscopy.
We found that the stereocilia protein of adult frogs and mice is remarkably stable, with less than 20 percent turnover over a period of five months. This is in contrast to the cell bodies, which show 20–60 percent turnover in 30 days, and is at odds with recent results suggesting that all the actin of a stereocilium is repolymerized in 23 days. Other structures of the inner ear with very low protein turnover are the tectorial membrane and pillar cells, structures that help convey the vibration of sound to the stereocilia. Thus the inner ear may construct its key mechanical structures to proper tolerances, and then leave them alone.
Some of these studies are supported by the National Institute on Deafness and Other Communication Disorders.
As of May 30, 2012