Protocadherin 15 as a critical component of the gating spring of human hearing

Project Summary Hearing loss is the most common sensory pathology in the United States, with one in five adults experiencing unilateral or bilateral hearing loss.

In the inner ear, hearing is mediated at the level of the hair cells: when a sound deflects the hair bundle, ion channels atop the stereocilia open, allowing for the mechanotransduction of sound.

The identity of the gating spring, the element that controls the opening of these channels, and thus the precision and sensitivity with which we hear, is unknown.

Connecting adjacent stereocilia is the filamentous tip link complex, which comprises a dimer of protocadherin 15 (PCDH15) and a dimer of cadherin 23.

Previous work in the laboratory showed that the monomer of PCDH15 is softer under physiological forces than predicted based on its structure alone, suggesting that it has the appropriate properties to serve as a component of the gating spring of hearing.

Using a high-speed optical trap, I have obtained preliminary evidence that the dimer of PCDH15 is stiffer than the monomer.

In Aim 1, I will examine the behavior of the PCDH15 dimer in response to force at different critical Ca2+ concentrations.

I will perform force-ramp experiments on the PCDH15 dimer, in which force is increased at a constant rate, in order to delineate its response to physiological levels of force.

There are multiple Ca2+ binding sites in the linker regions between extracellular cadherin (EC) domains in PCDH15, and previous work has shown Ca2+-dependent structural changes in the monomer of PCDH15.

I therefore hypothesize that the dimer will exhibit a similar Ca2+ dependence and will perform experiments at three Ca2+ levels to probe this.

In Aim 2, I will investigate how EC domain unfolding contributes to the overall response of the PCDH15 monomer to force.

Previous work on the monomer of PCDH15 revealed a class of unfolding events corresponding to the unfolding of an entire EC domain. I therefore hypothesize that EC domain unfolding is a critical mediator of tip-link tension.

I will probe this by performing force-ramp experiments on a PCDH15 construct in which each EC domain is prevented from unfolding.

In Aim 3, I will study how a mutation that results in non-syndromic deafness affects the mechanics of the PCDH15 monomer. Approximately 50 % of all congenital hearing loss stems from genetic causes. There are many mutations in PCDH15, such as the V507D mutation in EC5, that result in non-syndromic deafness.

In order to study how the mechanics of PCDH15 are affected in patients with this mutation, I will perform force-ramp experiments on the monomer of this construct.

I hypothesize that PCDH15 V507D will depend critically on Ca2+ concentration and will undergo more unfolding events than does the wildtype monomer.

Taken together, these studies will yield insight into the role of PCDH15 in normal and aberrant hearing and elucidate its ability to serve as a portion of the gating spring of hearing. These studies will be carried out with the direct mentorship of Dr. A. J.

Hudspeth in the group?s laboratory at The Rockefeller University, situated within the richly supportive environment of the Tri-Institutional MD-PhD Program. This proposal will greatly support my goal of becoming a physician-scientist.