A computational approach for understanding adaptation in vertebrate hair cells

Thesis (Ph. D.)--Harvard-MIT Program in Health Sciences and Technology, 2012.Pages 157 and 158 blank. Cataloged from PDF version of thesis.Includes bibliographical references (p. 150-156).Vertebrate hair cells respond to mechanical stimuli with an inward current that is carried by extracellular cations through mechanically-gated transmembrane ion channels called transduction channels, located in the hair cell's specialized apical surface called the stereocilia. The current is characterized as having a rapid onset and adapting exponentially with a fast and slow time constant. The fast component is usually attributed to calcium binding directly to the transduction channels to promote channel reclosure. Myosin-1C, an unconventional myosin motor protein that is also modulated by calcium, adjusts the tension applied to the transduction channel to cause slow adaptation. Neither adaptation typically acts completely to restore the transduction current back to the baseline level. Recent evidence has suggested that the transduction channel is further away from myosin-IC than previously believed, creating a spatial separation that changes the nature of the calcium feedback. I developed a computational model to explore the motion of vertebrate hair cells simultaneously with calcium diffusion within the cell. The model is also capable of simulating many other experimental techniques that are commonly applied to hair cells. The results of the model suggest a fundamentally different viewpoint for understanding adaptation in vertebrate hair cells. Calcium can create unique responses from different transduction channels within the same hair cell. The implications of these findings help to explain the incompleteness of adaptation as well as implicate myosin-1C for fast adaptation as well as slow adaptation. In addition, groundwork for better understanding stereocilia-based amplification in the mammalian cochlea was developed. Experimental predictions were created to test these theories.by Paul D. Niksch.Ph.D

Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

<div><p>Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.</p></div

Trpm2 is not required for hair cell mechanotransduction.

<p><b>(a)</b> FM1-43 accumulation by IHCs and OHCs from control <i>Trpm2</i>^fll+:<i>Gfi1</i>-Cre⁺ mice <i>(top)</i> and deleted <i>Trpm2</i>^fl/fl:<i>Gfi1</i>-Cre⁺ mice <i>(bottom)</i>. FM1-43 accumulation by hair cells was similar in control and <i>Trpm2</i>-deleted hair cells. Age: P5+2div. Scale bar = 20 μm. <b>(b)</b> A family of OHC transduction current recordings <i>(top traces)</i> in response to stereocilia bundle deflections <i>(bottom traces)</i> in WT <i>(left)</i> and <i>Trpm2</i>^fl/fl:<i>Gfi1</i>-Cre⁺ <i>(right)</i> mice. <b>(c)</b> Average peak transduction current (red traces in <b>b</b>) for control mice (WT and <i>Trpm2</i>^fl/+:<i>Gfi1</i>-Cre⁺) and <i>Trpm2</i>^fl/fl:<i>Gfi1</i>-Cre⁺ mice. <b>d,</b> ABR thresholds in response to pure tone stimuli. <i>Trpm2</i>^fl/fl:<i>Gfi1</i>-Cre⁺ mice show normal hearing. Data are mean ± sem; n as indicated.</p

Mice lacking single or multiple PKD genes show normal stereocilia bundle morphology and hearing function.

<p><b>(a-d)</b> SEM images of postnatal 4~6 weeks organ of Corti hair cells at low magnification in WT and PKD-deficient mice. Scale bar = 10 μm. <b>(e-l)</b> OHC bundles at high magnification. Scale bar = 1 μm. <b>(m)</b> ABR thresholds in response to pure tone stimuli in <i>Pkd2</i> and <i>Pkd2l1</i> single and double knockouts. <b>(n)</b> <i>Pkd2l1</i> and <i>Pkd1l3</i> single and double knockouts. <b>(o)</b> <i>Pkd2l2</i> knockouts. No functional deficit was observed in any combination tested. Data shown as mean ± sem.</p

Neither <i>Pkd2</i> nor <i>Pkd2l1</i> is required for hair cell mechanotransduction.

<p><b>(a)</b><i>In situ</i> hybridization in cochlear sections. (<i>Left</i>) no label is evident with a control sense probe. (<i>Middle</i>) no specific label is evident in the <i>Pkd2l1</i> knockout. (<i>Right</i>) <i>In situ</i> hybridization with an antisense probe shows label of inner hair cells (arrowhead), outer hair cells (arrows) and inner sulcus cells (asterisks) in the organ of Corti. Scale bar = 50 μm; age P2. <b>(b)</b> ABR thresholds in response to pure tone stimuli. <i>Pkd2l1</i>^-/- mice show normal hearing at age P31-P37. <b>(c)</b> <i>Pkd2</i>^-/-:<i>Atoh1</i>-Cre^+/-mice show normal hearing at age 4~6 weeks. Data are mean ± sem; n as indicated.</p

Primers used for genotyping and validation of gene deletion.

<p>Primers used for genotyping and validation of gene deletion.</p