Study of Mechanosensitive Transduction Channel on Hair Cell of Zebrafish

Hair cells reside in the auditory and vestibular system of the vertebrate inner ear and in the lateral line organ of fish. These cells carry out mechanotransduction, the conversion of a mechanical stimulus into an electrical response, which is then processed by the central nervous system. Because zebrafish possess a series of neuromasts on the lateral line of the body that are very similar in structure and function to the sensory patched of inner ear and is easy to observe and readily accessible for manipulation, zebrafish is recently used as a model system of hair cell. Mechanoelectrical transduction occurs in the hair bundle, which when moved towards its tallest edge opened mechanically gated ion channels near the tips of the component stereocilia. This allows an influx of Ca2+ ions that depolarize the hair cell. The channel is also permeable to small organic cations, even fluorescent styryl dyes like FM1-43 of molecular weight > 450Da. The transient receptor potential (TRP) family of ion channels was typified by a large conductance and a high Ca2+ permeability. Much of the circumstantial evidence indicates that these TRP channels potentially play significant roles in inner ear physiology. The expression of TRPN1, TRPV4 and TRPA1 has been found in the hair cell of C. elegant, drosophila and mouse. Morpholino knockdown experiments in zebrafish also show that TRPN1, TRPA1 is critical for the hair cell function. However controversial results also found in knockout mice experiments, more convincing experiments have to be investigated in the future. In my previous study, I had used the SIET technique (Scanning Ion-Selective Electrode Technique) to analysis the H+ flux in adult zebrafish inner ear. In this proposal, SIET will be used to analysis the Ca2+ flux of hair cell on lateral line after the morpholino knockdown of TRPN1, TRPA1 and TRPV4, respectively. Furthermore, the treatment of FM1-43 and inhibitor of mechanotransduction channel will also be used to examine the morphological and functional effect on hair cell. This proposal is designed as a three years project: The first year: In my preliminary data, I have successfully detected the Ca2+ influx of hair cell on the lateral line of zebrafish. In the first year, I will use SIET to analysis Ca2+ flux of hair cell during development and in different location (posterior or anterior lateral line). Amiloride and Gd3+ (inhibitor of mechanotransduction channel) will also be applied to examine the pharmacological effect on hair cell Ca2+ influx. Morpholino knockdown of TRPN1、TRPA1、TRPV4 also will be conducted to observed the effect on Ca2+ flux of hair cell and the hearing sense ability of embryo. The second year: The mRNA and protein expression of TRPN1、TRPA1、TRPV4 on zebrafish hair cell will be explored. the antibody of TRPN1、TRPA1、TRPV4 made in the first year will be used in the second year to analysis the localization of these three molecule. Double stain with Concanavalin A and FM1-43, which labeled the hair bundle and cell body, respectively, will show the location of the three TRP channel on the Hair cell precisely. The third year: Interaction of TRPN1、TRPA1、TRPV4 in the mechanotransduction mechanism of hair cell will be examined. The reciprocal regulation mechanism among TRPN1、TRPA1、TRPV4 will be conducted by knockdown one of the TRP channel and examined the expressing or functional change of the other two TRP channels. This proposal will combine the molecular and functional evidence to establish the zebrafish hair cell for a model of mechanotransduction mechanism.