MOLECULAR DELINEATION OF THE COCHLEAR HAIR CELLS

点击翻译按钮获取中文摘要

Our long term goal is to identify the voltage-gated ion channels expressed in the cochlear hair cells. The specific aims of this project are to identify and localize the expression of the voltage-gated potassium, calcium and sodium ion channels in rat inner hair cells (IHCs) and outer hair cells (OHCs). Once identified, these specific ion channels will be examined in a Xenopus oocyte expression system for their electrophysiologic and pharmacologic properties. The first specific goal of this project is to examine unidirectional lambdaZAP cDNA libraries, constructed from rat cochlear polyA+ RNA and from amplified polyA+ RNA from rat IHCc and OHCs, for the presence of cDNAs that represent transcripts of the voltage-gated ion channel proteins present in hair cells. Gene family-specific polymerase chain reaction (gfs-PCR) and gene specific-PCR will be used to characterize the isoform and/or subunit status of the potassium, calcium and sodium voltage-gated ion channels. Conserved gene family-specific primer motifs will be used to amplify cDNAs coding for proteins predicted to be present in the cochlear hair cells. Isoforms will be identified using a combination of PCR product size, restriction enzyme site differences (i.e., RFLP-PCR), hybridization studies, and dideoxy nucleotide sequence analyses. New or novel ion channel cDNA will be characterized by obtaining a complete full length sequence. The expressed isoforms will be analyzed by examining the PCR products containing the 3'NT and/or 5' ends of these messages for their DNA sequences that will be used to confirm their unique isoform status. Gene specific probes will be developed and used in Northern-blot analysis and in situ hybridization studies to distinguish the expression, tissue distribution, cellular localization of the IHC and/or OHC isoforms from other known isoforms. Once the isoforms and/or subunits expressed in the IHCs and OHCs are established, the next goal will be to confirm the existence of transcripts in the cochlear hair cells using in situ hybridization. Their precise cellular location and intracellular distribution will be determined using immunocytochemistry. The final goal will be to examine this ion channel for their electrophysiologic and pharmacologic properties in Xenopus oocytes. Collectively the molecular biology and expression studies will be used to define and establish the role of these specific ion channels in cochlear homeostasis and in the electrophysiology of IHCs and OHCs for acoustic signal transduction in the cochlea. These data will increase our understanding of the molecular processes underlying hearing and its disorders, especially regards to the functioning of cochlear hair cells as acoustic receptors.