MOLECULAR DELINEATION OF THE COCHLEAR HAIR CELLS

我们的长期目标是鉴定出表达的电压门控离子通道， 在耳蜗毛细胞中。 该项目的具体目标是 鉴定和定位电压门控钾的表达， 大鼠内毛细胞和外毛细胞的钙和钠离子通道 毛细胞（OHC）。 一旦确定，这些特定的离子通道将被 在非洲爪蟾卵母细胞表达系统中检测它们的 电生理学和药理学特性。 本项目的第一个具体目标是检查单向 从大鼠耳蜗polyA+ RNA和 来自大鼠IHCc和OHC的扩增polyA+ RNA， 代表电压门控离子通道蛋白转录本的cDNA 存在于毛细胞中。 基因家族特异性聚合酶链反应 （gfs-PCR）和基因特异性PCR将用于表征亚型 和/或钾、钙和钠电压门控的亚单位状态 离子通道 将使用保守的基因家族特异性引物基序 为了扩增编码预测存在于细胞中的蛋白质的cDNA， 耳蜗毛细胞 将使用以下组合鉴别亚型： PCR产物大小、限制酶位点差异（即，RFLP-PCR）， 杂交研究和双脱氧核苷酸序列分析。 新的或 新的离子通道cDNA的特征在于获得了一个完整的 长度序列 将通过检查表达的同种型来分析表达的同种型。 含有这些信息的3 'NT和/或5'末端的PCR产物， 它们的DNA序列将用于确认它们独特的同种型 status. 基因特异性探针的开发和Northern杂交的应用 分析和原位杂交研究以区分表达， IHC和/或OHC亚型的组织分布、细胞定位 与其他已知的同种型不同。 一旦同种型和/或亚基在细胞中表达， 在建立了国际卫生协调委员会和人道主义协调委员会之后，下一个目标将是确认 耳蜗毛细胞中转录本的存在 杂交方法 它们的精确细胞定位和细胞内 将使用免疫细胞化学确定分布。 最终目标 将检查这个离子通道的电生理学和 非洲爪蟾卵母细胞的药理学特性。 分子生物学和表达研究将共同用于 定义和建立这些特定离子通道在耳蜗中的作用， 稳态和电生理学的IHC和OHC的声学 耳蜗中的信号传导。 这些数据将增加我们的 理解听力的分子过程及其 疾病，特别是关于耳蜗毛细胞的功能， 听觉感受器

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.