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Functional analysis of ionic channel and transporter of the cochlear marginal cell using cellular physiology and molecular biology

利用细胞生理学和分子生物学对耳蜗边缘细胞离子通道和转运蛋白进行功能分析

基本信息

批准号：
09470367
负责人：
IKEDA Katsuhisa
金额：
$ 7.62万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

A variety of ionic channels and transportes are known to be related to inner ear function. We have so far found Cl voltage-dependent chloride channels (ClC), Na-K-2C1 cotransporter (CCC), and Na-Ca exchanger (NCX), and Na-H exchange (NHE) in the cochlea by physiological methods. Furthermore, recent advances in molecular biology enabled us to prove the definite evidence of molecular characterization of these ion chanells and transportes. We used the reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of various isoforms and the non-radioactive in situ hybridization (ISH) to examine their localization. Among CIC family, ClC-2 and -3 were significantly present in the inner ear tissue. BSC2 transcript alone was detected in the cochlea and intensively located in the strial marginal cells and spiral ligament fibrocytes, indicating a good explanation of loop-diuretics cochleotoxicity. All of three isoforms of NCX (1-3) were detected in the inner ear. Unique spicing varients of NCX-1 were found in the cochlear modiolus. Although NHE-2, -3, and -4 isoform mRNAs could be detected in the cochlear tissue, NHE-1 message was clearly dominant. NHE-1 isoform expression was distributed in the hair cells, marginal cells, and spiral ligament fibrocytes, and ganglion cells.

已知多种离子通道和传输与内耳功能相关。迄今为止，我们通过生理学方法在耳蜗中发现了Cl电压依赖性氯离子通道（ClC）、Na-K-2C1协同转运蛋白（CCC）、Na-Ca交换器（NCX）和Na-H交换（NHE）。此外，分子生物学的最新进展使我们能够证明这些离子通道和传输的分子特征的明确证据。我们使用逆转录聚合酶链反应（RT-PCR）来评估各种异构体的表达，并使用非放射性原位杂交（ISH）来检查它们的定位。在CIC家族中，ClC-2和-3在内耳组织中显着存在。在耳蜗中单独检测到 BSC2 转录物，并且集中位于纹状体边缘细胞和螺旋韧带纤维细胞中，这表明袢利尿剂耳蜗毒性得到了很好的解释。在内耳中检测到了所有三种 NCX (1-3) 亚型。在耳蜗蜗轴中发现了 NCX-1 的独特香料变体。尽管可以在耳蜗组织中检测到 NHE-2、-3 和 -4 同工型 mRNA，但 NHE-1 信息明显占主导地位。 NHE-1亚型表达分布于毛细胞、边缘细胞、螺旋韧带纤维细胞和神经节细胞。