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

已知多种离子通道和转运体与内耳功能有关。到目前为止，我们已经通过生理学方法在耳蜗肌中发现了氯离子电压依赖性氯离子通道(CLC)、Na-K-2c1共转运体(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转运蛋白的分子特征的确凿证据。用逆转录聚合酶链式反应(RT-PCR)检测各种异构体的表达，用非放射性原位杂交(ISH)检测它们的定位。在CIC家族中，ClC-2和ClC-3显著存在于内耳组织中。BSC2转录本仅在耳蜗组织中检测到，且密集分布于耳蜗纹边缘细胞和螺旋韧带成纤维细胞中，提示BSC2转录本可以很好地解释环状利尿剂的耳蜗毒性。Ncx(1-3)的3种亚型均在内耳中检测到。在耳蜗蜗壳中发现了NCX-1独特的香料变体。虽然NHE-2、-3和-4亚型的mRNAs可以在耳蜗组织中检测到，但NHE-1的信息明显占优势。NHE-1亚型表达主要分布于毛细胞、边缘细胞、螺旋韧带纤维细胞和神经节细胞。

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.