Park H J, Niedzielski A S, Wenthold R J
Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, MD 20892, USA.
Hear Res. 1997 Oct;112(1-2):95-105. doi: 10.1016/s0378-5955(97)00111-1.
Acetylcholine is a major neurotransmitter of the cochlear efferent system. Based on its high level of expression in hair cells, the recently cloned nicotinic receptor subunit, alpha9 [Elgoyhen et al., Cell 79 (1994) 705-715], is likely to be the postsynaptic receptor for acetylcholine in hair cells either as a homomeric complex or with other subunits yet to be identified. To further study this receptor, we cloned and sequenced alpha9 cDNA from the guinea pig organ of Corti library [Wilcox and Fex, Hear. Res. 62 (1992) 124-126]. The sequence of the guinea pig alpha9 cDNA is similar to that of the rat, with identities of 85% and 89% at the nucleotide and amino acid levels, respectively. Most differences are in the cytoplasmic loop domain between the transmembrane segments 3 and 4. We also observed minor differences in the putative ligand binding regions. Pharmacological differences between acetylcholine receptors on outer hair cells of rat and guinea pig have been reported, and the minor structural changes we observe could account for these differences. Reverse transcription-polymerase chain reaction analysis showed a high expression of alpha9 in the organ of Corti while expression was low or not detected in the spiral ganglion. In situ hybridization histochemistry showed expression of alpha9 mRNA in both inner and outer hair cells, with much higher expression in outer hair cells than in inner hair cells. In the inner hair cell, silver grains were more abundant over the basal part of the cell than over the apical part. Immunocytochemistry showed a pattern of distribution of the alpha9 protein similar to that seen for mRNA with in situ hybridization. Immunolabeling was most intense at the bases of both inner and outer hair cells. To determine the effect of hair cell loss on alpha9 expression, hair cells were destroyed by either systemic or local application of kanamycin. This treatment led to a down regulation of alpha9 in hair cells; this down regulation appeared to precede hair cell degeneration. In the spiral ganglion, a transient up regulation of alpha9, as determined by RT-PCR, was seen 4-6 weeks after kanamycin treatment.
乙酰胆碱是耳蜗传出系统的一种主要神经递质。基于其在毛细胞中的高表达水平，最近克隆的烟碱样受体亚基α9[埃尔戈伊亨等人，《细胞》79(1994)705 - 715]，很可能是毛细胞中乙酰胆碱的突触后受体，要么作为同聚体复合物，要么与其他有待鉴定的亚基结合。为了进一步研究该受体，我们从豚鼠柯蒂氏器文库[威尔科克斯和费克斯，《听觉研究》62(1992)124 - 126]中克隆并测序了α9 cDNA。豚鼠α9 cDNA的序列与大鼠的相似，在核苷酸和氨基酸水平上的同源性分别为85%和89%。大多数差异存在于跨膜区段3和4之间的胞质环结构域。我们还在假定的配体结合区域观察到微小差异。已有报道大鼠和豚鼠外毛细胞上乙酰胆碱受体的药理学差异，我们观察到的微小结构变化可能解释了这些差异。逆转录 - 聚合酶链反应分析显示α9在柯蒂氏器中高表达，而在螺旋神经节中表达低或未检测到。原位杂交组织化学显示α9 mRNA在内毛细胞和外毛细胞中均有表达，在外毛细胞中的表达远高于内毛细胞。在内毛细胞中，银颗粒在细胞基部比在顶部更丰富。免疫细胞化学显示α9蛋白的分布模式与原位杂交检测到的mRNA分布模式相似。免疫标记在内毛细胞和外毛细胞的基部最为强烈。为了确定毛细胞损失对α9表达的影响，通过全身或局部应用卡那霉素破坏毛细胞。这种处理导致毛细胞中α9的下调；这种下调似乎先于毛细胞变性。在螺旋神经节中，卡那霉素处理4 - 6周后，通过逆转录 - 聚合酶链反应测定，可见α9短暂上调。
