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Development of in vitro bitter-and sweet-tastes sensors and screening of taste modulators.

体外苦味和甜味传感器的开发以及味觉调节剂的筛选。

基本信息

批准号：
13558114
负责人：
UGAWA Shinya
金额：
$ 9.02万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Acid-sensing ion channel-2a (ASIC2a) is an amiloride-blockable proton-gated action channel, probably contribution to sour-taste detection in rat taste cells. To isolate another subtype of the sour-taste receptor, we screened a rat circumvallate papilla cDNA library and identified ASIC2b, an N-terminal splice variant of ASICA2a. Reverse transcription-polymerase chain reaction analyses confirmed the expression of ASIC2b transcripts in the circumvallate papilla and, moreover, demonstrated its expression in the foliate and fungiform papillae. Immunohistochemical analyses revealed that ASIC2b, as well as ASIC2a, was expressed in a subpopulation of taste cells in the circumvallate, foliate, and fungiform papillae, and some of the cells displayed both ASIC2a-and ASIC2b-immunoreactivities. Subsequent co-immunoprecipitation studies with circumvallate papillae extracts indicated that ASIC2b associated with ASIC2a to form assemblies, and, together with our immunohistochemical findings, strongly suggested that both ASIC2 subunits formed heteromeric channels in taste cells in the circumvallate, foliate, and fungiform papillae. Oocyte electro-physiology demonstrated that the ASIC2a/ASIC2b channel generated maximal inward currents at pH 2.0, which is in good agreement with the in vivo pH-sensitivity of rat taste cells, and that the amiloride-sensitivity of the heteromer decreased with decreasing pH and was almost completely abolished at pH 2.0. These findings provide persuasive explanations for the amiloride-insensitivity of acid-induced responses of rat taste cells.

酸敏感离子通道-2a（ASIC 2a）是一种可被阿米洛利阻断的质子门控通道，可能参与大鼠味觉细胞的酸味检测。为了分离酸味受体的另一种亚型，我们筛选了大鼠轮廓乳头cDNA文库，并确定了ASIC 2b，ASICA 2a的N-末端剪接变体。逆转录-聚合酶链反应分析证实了ASIC 2b转录本在轮廓乳头的表达，而且，证明了其在叶状和菌状乳头的表达。免疫组织化学分析显示，ASIC 2b，以及ASIC 2a，在轮廓，叶状，菌状乳头的味觉细胞的亚群中表达，和一些细胞显示ASIC 2a和ASIC 2b的免疫反应。随后的免疫共沉淀研究与轮廓乳头提取物表明，ASIC 2b与ASIC 2a形成组件，并与我们的免疫组织化学研究结果一起，强烈表明，这两个ASIC 2亚基形成异聚体通道的味细胞中的轮廓，叶状，和菌状乳头。卵母细胞电生理学表明，ASIC 2a/ASIC 2b通道在pH 2.0时产生最大内向电流，这与大鼠味觉细胞的体内pH敏感性非常一致，并且异聚体的阿米洛利敏感性随着pH的降低而降低，并且在pH 2.0时几乎完全消除。这些发现提供了有说服力的解释阿米洛利不敏感的酸诱导反应的大鼠味觉细胞。