Cloning and functional analysis of taste receptors for sourness and saltiness

本研究采用同源性分析和功能表达相结合的方法，从大鼠轮廓乳头cDNA文库中鉴定出一个编码酸味味觉受体（MDEG 1）的cDNA。当在非洲爪蟾卵母细胞中表达时，MDEG1表现出质子门控阿米洛利敏感阳离子通道的特征。有趣的是，在相同的pH值下，醋的主要成分醋酸的酸性刺激比盐酸产生更大的内向电流。原位杂交显示，MDEG1的mRNA只定位于味蕾。MDEG 1在味蕾细胞的细胞表面和顶端部分均有表达，尤其是在味觉感受器细胞III型细胞中。这是第一个被克隆的味觉受体cDNA。为了分离酸味受体的另一个亚基，我们筛选了大鼠轮廓乳头cDNA文库并鉴定了MDEG 2。免疫组织化学和电生理研究表明，MDEG 2与MDIEG 1在单个味细胞的顶端部分形成异源寡聚通道，并改变了MDEG 1通道的脱敏作用。在不改变其他通道特性的情况下，通过MDEG2中Gly481的单个氨基酸取代显著改变了异源寡聚体的脱敏速率。这些发现表明，MDEG2调节酸味受体脱敏有助于酸味适应，MDEG2中的Gly481是控制酸味感觉持续时间的关键氨基酸残基。

We have identifieda cDNA encoding a taste receptor for sourness (MDEG1) from a rat circumvallate papilla cDNA library by combined cDNA homology and functional expression approaches. When expressed in Xenopus laevis oocytes, MDEG1 showed characteristics of a proton-gated amiloride-sensitive cation channel. Interestingly, acidic stimulation by acetic acid, the chief ingredient of vinegar, generatedlarger inwardcurrents than that by hydrochloric acidat equal pH.In situ hybridization revealed that the mRNA of MDEG1 was exclusively localized in taste buds. MDEG1 immunoreactivity was found on the cell surface and in the apical portion of taste bud cells, especially in type III cells which are classifiedas gustatory receptor cells. This is the first taste receptor cDNA to be cloned. To isolate another subunit of the sour-taste receptor, we screened a rat circumvallate papilla cDNA library and iden tified MDEG2. Immunohistochemical and electrophysiological studies demonstrated that MDEG2 formed heterooligomeric channels with MDIEG1 at the apical portion of single taste-cells and modified the desensitization of MDEG1 channel. The desensitization rate of the heterooligomer was dramatically altered by a single amino acid substitution of Gly481 in MDEG2 without changing other channel properties. These findings suggest that MDEG2 regulates the sour-taste receptor desensitization contributing to sour-taste adaptation and that Gly481 in MDEG2 is the key amino acidresidue that controls the duration of sourtaste sensation.