Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates
Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates
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DOI：
10.1074/jbc.m111.305698
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发表时间：
2012-01-20
影响因子：
4.8
通讯作者：
Ohta, Toshio
中科院分区：
文献类型：
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作者：
Ohkita, Masashi;Saito, Shigeru;Ohta, Toshio
温敏瞬时受体电位（TRP）通道在进化背景下的功能差异引起了人们的关注，但迄今为止关于两栖动物TRP香草酸1（TRPV 1）功能的信息很少，而两栖动物最早从陆生脊椎动物谱系中分化出来。在这项研究中，我们克隆了热带爪蟾青蛙TRPV 1（xtTRPV 1），并使用异源表达xtTRPV 1的HeLa细胞（xtTRPV 1-HeLa）和从X分离的背根神经节神经元进行功能表征。通过测量细胞内钙浓度（[Ca 2 +]（i））的变化，观察了xtDRG神经元对Tropicalis细胞内钙离子浓度的影响。在表达xtTRPV 1的非洲爪蟾卵母细胞中也观察到通道活性。此外，我们测试了辣椒素和热诱导的青蛙X的伤害反应行为。tropicalis in vivo.在氨基酸水平上，xtTRPV 1与其他陆生脊椎动物TRPV 1直系同源物的序列同源性接近60%。辣椒素诱导xtTRPV 1-HeLa和xtDRG神经元内[Ca ~（2+）]（i）增加，并诱发X.热带植物然而，与哺乳动物直系同源物相比，其灵敏度极低。低细胞外pH和热激活xtTRPV 1-HeLa和xtDRG神经元。热也诱发了伤害反应行为。在卵母细胞表达xtTRPV 1，内向电流引起的热量和低细胞外pH值。突变分析表明，两个氨基酸（酪氨酸523和丙氨酸561）负责辣椒素的低敏感性。两者合计，我们的研究结果表明，xtTRPV 1功能作为一个多模态受体类似于其哺乳动物的直系同源物。目前的研究表明，TRPV 1功能作为一个热和酸敏感通道的祖先陆生脊椎动物。由于可以在体外和体内检测香草素和热敏感性，因此X。热带小蠊是研究TRPV 1功能的理想实验动物。
The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca2+](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin-and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays similar to 60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca2+](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat-and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.