Towards understanding physiological function of TRPM6/7 like channel in a HCO_3-secreting epithelium

反刍动物的腮腺即使在休息时也会分泌大量富含hco_3的唾液，但其分子机制尚不清楚。反刍动物腮腺分泌细胞含有多种离子通道，可通过细胞内Ca^<2+>和Mg^<2+>进行调节，这些二价阳离子的静息细胞稳态也可能在HCO_3的持续分泌中发挥重要作用。为了更好地理解腮腺HCO_3分泌的机制，本项目将重点研究TRPM6和TRPM7，这两个已知的阳离子通道可以渗透到Ca^<2+>和Mg^<2+>等二价阳离子。利用电生理和分子生物学技术发现，1)牛腮腺腺泡细胞表现出对细胞内Mg^<2+>敏感的膜电流，如报道的重组TRPM7和TRPM7电流；2)牛腮腺细胞表达TRPM6和TRPM7的转录本；3)从牛腮腺中克隆的TRPM7在异源表达系统中具有功能。这些基础结果将为进一步研究牛腮腺腺泡细胞天然TRPM6/7样电导的分子基础和细胞调控，以及TRPM6和TRPM7在上皮细胞HCO_3分泌中的作用奠定基础。

Ruminant parotid glands secrete continuously large volumes of HCO_3-rich saliva even at resting, but the underlying molecular mechanism remains unclear. Since ruminant parotid secretory cells contain several ion channels that can be regulated by intracellular Ca^<2+> and Mg^<2+>, resting cellular homeostasis of these divalent cations might also play an important role in the continuous HCO_3 secretion. Towards better understanding the mechanism of the parotid HCO_3 secretion, this project focused on TRPM6 and TRPM7 that are known to be cation channels permeable to divalent cations such as Ca^<2+> and Mg^<2+>. Using electrophysiological and molecular biological techniques, it was found that 1) bovine parotid acinar cells exhibit a membrane current, which is sensitive to intracellular Mg^<2+> as reported for recombinant TRPM7 and TRPM6 currents, 2) bovine parotid cells express transcripts of TRPM6 and TRPM7, 3) TRPM7 that was cloned from bovine parotid was functional in a heterologous expression system. These basic results would be useful for future studies to address the issues as to not only the molecular basis and cellular regulation of the native TRPM6/7-like conductance in bovine parotid acinar cells, but also the role of TRPM6 and TRPM7 in epithelial HCO_3 secretion.