Sinkins W G, Vaca L, Hu Y, Kunze D L, Schilling W P
Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998, USA.
J Biol Chem. 1996 Feb 9;271(6):2955-60. doi: 10.1074/jbc.271.6.2955.
Previous studies have shown that the Drosophila cation channels designated Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.
先前的研究表明，果蝇中名为Trp和Trpl的阳离子通道可以利用杆状病毒表达载体在Sf9昆虫细胞中实现功能表达。trp基因编码一种Ca2+通透通道，该通道可被毒胡萝卜素激活，被低微摩尔浓度的Gd3+阻断，并且对Ca2+相对于Na+和Ba2+具有相对选择性。相比之下，trpl编码一种Ca2+通透阳离子通道，该通道组成性激活，不受毒胡萝卜素影响，被高微摩尔浓度的Gd3+阻断，并且对Ca2+、Na+和Ba2+无选择性。Trp和Trpl之间序列一致性最低的区域位于COOH末端结构域。为了验证该区域负责这些通道对毒胡萝卜素的不同敏感性这一假说，构建了Trp和Trpl的嵌合构建体，其中每种蛋白质的COOH末端尾部区域进行了交换。带有Trpl COOH尾部的Trp构建体组成性激活，对毒胡萝卜素不敏感，但对Ca2+相对于Na+和Ba2+仍保持选择性。相比之下，带有Trp COOH尾部的Trpl构建体不是组成性激活，可被毒胡萝卜素激活，但对Ca2+、Ba2+和Na+仍无选择性。这些结果表明，Trpl的COOH末端结构域在决定组成性活性方面起重要作用，而Trp的COOH末端区域包含被毒胡萝卜素激活所需的结构特征。
