Gamo Sumiko, Tomida Junya, Dodo Katsuyuki, Keyakidani Dai, Matakatsu Hitoshi, Yamamoto Daisuke, Tanaka Yoshiharu
Department of Earth and Life Sciences, Osaka Prefecture University, Sakai, Japan.
Anesthesiology. 2003 Oct;99(4):867-75. doi: 10.1097/00000542-200310000-00019.
Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity.
Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311).
Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains.
ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.
多种物种，如秀丽隐杆线虫、黑腹果蝇和小鼠，已被用于在体内探索全身麻醉药的作用机制。作者分离出一种对乙醚高度敏感的果蝇突变体ethas311，并将钙网蛋白（crc）基因鉴定为麻醉敏感性改变的候选基因。
对crc进行分子分析，包括cDNA的克隆和测序、Northern印迹分析以及原位杂交，以完成该基因及其突变的功能研究。对于麻醉表型分析，测定了ethas311、回复突变体和双突变体菌株（野生型crc转基因加上ethas311）的50%麻醉浓度。
在所有发育阶段，突变体ethas311中crc 1.4-kb转录本的表达均低于野生型。在野生型蛹化后19小时，大脑中观察到最高表达，但在该阶段突变体中的表达仍然很低。该突变体对异氟烷有抗性，对乙醚高度敏感，而对氟烷表现出野生型表型。在回复突变体和双突变体菌株中，两种突变体表型均恢复为野生型。
ethas311是果蝇钙网蛋白基因低表达的一种突变。作者证明，对乙醚的高度敏感和对异氟烷的抗性与该基因的低表达有关。在果蝇中，钙网蛋白似乎介导了这些麻醉敏感性，并且它可能是乙醚和异氟烷的作用靶点，尽管基于许多体外和体内研究预测的麻醉靶点是膜蛋白，如离子通道和受体。
