Having an Anesthetic without adverse effects : The binding sites of dexmedetomidine on adrenoceptor

右美托咪定（DEX）是一种亚型非选择性α 2肾上腺素能激动剂，因其镇静/催眠、麻醉保留、镇痛和交感神经阻滞特性而开发用于麻醉管理。这些特性中的每一个被认为是由α 2A肾上腺素受体亚型的激活介导的。我们试图确定负责与DEX结合的人α 2A肾上腺素受体亚型上的TMD，以设计新的亚型选择性α 2A激动剂。构建嵌合受体以用来自人β 2肾上腺素受体的同源序列取代野生型人α 2A肾上腺素受体的限定TMD，所述人β 2肾上腺素受体本身对DEX没有亲和力。用12种新的构建体以及野生型人α 2A和人β 2肾上腺素受体转染COS-7细胞。收获细胞并制备用于放射性标记的配体结合的膜，使用3 H-阿替美唑和DEX作为置换配体。TMD#7对于DEX结合是关键的，因为CRS 25（α 2 TMD#7）具有> 10，000的亲和力，比CRT 3（β 2 TMD#7）更高。TMD#2在较小程度上参与DEX结合，因为CRS 16（α 2 TMD#2）对DEX的亲和力比β 2肾上腺素受体高>100倍。TMD#6可能参与DEX结合，因为CRS 25（α 2 TMD#6）对DEX的亲和力比CR 8（β 2 TMD#6）高10倍。为了更好地了解负责亚型选择性的氨基酸残基，需要进行三种肾上腺素受体亚型之间TMD#7和2（和6）中氨基酸残基差异的进一步定点诱变研究。这将促进用于麻醉的新型α 2A亚型选择性激动剂的基于靶点的药物设计。

Dexmedetomidine (DEX) is a subtype non-selective, alpha2 adrenergic agonist developed for anesthetic management because of its sedative/hypnotic, anestheticsparing, analgesic and sympatholytic properties. Each of these properties are thought to be mediated by activation of the alpha2A adrenoceptor subtype. We sought to determine the TMDs on the human alpha2A adrenoceptor subtype which are responsible for binding to DEX in order to design novel subtype selective alpha2A agonists. Chimeric receptor were constructed to replace defined TMDs of the wild-type human alpha2A adrenoceptor with homologous sequence from the human beta2 adrenoceptor which itself has no affinity for DEX.Chimerae of the human alpha2A and human beta2 adrenoceptors were constructed from genes encoding human alpha2A (or C10) and beta adrenoceptors. COS-7 cells were transfected with 12 new constructs as well as the wild-type human alpha2A and human beta2 adrenoceptors. Cells were harvested and membranes prepared for radiolabeled ligand binding using 3H-atipamezole and DEX as the displacing ligand.TMD#7 is pivotal for DEX binding since CRS 25 (alpha2 TMD#7) has > 10,000 for higher affinity than CRT3 (beta2 TMD#7). To a lesser extent TMD#2 is involved in DEX binding since CRS16 (alpha2 TMD#2) has >100 fold higher affinity for DEX than does beta2 adrenoceptor. TMD#6 may be involved in DEX binding since CRS 25(alpha2 TMD#6) has 10 fold higher affinity for DEX than does CR8 (beta2 TMD#6).The next generation of alpha2 adrenergic agonists needs to be alpha2A subtype selective. To better understand the amino acid residues responsible for subtype selectivity, further site-directed mutagenesis studies of the differences in amino acid residues in TMD#7 and 2 (and 6) between the three adrenoceptor subtypes need to be undertaken. This will facilitate target-based drug design of the novel alpha2A subtype selective agonists for use in anesthesia.