MUTANT ANALYSIS OF GENES CONTROLLING ANESTHETIC ACTION

挥发性麻醉剂是临床上重要的一类药物，其作用机制尚不清楚。除了有可能合理设计更安全、更有效的麻醉剂外，阐明麻醉机制应该影响神经生物学的基本领域，如突触传递、意识和记忆。遗传学是确定哪些分子事件实际上负责麻醉引起的神经系统抑郁的重要工具。秀丽隐杆线虫是一种强大的遗传模型，它有一个简单的神经系统，利用与人类相同的基本突触机制。我们发现，在高度保守的syntaxin基因编码中，一个不寻常的突变使秀丽隐杆线虫对临床浓度的挥发性麻醉剂产生抗性。我们还发现，麻醉剂异氟醚与大鼠SNAP-25结合，SNAP-25是一种与syntaxin紧密相互作用的蛋白质。我们提出的实验旨在通过以下方式来探索这一惊人的发现：1)确定产生麻醉抗性所需和足够的突变syntaxin区域。2)鉴定与syntaxin一起调节麻醉作用的基因，并试图在SNAP-25中发现对麻醉剂有抗性的突变体。3)确定这些基因介导麻醉作用的途径和细胞。4)确定麻醉剂与syntaxin复合物时是否与SNAP-25结合，并通过诱变确定麻醉剂在SNAP-25上的结合位点。挥发性麻醉药的作用机制将被提出的工作所定义，可用于解释和指导脊椎动物实验，如转基因小鼠敲除。无脊椎动物和脊椎动物遗传学以及体外研究的结合应该最终使我们能够达到了解这类临床和科学上重要的药物的作用机制的目标。

Volatile anesthetics are a clinically important class of drugs whose mechanism of action is not understood. Besides the potential to rationally design safer and more effective anesthetics, elucidating anesthetic mechanisms should impact fundamental areas of neurobiology such as synaptic transmission, consciousness, and memory. Genetics is an essential tool for determining which molecular events are actually responsible for anesthetic-induced nervous system depression. The nematode Caenorhabditis elegans is a powerful genetic model that has a simple nervous system utilizing the same fundamental synaptic machinery as humans. We have found that an unusual mutation in the highly conserved gene coding for syntaxin renders C. elegans resistant to clinical concentrations of volatile anesthetics. We have also found that the anesthetic isoflurane binds to rat SNAP-25, one of the proteins that tightly interacts with syntaxin. Our proposed experiments are designed to explore this astonishing discovery by: 1) Identifying the region of mutant syntaxin necessary and sufficient to produce anesthetic resistance. 2) Identifying genes that function along with syntaxin to regulate anesthetic action and attempt to find mutants in SNAP-25 that are resistant to anesthetics 3) Determine the pathway and cells through which these genes act to mediate anesthetic effects 4) Determine whether anesthetics bind to SNAP-25 when in complex with syntaxin and define by mutagenesis the binding site for anesthetics on SNAP-25. The mechanism of action of volatile anesthetics that will be defined by the proposed work can be used to interpret and guide vertebrate experiments such as transgenic mice knockouts. The combination of invertebrate and vertebrate genetics and in vitro studies should finally allow us to reach our goal of understanding the mechanism of action of this clinically and scientifically important class of drugs.