PHYSIOLOGY OF DROSPHILA EXCITABLE MEMBRANE AND SYNAPSE

果蝇兴奋膜和突触的生理学

• 批准号: 3398532
• 负责人: CHUN-FANG WU
• 金额: $ 17.56万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3398532
• 关键词: Drosophilidae; RNA splicing; action potentials; adenylate cyclase; alleles; cell membrane; cell type; complementary DNA; cyclic AMP; electrical potential; electrophysiology; gene expression; gene mutation; genetic recombination; genetically modified animals; in situ hybridization; ion transport; larva; membrane channels; mutant; myofibrils; neuronal guidance; phosphoester ligase; potassium channel; synapses; voltage /patch clamp

The rich diversity of neurons with distinct signalling capability reflects cellular differences in the expression of ion channel types, their distribution and their modulation. This biological complexity can be dissected by mutations of the genes involved. By using a collection of Drosophila mutants, the proposed experiments examine the distribution of different K+ channels and their roles in subcellular regions of the neuron. In addition, we will study the genetic control of expression, assembly and modulation of K+ channels. Electrophysiological studies comparing different cellular regions have been hampered by the small size of Drosophila neurons. We recently developed a culture system of "giant" Drosophila neurons differentiated from cell division-arrested neuroblasts. The cells exhibit the various ion currents and thickened neurites, permitting patch-clamp recordings from cell bodies, neurites and terminals. It is also possible to correlate voltage-clamp and current-clamp data from the same neurons in this new system. Individual K+ currents can be eliminated to examine how they shape action potentials and firing patterns by using drugs or mutations, such as Sh (Shaker) and slo (slowpoke) that separately affect two different K+ channels. The Sh gene produces multiple messages by alternative exon splicing. The in situ expression of different Sh cDNAs will be studied in transgenic flies. The expressed Sh channels will be characterized in larval muscles, which permits precise voltage-clamp measurements. Interactions between the product of the transformed cDNA and that of the native Sh gene in the transformant will provide clues to the channel subunit assembly. Mutations of the dunce (dnc) and rutabaga (rut) genes separately affect phosphodiesterase II and adenylate cyclase, leading to altered cAMP levels and learning deficiencies. These mutants offer a unique opportunity to study cAMP-dependent channel modulation to complement the current information based on pharmacological studies. The possibility that another gene ether a go-go (eag) is involved in channel modulation will be examined in larval muscle fibers. Its unusual allele-dependent effect on different K+ currents will be correlated with recombinant DNA analysis of the structural alterations. Analysis of K+ currents in double-mutant combinations of eag, Sh and dnc will yield information about how the eag product affects Sh channels and whether it is mediated by the cAMP pathway.

具有独特信号传导能力的神经元的丰富多样性反映了 离子通道类型表达的细胞差异及其 分布及其调制。 这种生物复杂性可以是 通过相关基因的突变进行剖析。 通过使用集合 果蝇突变体，拟议的实验检查了 不同的 K+ 通道及其在神经元亚细胞区域中的作用。 此外，我们还将研究表达、组装和表达的遗传控制。 K+通道的调制。 比较不同细胞区域的电生理学研究 由于果蝇神经元尺寸较小而受到阻碍。 我们最近开发了一个 细胞分化的“巨型”果蝇神经元培养系统 分裂停滞的神经母细胞。 细胞表现出各种离子电流 和增厚的神经突，允许从细胞体进行膜片钳记录， 神经突和末端。 还可以将电压钳位和 来自这个新系统中相同神经元的电流钳数据。 个人K+ 可以消除电流以检查它们如何塑造动作电位和 使用药物或突变的激发模式，例如 Sh (Shaker) 和 slo (slowpoke) 分别影响两个不同的 K+ 通道。 Sh 基因通过选择性外显子剪接产生多种信息。 这 将在转基因中研究不同Sh cDNA的原位表达 苍蝇。 表达的 Sh 通道将在幼虫肌肉中表征， 这允许精确的电压钳测量。 之间的相互作用 转化的 cDNA 和天然 Sh 基因的产物 转化体将为通道亚基组装提供线索。 dunce (dnc) 和 rutabaga (rut) 基因的突变分别影响 磷酸二酯酶 II 和腺苷酸环化酶，导致 cAMP 水平改变 以及学习上的不足。 这些突变体提供了独特的机会 研究cAMP依赖性通道调制以补充当前的 基于药理学研究的信息。 另一个可能性是 基因以太a go-go (eag)参与通道调制将被检查 在幼虫的肌纤维中。 其不同寻常的等位基因依赖性效应 K+电流将与重组DNA分析相关 结构改变。 双突变体中K+电流的分析 eag、Sh 和 dnc 的组合将产生有关 eag 如何进行的信息 产品影响 Sh 通道以及它是否由 cAMP 途径介导。