MOLECULAR IDENTIFICATION OF FUNCTIONAL K+ CHANNELS

非洲爪哇为研究非洲爪哇提供了一个理想的动物模型。 脊椎动物神经系统的发育阶段 物种的特征已经非常好了。此外，还使用了 标准的体外受精技术可以生产胚胎 任何年龄的人，其神经元可以被检查以获得电 兴奋性。两栖类脊髓神经元首次显示出动作电位 在神经板阶段(受精后22小时)。这些冲动是 依赖钙，且持续时间长。在接下来的24小时内， 动作电位的形式发生了显著的变化，变成了短暂的钠- 成熟神经元的驱动棘波特征。以前的工作已经表明 从成熟的波形到未成熟的波形的转变主要是 由于延迟整流钾电流的成熟。在.期间 这一发展思路转变，钙电流变化不大，而 钠电流密度加倍。然而，钾电流是原来的三倍 在密度上，也表现出更快的动力学。数学 动作电位的重建也表明，动作电位的变化 冲量的形状可以用钾的变化来解释 当前属性。这些刻板印象背后的分子基础 到目前为止，变化仍然是未知的。我的项目的一个目标是与 迄今为止在非洲爪哇克隆的钾通道的分子同一性 随着从发育中记录到的功能性钾通道种群 两栖类脊髓神经元。已知的四个阿尔法中的两个 非洲爪哇、Kv1.1(Shaker-like亚家族成员)和 Kv2.2(Shab Like亚家族的一个成员)，已被原位显示 杂交和单细胞逆转录酶聚合酶链式反应将出现在 发育中的两栖动物脊髓。此外，这些渠道还会诱导 非洲爪哇卵母细胞表达延迟整流钾电流。 因此，Kv1.1和Kv2.2基因产物可能是诱导的候选基因 导致动作的缩短和成熟的洋流 潜力。 上面概述的工作应该能够鉴定内源 含有Kvc1和/或Kv2亚基的功能单一通道。这是 值得注意的是，到目前为止，还没有实验能与分子同一性相匹配 从发育过程中记录的具有功能通道群体的基因 脊髓。下一步将是阐明这些机制。 参与感兴趣基因的转录--即 确定为什么该特定基因在特定的 处于一定发育阶段的神经元亚群。最终是这样的 知识将提供对所涉及事件的更完整的了解 在获得电的兴奋性方面，因此有可能 设计更有效的治疗方法来治疗儿童精神障碍 发展中的神经系统和那些因不当而导致的障碍 不适当的兴奋，如癫痫。

Xenopus laevis provides an ideal animal model for the study of the developing vertebrate nervous system as the developmental stages in this species have been extremely well characterized. In addition, the use of standard in vitro fertilization techniques allows the production of embryos of any age whose neurons can be examined for the acquisition of electrical excitability. Amphibian spinal neurons first demonstrate action potentials at the neural plate stage (22 hr after fertilization). These impulses are calcium-dependent and of long duration. During the following 24 hours, the form of the action potential changes markedly, becoming the brief, sodium- driven spike characteristic of mature neurons. Previous work has shown that the transition from the mature to the immature wave-form is primarily due to the maturation of a delayed rectifier potassium current. During this develop mental transition the calcium current changes little while the sodium current doubles in density. The potassium current, however, triples in density and also demonstrates faster kinetics. Mathematical reconstruction of action potentials has also shown that the change in the shape of the impulse can be accounted for by the alternation in potassium current properties. The molecular bases underlying these stereotyped changes remain, as yet, unknown. A goal of my project is to match the molecular identities of the potassium channels cloned thus far in Xenopus with the functional potassium channel populations recorded from developing amphibian spinal neurons. Transcripts from two of the four known alpha subunit genes in Xenopus, Kv1.1 (a member of the Shaker-like subfamily) and Kv2.2 (a member of the Shab like subfamily), have been shown by in situ hybridization and single-cell reverse transcriptase PCR to be present in the developing amphibian spinal cord. In addition, these channels induce delayed rectifier potassium currents when expressed in the Xenopus oocyte. Kv1.1 and Kv2.2 gene products are thus likely candidates for the induction of currents leading to the shortening and maturation of the action potential. Thw work outlined above should allow the identification of endogenous functional single channels that contain Kvc1 and/or Kv2 subunits. This is significant as to date, no experiments have matched the molecular identity of gene with a functional channel population recorded from the developing spinal cord. The next step would be an elucidation of the mechanisms involved in the transcription of the gene of interest - i.e. a determination of why that particular gene is transcribed in a specific neuronal subset at a certain developmental stage. Ultimately such knowledge will provide a more complete understanding of the events involved in the acquisition of electrical excitability and hence the possibility of designing more effective therapies for the treatment of disorders of the developing nervous system and those disorders due to improper due to improper excitation such as epilepsy.