MOLECULAR IDENTIFICATION OF FUNCTIONAL K+ CHANNELS

非洲爪蟾为研究该疾病提供了理想的动物模型。 发展中的脊椎动物神经系统的发展阶段， 物种已经被很好地描述了。 此外，使用 标准的体外受精技术允许生产胚胎 任何年龄的人，其神经元可以被检查是否获得电 兴奋性 两栖类脊髓神经元首次显示动作电位 受精后22小时的神经板期。 这些冲动是 钙依赖性和持续时间长。 在接下来的24小时内， 动作电位的形式发生了明显的变化，成为简短的，钠- 成熟神经元的驱动性尖峰特征。 以前的工作表明 从成熟到不成熟的波形转变主要是 这是由于延迟整流钾电流的成熟。 期间 这种发展的心理过渡，钙电流变化不大，而 钠电流密度加倍。 然而钾离子流却增加了三倍 密度更高，动力学也更快。 数学 动作电位的重建也表明， 脉冲的形状可以用钾离子的变化来解释 当前属性 这些刻板印象背后的分子基础 变化仍然未知。 我的项目的一个目标是匹配 到目前为止在非洲爪蟾中克隆的钾通道的分子身份 与功能性钾通道群体记录从发展 两栖类脊髓神经元 四个已知阿尔法战士中的两个 非洲爪蟾中的Kv1.1亚基基因（Shaker样亚家族的成员）和 Kv2.2（Shab样亚家族的成员）已通过原位显示 杂交和单细胞逆转录酶PCR将存在于 正在发育的两栖动物脊髓 此外，这些通道诱导 延迟整流钾电流在非洲爪蟾卵母细胞中表达。 因此，Kv1.1和Kv2.2基因产物可能是诱导的候选物 电流导致动作的缩短和成熟 潜力 通过上述工作，应能确定 含有Kvc 1和/或Kv 2亚基的功能性单通道。 这是 重要的是，到目前为止，没有实验符合分子身份 基因的功能通道人口记录从发展中国家 脊髓 下一步将是阐明这些机制 参与感兴趣的基因的转录-即， 确定为什么特定的基因在特定的 神经元亚群在某个发育阶段。 最终这样 知识将提供对所涉及的事件的更完整的理解 在获得电兴奋性的过程中， 设计更有效的治疗方法， 发展神经系统和那些疾病由于不适当的， 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.