Genetic Studies of the Synapse

描述(由申请人提供)：长期目标是了解单个钾(K+)通道是如何调节神经元兴奋性的，使用生理学和分子相结合的方法，从1987年第一个电压门控K+通道(每个亚单位有六个跨膜片段)的位置克隆开始，到1993年对前两个内向整流性K+通道之一(每个亚单位有两个TM片段)的表达克隆。现在众所周知，6-Tm Kv通道和2-Tm Kir通道构成了两个非常大的结构相关的K*通道家族。K+通道与人类大脑、耳朵、心脏、肌肉和其他器官的疾病有关，这一事实证明了该项目与健康有关。事实上，K+通道阻滞剂和开放剂已被开发用于药物用途，用于治疗癫痫、中风、偏头痛、心律失常、糖尿病、高血压、神经病理性疼痛和焦虑相关疾病。在使用了更简单的2-TM KIR通道来开发新的方法后，例如依赖随机突变的K+通道的酵母筛选来推断TM螺旋如何在K+通道中排列的无偏方法-这种方法得到了1999年基于哺乳动物Kir2.1通道的酵母研究做出的预测与2003年报道的细菌KirBad.1结构之间的良好一致性的验证，我们计划应用这些新方法来研究6个TM螺旋在Kv通道中是如何排列的。长期以来，神经元如何控制K+通道的数量和位置，从而使这些通道发挥其生理功能，我们将利用最近在通道运输和靶向研究中取得的概念和技术进展，探讨以下问题：(1)培养的海马神经元和皮质神经元中KV1通道蛋白水平如何调节？利用我们开发的翻译报告进行的初步研究已经确定了这一调控的一条信号通路。(2)是什么介导了KV1通道的轴突靶向？我们将寻求通过我们已经制定的分析方法获得的新线索，以量化培养的海马神经元轴突和树突表面K+通道的相对丰度。

DESCRIPTION (provided by applicant): The long-term objectives are to understand how individual potassium (K+) channels regulate neuronal excitability, using a combination of physiological and molecular approaches starting with positional cloning of the first voltage-gated K+ channel (with six transmembrane segments per subunit) in 1987 and expression cloning of one of the first two inwardly rectifying K+ channels (with two TM segments per subunit) in 1993. It is now well known that the 6-TM Kv channels and 2-TM Kir channels constitute two very large families of structurally related K* channels. The health relatedness of the project is evident from the fact that K+ channels are linked to human diseases of the brain, ear, heart, muscle and other organs. Indeed, K+ channel blockers and openers have been developed for pharmaceutical purposes, for the treatment of epilepsies, stroke, migraine, arrhythmias, diabetes, hypertension, neuropathic pain, and anxiety-related disorders. Having used the simpler 2-TM Kir channels to develop new methodologies, such as the unbiased approach of relying on yeast screens of randomly mutagenized K+ channels to deduce how TM helices are arranged in a K+ channel-an approach validated by the excellent agreement between predictions made in 1999 based on yeast studies of mammalian Kir2.1 channels and the bacterial KirBad.1 structure reported in 2003, we plan to apply these new methods to study how the 6 TM helices are arranged in a Kv channel. With a long-standing interest in the question how neurons control the number and location of their K+ channels, thereby allowing these channels to fulfill their physiological functions, we will make use of the conceptual and technological advances made in recent studies of channel trafficking and targeting, and pursue the following questions: (1) How might the Kv1 channel protein level be regulated in cultured hippocampal neurons and cortical neurons? Preliminary studies using a translation reporter we developed have identified one signaling pathway for this regulation. (2) What mediates the axonal targeting of Kv1 channels? We will pursue new leads obtained with the assays we have worked out to quantify the relative abundance of K+ channels on the surface of axons versus dendrites of cultured hippocampal neurons.