Genetic Studies of the Synapse

描述（申请人提供）：长期目标是了解单个钾（K+）通道如何调节神经元兴奋性，使用生理学和分子方法的组合，从第一个电压门控K+通道的定位克隆开始，（每个亚基有六个跨膜片段）和表达克隆了前两个内向整流钾通道之一（每个亚基有两个TM片段）。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。该项目的健康相关性从K+通道与大脑，耳朵，心脏，肌肉和其他器官的人类疾病有关的事实中显而易见。实际上，K+通道阻断剂和开放剂已被开发用于药物目的，用于治疗癫痫、中风、偏头痛、心律失常、糖尿病、高血压、神经性疼痛和焦虑相关病症。使用更简单的2-TM Kir通道开发了新的方法，例如依靠随机诱变的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--1999年基于哺乳动物Kir2.1通道的酵母研究所做的预测与2003年报道的细菌KirBad.1结构之间的极好一致性验证了该方法，我们计划应用这些新的方法来研究6个TM螺旋在Kv通道中是如何排列的。长期以来，我们一直对神经元如何控制其K+通道的数量和位置的问题感兴趣，从而使这些通道能够实现其生理功能，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步，并探讨以下问题：（1）在培养的海马神经元和皮层神经元中，Kv 1通道蛋白水平可能如何调节？使用我们开发的翻译报告的初步研究已经确定了这种调节的一个信号通路。(2)什么介导Kv 1通道的轴突靶向？我们将继续寻找新的线索，我们已经制定了定量的相对丰富的K+通道的表面上的轴突与树突的培养海马神经元的测定。

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.