Genetic Studies of the Synapse

描述（由申请人提供）：长期目标是了解单个钾（K+）通道如何调节神经元兴奋性，使用生理和分子方法的结合，从1987年的第一个电压门控K+通道（每个亚基有六个跨膜段）的位置克隆开始，以及1993年的前两个内向纠正K+通道之一（每个亚基有两个TM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。从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可以明显看出，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制药目的，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疾病。利用更简单的2-TM Kir通道开发了新的方法，例如依靠酵母随机诱变K+通道的筛选来推断TM螺旋如何在K+通道中排列的无偏倚方法——1999年基于酵母对哺乳动物Kir2.1通道的研究和细菌KirBad的预测非常一致，这一方法得到了验证。我们计划应用这些新方法来研究6个TM螺旋如何在Kv通道中排列。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，从而使这些通道实现其生理功能的问题感兴趣，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，并追求以下问题：(1)如何调节培养的海马神经元和皮质神经元中的Kv1通道蛋白水平？使用我们开发的翻译报告器进行的初步研究已经确定了这种调节的一个信号通路。(2)是什么介导了Kv1通道的轴突靶向？我们将继续进行新的研究，以量化培养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.