REGULATION OF ION CHANNELS IN NERVE CELLS

我建议继续我们正在进行的神经细胞离子通道调控的研究。由于离子通道负责神经元(和其他细胞)的电活动，因此有关其调节的分子细节的信息对于了解正常和病理的大脑功能是至关重要的。主要焦点将放在大电导钙依赖钾通道上，这种通道普遍存在于神经、肌肉和其他细胞中。这些通道特别令人感兴趣，因为它们是整合各种信号的分子位置，包括细胞内钙浓度、其他细胞内信使和膜电位。我们将深入探讨离子通道作为调节复合体的一部分存在于细胞膜中的主题，其中包括蛋白激酶和其他可以影响通道特性的信号蛋白。这将建立在我们的演示基础上，在当前的授权期内，几种不同的信号蛋白直接结合到钙依赖钾(以及其他)通道上。更具体地说，我们将研究钙依赖型钾通道与以下物质相互作用的分子机制和生理意义：(I)蛋白激酶，最显著的是环AMP依赖性蛋白激酶的催化亚基；(Ii)Slob，我们克隆的一种新的通道结合蛋白，可以直接影响通道的特性，也可以招募普遍存在的信号蛋白支架14-3-3与通道形成复合体。我们将结合分子、生化、遗传和电生理技术来表征相互作用的分子细节，设计干扰相互作用的工具，并使用这些工具来探索相互作用在神经生理学中的作用。与离子通道相关的调节蛋白复合体的存在对神经元信号转导具有重要意义。

I propose to continue our ongoing studies of the regulation of ion channels in nerve cells. Because ion channels are responsible for the electrical activity of neurons (and other cells), information about the molecular details of their regulation is of fundamental importance for understanding normal and pathological brain function. The major focus will be on large conductance calcium- dependent potassium channels, which are ubiquitous in nerve, muscle and other cells. These channels are of particular interest because they are a molecular locus for integration of a variety of signals, including the intracellular calcium concentration, other intracellular messengers, and the membrane potential. We will pursue in depth the theme that ion channels exist in the cell membrane as part of a regulatory complex, that includes protein kinases and other signaling proteins that can influence channel properties. This will build on our demonstration, during the current grant period, of the direct binding of several different signaling proteins to calcium-dependent potassium (as well as to other) channels. More specifically, we will investigate the molecular mechanisms and physiological significance of the interaction of calcium-dependent potassium channels with: (i) protein kinases, most notably the catalytic subunit of the cyclic AMP-dependent protein kinase; and (ii) Slob, a novel channel- binding protein we cloned that can influence channel properties directly, and also can recruit the ubiquitous signaling protein scaffold, 14-3-3, into a complex with the channel. We will use a combination of molecular, biochemical, genetic and electrophysiological techniques to characterize the molecular details of the interactions, design tools to interfere with the interactions, and use these tools to explore the role of the interactions in neuronal physiology. The existence of regulatory protein complexes associated with ion channels has fundamental implications for neuronal signaling.