NEUROMODULATION OF ION CHANNELS

离子通道不是简单的被动孔，而是动态实体 经受可以持续数秒、数分钟甚至数小时的调制。 我建议研究磷酸化的机制， 离子通道产生这种长期调节。 特别是我会 检查离子通道是否可以作为调节复合物存在 与调节蛋白有关。 该研究将侧重于一组 大电导Ca 2+激活的K+通道，因为许多 这些通道的磷酸化可以调节， 通道本身，或密切相关的蛋白质。 具体目标 是：1）探索离子通道是否可以驻留在膜中， 蛋白质复合物，与调节成分紧密相关， 蛋白激酶和蛋白磷酸酶; 2）检查 离子通道门控上的磷酸化位点， 被多重磷酸化; 3）研究 作用于单个离子通道的不同蛋白激酶;以及4） 定义蛋白磷酸酶在调节 通道选通 这些实验将依赖于两种互补的实验方法。 首先，将使用平面脂质双层来重构单个离子 通道与所有细胞质的影响分开。 的影响 纯化的蛋白激酶和磷酸酶对通道门控的作用将被 考察 第二，天然膜和完整膜中的通道调节 细胞将通过膜片钳培养的神经元来研究。 而 有很多关于神经元如何对 细胞外信号通过改变它们的电活动， 关于这些调节剂改变的机制知之甚少 通道选通 这项工作的目的是提供这样的机制 信息，特别是在离子通道/激酶的背景下 磷酸酶复合物 这样的复合物可以代表用于治疗癌症的共同靶标。 多个第二信使途径，并在整合中发挥作用， 甚至是细胞外物质携带的信息的储存 信号.

Ion channels are not simply passive pores, but are dynamic entities subject to modulation that can last for seconds, minutes, or even hours. I propose to investigate the mechanisms by which the phosphorylation of ion channels produces such long-term modulation. In particular I will examine whether ion channels can exist as regulatory complexes tightly associated with modulatory proteins. The study will focus on a group of large conductance Ca2+-activated K+ channels from rat brain, because many of these channels can be modulated by phosphorylation of either the channel itself, or of closely associated proteins. The specific aims are: 1) to explore whether ion channels can reside in membranes as protein complexes, tightly associated with regulatory components such as protein kinases and protein phosphatases; 2) to examine the contribution of individual phosphorylation sites on the gating of ion channels that are multiply phosphorylated; 3) to investigate the interactions between different protein kinases that act on a single ion channel; and 4) to define the role and specificity of protein phosphatases in modulating channel gating. These experiments will rely on two complementary experimental approaches. First, planar lipid bilayers will be used to reconstitute individual ion channels separated from all cytoplasmic influences. The effect of purified protein kinases and phosphatases on channel gating will be examined. Second, channel modulation in native membranes and intact cells will be investigated by patch-clamping cultured neurons. While there is much descriptive information about how neurons respond to extracellular signals by an alteration in their electrical activity, little is known about the mechanisms by which these modulators alter channel gating. This work will aim to provide such mechanistic information, particularly in the context of ion channel / kinase phosphatase complexes. Such complexes may represent a common target for multiple second messenger pathways, and play a role in the integration, and perhaps even the storage, of information carried by extracellular signals.