Targeting of Voltage-gated K+ Channels to Lipid Rafts

描述：神经元和肌肉膜内的离子通道调节是一种神经元和肌肉膜内的离子通道调节。 神经系统电兴奋性的重要决定因素， 心血管系统、骨骼肌、胃肠道和子宫。电压-门控K+ Kv通道在静息电位的设定中起重要作用 并决定这些功能多样的系统中的复极。最近 有证据表明，通常被称为脂质的特殊微区 筏存在于大多数质膜的平面内。这些结构域 富含胆固醇和鞘脂， 转导分子在初步数据部分，我们证明， 电压门控K+通道Kv2.1、Kv1.1、Kv1.5和Kv1.4，但不包括Kv4.2， 靶向于异源表达系统和大鼠脑中脂筏。在 此外，Kv2.1和Kv1.5可能位于不同的筏舱中。 细胞胆固醇的消耗改变了Kv2.1相关蛋白的浮力。 筏并将Kv2.1失活的中点移动30-40 mV， 影响峰值电流密度或沟道激活。Kv2.1的孵育 用伏马菌素B（一种神经酰胺合成酶抑制剂）表达细胞， 失活曲线的类似移动。神经酰胺既是筏的组成部分， 一种细胞内信号分子因此，筏协会在功能上是 重要的是，这种失活的转变将导致大的 Kv2.1通道在以下范围内功能性沉默的百分比 生理膜电位此外，初步数据显示， 伏马菌素B诱导Kv2.1从细胞体到远端的错误启动 树突在培养的神经元，这表明筏相关的信号机制是 参与了Kv2.1的定位具体目标将：（1）解决机制问题 参与Kv2.1靶向脂筏，重点放在 亚基组成和通道跨膜结构域; 2）检查 Kv2.1与脂筏结构域相关的功能意义 强调神经酰胺信号通路，3）检查 神经元中的筏缔合、神经酰胺信号传导和细胞表面定位; 和4）开始旨在纯化含Kv 2.1的脂质的初步工作 从大脑中漂流出来。 这项拟议的研究探讨了一个新的领域，千伏通道的研究，将 在多个组织系统中具有重要意义。

DESCRIPTION: Ion channel regulation within neuronal and muscle membranes is an important determinant of electrical excitability in the nervous and cardiovascular systems, skeletal muscle, GI tract, and uterus. Voltage-gated K+ channels (Kv channels) play an important role in setting the resting potential and determining repolarization in these functionally diverse systems. Recent evidence suggests that specialized microdomains commonly referred to as lipid rafts exist within the plane of most plasma membranes. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction molecules. In the Preliminary Data section, we demonstrate that the voltage-gated K+ channels, Kv2.1, Kv1.1, Kv1.5, and Kv1.4, but not Kv4.2, target to lipid rafts in both heterologous expression systems and rat brain. In addition, Kv2.1 and Kv1.5 probably reside in different raft compartments. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1-associated rafts and shifts the midpoint of Kv2.1 inactivation by 30-40 mV without affecting peak current density or channel activation. Incubation of Kv2.1 expressing cells with fumonisin B, an inhibitor of ceramide synthase, causes a similar shift in the inactivation curve. Ceramide is both a raft component and an intracellular signaling molecule. Thus, raft association is functionally significant, for such a shift in the inactivation will result in a large percentage of the Kv2.1 channels being functionally silenced in the range of physiological membrane potentials. In addition, the preliminary data suggest fumonisin B induces mistargeting of Kv2.1 from the cell body to the distal dendrites in cultured neurons, suggesting raft-related signaling mechanisms are involved in Kv2.1 targeting. The Specific Aims will 1) address the mechanisms involved in the targeting of Kv2.1 to lipid rafts, with emphasis placed on subunit composition and channel transmembrane domains; 2) examine the functional significance of Kv2.1 association with lipid raft domains with emphasis on ceramide signaling pathways, 3) examine the relationship between raft association, ceramide signaling, and cell surface localization in neurons; and 4) begin initial work aimed at purification of Kv 2.1-containing lipid rafts from brain. This proposed research examines a new area in Kv channel research that will have important implications in multiple tissue systems.