Molecular Modulation of Hippocampal Neuron K+ Channels

海马神经元 K 通道的分子调节

• 批准号: 6831169
• 负责人: John David Sweatt
• 金额: $ 32.8万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6831169
• 关键词: Xenopus; Xenopus oocyte; biological signal transduction; cell line; electrophysiology; hippocampus; laboratory mouse; laboratory rat; molecular site; neural transmission; neuroregulation; phosphorylation; posttranslational modifications; potassium channel; protein kinase; protein protein interaction; protein structure function; pyramidal cells; voltage gated channel; western blottings

This proposal is to investigate the molecular basis for activity-dependent and neurotransmitter-dependent modulation of the biophysical and biochemical properties of potassium channels. We are testing the hypothesis that short-term and long-term regulation of the Kv4.2 channel is mediated by phosphorylation. Specifically we hypothesize that protein kinase-dependent regulation of Kv4.2 is mediated by direct phosphorylation of the pore-forming alpha subunit. In our studies over the last few years we have identified 9 different phosphorylation sites on Kv4.2, mediated by 4 different second-messenger-regulated kinases. Moreover, we have found that PKC phosphorylation of the Kv4.2 C-terminal cytoplasmic domain can regulate the capacity of ERK MAP Kinase to phosphorylate this same channel subregion. Addressing the mechanism for this PKC/ERK interaction will be the first Specific Aim of this project: To determine the protein structure/function relationships underlying PKC modulation of ERK phosphorylation of the C-terminal cytoplasmic domain of Kv4.2. Another example of the complexity of phosphorylationdependent regulation of Kv4.2 is illustrated by considering PKA regulation of Kv4.2. In recent studies we found that PKA regulation of Kv4.2-encoded currents required the presence of a KChlP ancillary subunit. Despite the ability of PKA to phosphorylate the Kv4.2 alpha subunit in the absence of KChlP3, PKA was unable to alter channel biophysical properties by this mechanism alone. Thus, we propose Specific Aim 2 of the project: To determine the structure/function relationships for KChlP modulation of phosphoregulation of Kv4.2. Our final Specific Aim will focus on Calcium/calmodulin-dependent Protein Kinase II (CaMKII) regulation of Kv4.2. We have identified two sites of CaMKII phosphorylation on Kv4.2 and determined that phosphorylation at one of these sites (T438) is necessary and sufficient for CaMKII stabilization and enhanced surface expression of Kv4.2 in COS cells. Additional preliminary results indicate that CaMKII regulation of Kv4.2 surface expression occurs in hippocampal pyramidal neurons as well. We will test the hypothesis that phosphorylation-dependent protein-protein interactions mediate this effect in the final Specific Aim: To investigate the mechanism of CaMKII regulation of Kv4.2 expression and protein stabilization. These studies should allow for the first time the definition of specific phosphorylation events, of known physiologic consequence, as mechanisms for Kv4.2 A-type potassium channel regulation.

本研究旨在探讨钾离子通道活性依赖和神经递质依赖的生物物理和生化特性调控的分子基础。我们正在验证一种假说，即Kv4.2通道的短期和长期调节是由磷酸化介导的。 具体地说，我们假设Kv4.2的蛋白激酶依赖的调节是通过直接磷酸化形成孔的阿尔法亚单位来介导的。在过去几年的研究中，我们已经确定了Kv4.2上的9个不同的磷酸化位点，它们是由4个不同的第二信使调节的激酶介导的。 此外，我们还发现，Kv4.2 C末端胞质结构域的PKC磷酸化可以调节ERK MAP Kinase对同一通道亚区的磷酸化能力。解决这种PKC/ERK相互作用的机制将是本项目的第一个具体目标：确定Kv4.2 C末端细胞质结构域ERK磷酸化的PKC调控下的蛋白质结构/功能关系。另一个例子说明了Kv4.2的磷酸化依赖调控的复杂性，通过考虑Kv4.2的PKA调控。在最近的研究中，我们发现PKA对Kv4.2编码电流的调节需要KChlP辅助亚基的存在。 尽管在没有KChlP3的情况下，PKA能够磷酸化Kv4.2α亚基，但PKA不能单独通过这一机制改变通道的生物物理特性。因此，我们提出了该项目的具体目标2：确定KChlP调控Kv4.2的结构/功能关系。我们最终的具体目标将集中在Kv4.2的钙/钙调蛋白依赖蛋白激酶II(CaMKII)的调控上。我们已经确定了Kv4.2上的两个CaMKII磷酸化位点，并确定其中一个位点(T438)的磷酸化是稳定CaMKII和增强Kv4.2在COS细胞表面表达的必要条件和充分条件。另外的初步结果表明，CaMKII对Kv4.2表面表达的调节也发生在海马锥体神经元中。我们将检验这一假设，即依赖于磷酸化的蛋白质-蛋白质相互作用在 目的：探讨CaMKII对Kv4.2基因表达的调控机制。 蛋白质稳定。这些研究应该首次允许定义特定的磷酸化事件，已知的生理后果，作为Kv4.2A型钾通道调节的机制。