Mechanisms of HCN regulation by accessory subunit Trip8b using fluorescence and e

利用荧光和 e 辅助亚基 Trip8b 调节 HCN 的机制

• 批准号: 8335533
• 负责人: John Bankston
• 金额: $ 5.39万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-16 至 2014-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335533
• 关键词: Address; Alternative Splicing; Amino Acids; Binding; Biological Assay; Biophysics; Brain; C-terminal; Cardiac; Cations; Cell surface; Complex; Cyclic AMP; Cyclic Nucleotides; Cytoplasmic Protein; DNA Sequence Rearrangement; Data; Dendrites; Dendritic Cells; Dependence; Disease; Distal; Electrophysiology (science); Epilepsy; Figs - dietary; Fluorescence; Fluorometry; Goals; Heart; Homeostasis; Individual; Ion Channel; Lead; Ligand Binding; Long-Term Potentiation; Longitudinal Studies; Measures; Membrane; Membrane Potentials; Mental Depression; Methods; Mind; Movement; Mutagenesis; Neurons; Pacemakers; Parkinson Disease; Pattern; Peptides; Peripheral; Phosphorylation; Physiological; Play; Protein Binding; Protein Kinase; Proteins; Publishing; RNA Splicing; Regulation; Rest; Role; Signal Transduction; Site; Specific qualifier value; Status Epilepticus; Stretching; Structure; Surface; Synaptic Transmission; System; Techniques; Testing; Tissues; Variant; Work; base; cyclic-nucleotide gated ion channels; density; fluorophore; hippocampal pyramidal neuron; improved; in vivo; infancy; interest; neuronal cell body; painful neuropathy; patch clamp; receptor; relating to nervous system; single molecule; small molecule; stoichiometry; trafficking; voltage

Project Summary HCN channels play a critical physiological role in many tissues including the brain and heart. These channels are responsible for pacemaker activity in both cardiac and neuronal cells, dendritic integration, and setting resting membrane potentials[1]. HCN channels have also been implicated in many pathophysiological conditions including epilepsy, peripheral neuropathic pain and Parkinson¿s disease[2-5]. In 2004, an accessory protein of HCN channels, termed Trip8b, was discovered by Santora and Colleagues[6]. More recently, three simultaneous studies were published that showed that Trip8b was highly alternatively spliced and that the variants had different effects on trafficking HCN channels to the cell surface[7-9]. In addition, these groups showed that all of the variants studied were able to blunt the effect of cAMP on the channel. Gating of HCN channels is regulated by cAMP in a direct, protein kinase or phosphorylation, independent manner, but in the presence of Trip8b that regulation is greatly reduced. In neurons, little is known about the diversity of expression and function of HCN channels. It is known that the trafficking and gating of the channel is different in neurons than in expression systems. HCN channels show a highly specified pattern of expression in neurons, for example, in CA1 pyramidal neurons HCN channels are expressed in a gradient of increasing density with increasing distance from the soma. Given its diversity of function in vivo, Trip8b is an attractive candidate for regulation of HCN channels in vivo. With that in mind, I plan to study the biophysics of the interaction of HCN2 channels and Trip8b. What residues are critical for the interaction for these two proteins? There is increasing evidence that there is a second interaction site in the cyclic nucleotide binding domain (CNBD)[7, 10]. Are both of these interactions important for the physiological role of Trip8b? What is the stoichiometry of that complex? How does the Trip8b/HCN interaction alter the cyclic nucleotide dependence of channel gating? I plan to address these questions using a combination of fluorescence and electrophysiology that will include patch clamp fluorometry, single molecule fluorescence, and targeted mutagenesis based on crystal structures. In addition to the information provided about Trip8b, I believe over the long term this study will help elucidate the important structures and rearrangements that occur during ¿normal¿ HCN channel gating and ligand binding. Also, these finding will be of general interest towards the understanding of gating and ligand binding movements for many different types of ion channel and receptors.

项目摘要 HCN通道在包括脑和心脏在内的许多组织中发挥关键的生理作用。这些通道负责心脏和神经元细胞中的起搏器活动、树突整合和设置静息膜电位[1]。HCN通道还涉及许多病理生理学病症，包括癫痫、周围神经性疼痛和帕金森病[2-5]。 2004年，Santora及其同事发现了HCN通道的辅助蛋白Trip 8b [6]。最近，同时发表了三项研究，表明Trip 8b是高度可变剪接的，并且变体对将HCN通道运输到细胞表面具有不同的影响[7-9]。此外，这些研究组表明，所有研究的变体都能够减弱cAMP对通道的影响。HCN通道的门控由cAMP以直接的、蛋白激酶或磷酸化独立的方式调节，但在Trip 8b存在下，该调节大大降低。 在神经元中，对HCN通道的表达和功能的多样性知之甚少。已知通道的运输和门控在神经元中与在表达系统中不同。HCN通道在神经元中显示出高度特异性的表达模式，例如，在CA 1锥体神经元中，HCN通道以随着与索马的距离增加而密度增加的梯度表达。鉴于其在体内的功能多样性，Trip 8b是体内HCN通道调节的有吸引力的候选者。考虑到这一点，我计划研究HCN 2通道和Trip 8b相互作用的生物物理学。哪些残基对这两种蛋白质的相互作用至关重要？越来越多的证据表明，在环核苷酸结合结构域（CNBD）中存在第二个相互作用位点[7，10]。这两种相互作用对Trip 8b的生理作用重要吗？这个络合物的化学计量是多少？Trip 8b/HCN相互作用如何改变通道门控的环核苷酸依赖性？我计划使用荧光和电生理学的结合来解决这些问题，其中包括膜片钳荧光测定法、单分子荧光和基于晶体结构的靶向诱变。除了提供有关Trip 8b的信息外，我相信从长远来看，这项研究将有助于阐明在正常HCN通道门控和配体结合期间发生的重要结构和重排。此外，这些发现将对许多不同类型的离子通道和受体的门控和配体结合运动的理解具有普遍意义。