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

描述（由申请人提供）：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。最近，同时发表的三项研究表明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。 公共卫生相关性：本项目的长期目标是通过应用结构和功能分析的组合来理解新发现的辅助亚基Trip 8b调节HCN通道的结构机制。HCN通道负责许多生理学上重要的功能，包括脑和心脏中的起搏活动、突触传递的调节以及树突中的电信号的微调。鉴于Trip 8b潜在的关键生理作用，阐明这种相互作用将有助于理解健康个体中HCN通道的功能及其在癫痫和帕金森病等疾病中的失调。

DESCRIPTION (provided by applicant): 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 potentials1. HCN channels have also been implicated in many pathophysiological conditions including epilepsy, peripheral neuropathic pain and Parkinson's disease2-5. In 2004, an accessory protein of HCN channels, termed Trip8b, was discovered by Santora and colleagues6. 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 surface7- 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. PUBLIC HEALTH RELEVANCE: The long term goal of this project is to understand the structural mechanisms of the regulation of HCN channels by the newly discovered accessory subunit Trip8b by applying a combination of structural and functional assays. HCN channels are responsible for many physiologically important functions including pacemaking activity in the brain and heart, modulation of synaptic transmission, and fine tuning the electrical signals in dendrites. Given Trip8b's potentially critical physiological role, elucidating this interaction will be instrumental in understanding HCN channel function in healthy individuals and its misregulation in diseases such as epilepsy and Parkinson's disease.