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

描述(申请人提供)：HCN通道在包括大脑和心脏在内的许多组织中起着关键的生理作用。这些通道负责心脏和神经细胞的起搏器活动、树突整合和设置静息膜电位1。HCN通道还与许多病理生理状况有关，包括癫痫、周围神经病理性疼痛和帕金森氏病2-5。2004年，Santora及其同事发现了一种HCN通道的辅助蛋白，称为Trip8b。最近，三项同时发表的研究表明，Trip8b是高度选择性剪接的，并且这些变体对将HCN通道运输到细胞表面7-9具有不同的影响。此外，这些小组还表明，所有研究的变体都能够减弱cAMP对通道的影响。HCN通道的门控是由cAMP以直接的、蛋白激酶或磷酸化的独立方式调节的，但在Trip8b存在的情况下，这种调节大大减少。在神经元中，对HCN通道的表达和功能的多样性知之甚少。众所周知，在神经元和表达系统中，该通道的运输和门控是不同的。HCN通道在神经元中表现出高度特异性的表达模式，例如，在CA1锥体神经元中，随着距离胞体的距离增加，HCN通道以密度递增的梯度表达。鉴于其在体内的功能多样性，Trip8b是体内调节HCN通道的一个有吸引力的候选基因。考虑到这一点，我计划研究HCN通道和Trip8b相互作用的生物物理学。对于这两种蛋白质的相互作用，哪些残基是关键的？越来越多的证据表明，在环核苷酸结合域(CNBD)7、10上存在第二个相互作用部位。这两个相互作用对Trip8b的生理作用重要吗？这个复合体的化学计量比是多少？Trip8b/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相结合的方法来解决这些问题，包括膜片钳荧光测量、单分子荧光和基于晶体结构的定向突变。除了提供的关于Trip8b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”的HCN通道门控和配体结合过程中发生的重要结构和重排。此外，这些发现将对理解许多不同类型的离子通道和受体的门控和配体结合运动具有普遍的兴趣。 公共卫生相关性：该项目的长期目标是通过应用结构和功能分析相结合的方法，了解新发现的辅助亚单位Trip8b调节HCN通道的结构机制。HCN通道负责许多重要的生理功能，包括大脑和心脏的起搏活动，突触传递的调制，以及树突电信号的微调。鉴于Trip8b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的HCN通道功能及其在癫痫和帕金森病等疾病中的错误调节。

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.