Molecular physiology and biophysics of cyclic nucleotide-gated channels

项目摘要 在光感受器中，环核苷酸门控(CNG)通道将化学信号转换为电信号 和嗅觉感受器细胞，对视觉和嗅觉是必不可少的。CNG通道基因突变导致 色素性视网膜炎和色觉减退。细胞内cGMP或cGMP协同激活CNG通道 夏令营。虽然对CNG通道的广泛功能研究已经获得了丰富的知识， CNG通道特性和机制的结构基础是有限的。最近，关闭-和开放- 同源线虫Tax-4 CNG通道和人CNGA1通道的状态冷冻-EM结构 已经上报了。这些结构揭示了许多有趣和独特的特征，解释了一些CNG通道 属性。然而，这些结构并不能忠实地代表天然的哺乳动物CNG通道，这些通道是 CNGA1和CNGB1与CNGA3和CNGB3在杆状感光器中形成的异构体复合体 在视锥感光器中。疾病相关突变(DAM)如何改变CNG通道也不清楚 结构和功能。我们最近获得了一个3.5？分辨率的全身人的冷冻-EM结构 CNGA3/CNGB3通道处于载脂蛋白关闭状态。结构表明CNGA3/CNGB3通道是 由3个CNGA3和1个CNGB3组成，总体结构与Tax-4和CNGA1相似，但 值得注意的是，CNGB3的R403(位于毛孔螺旋)和R442(位于S6的细胞质末端)突出 进入毛孔。我们还发现，CNGA3中的DAMA R410W在没有cGMP的情况下打开了这条通道。 在这些进展的基础上，我们提出了三个具体的目标来阐明分子机制。 CNGA3/CNGB3和TAX-4通道的变构、协同和通道病变。(1)我们将获得高- CNGA3/CNGB3通道在闭合和开放状态下的分辨结构 CA2.这些结构可能会阐明CNGB3的独特作用，以及 为什么天然锥体CNG通道有CNGB3亚基。(2)我们将阐明其分子机制。 变构cGMP通过功能和结构激活CNGA3/CNGB3和TAX-4通道 部分连接通道的表征。部分cGMP占有率将通过混合CNGA3或 CNGB3突变的CNGB3或CNGA3亚基不能结合cGMP，通过连接CNGA3和/或 将含有不能结合cGMP的亚基的CNGB3串联成二聚体，并将Tax-4串联到 含有不同数量的WT TAX-4和TAX-4_EA突变体的串联二聚体和串联四聚体 不能结合cGMP的亚基。(3)探讨CNG经络病的分子机制。 重点系统研究S4中的DMAS和孔道螺旋对结构和功能的影响 CNGA3/CNGB3和TAX-4通道。这些研究将加深我们对CNG通道离子的理解 渗透、门控和经络病，并为制定治疗策略提供指导 治疗退行性视力障碍。

Project Summary Cyclic nucleotide-gated (CNG) channels transduce chemical signals to electrical signals in photoreceptors and olfactory receptor cells and are essential for vision and smell. Mutations in CNG channel genes cause retinitis pigmentosa and achromatopsia. CNG channels are activated cooperatively by intracellular cGMP or cAMP. Although rich knowledge has been gained from extensive functional studies of CNG channels, structural underpinnings of CNG channel properties and mechanisms are limited. Recently, closed- and open- state cryo-EM structures of the homomeric C. elegans TAX-4 CNG channel and human CNGA1 channel have been reported. These structures reveal many interesting and unique features that explain some CNG channel properties. However, these structures do not faithfully represent native mammalian CNG channels, which are heteroterameric complexes formed by CNGA1 and CNGB1 in rod photoreceptors and by CNGA3 and CNGB3 in cone photoreceptors. It is also unclear how disease-associated mutations (DAMs) alter CNG channel structure and function. We have recently obtained a 3.5 Å-resolution cryo-EM structure of the full-length human CNGA3/CNGB3 channel in the apo closed state. The structure shows that the CNGA3/CNGB3 channel is composed of 3 CNGA3 and 1 CNGB3, with an overall structure similar to that of TAX-4 and CNGA1, but strikingly, R403 (located in the pore helix) and R442 (located at the cytoplasmic end of S6) of CNGB3 protrude into the pore. We have also found that R410W, a DAM in CNGA3, opens the channel in the absence of cGMP. Building on these advances, we propose three specific aims to elucidate the molecular mechanisms of allostery, cooperativity and channelopathy of CNGA3/CNGB3 and TAX-4 channels. (1) We will obtain high- resolution structures of CNGA3/CNGB3 channels in closed and open states, in the absence and presence of Ca2+. These structures are likely to shed light on the unique role of CNGB3 and on the fundamental question of why native cone CNG channels have a CNGB3 subunit. (2) We will elucidate the molecular mechanisms of allosteric cGMP activation of CNGA3/CNGB3 and TAX-4 channels through functional and structural characterization of partially liganded channels. Partial cGMP occupancy will be achieved by mixing CNGA3 or CNGB3 with a mutant CNGB3 or CNGA3 subunit unable to bind cGMP, by concatenating CNGA3 and/or CNGB3 into tandem-dimers that contain a subunit unable to bind cGMP, and by concatenating TAX-4 into tandem-dimers and tandem-tetramers that contain varying numbers of WT TAX-4 and TAX-4_EA, a mutant subunit incapable of binding cGMP. (3) We will investigate the molecular mechanisms of CNG channelopathy, focusing on a systematic examination of the effect of DMAs in S4 and pore helix on the structure and function of CNGA3/CNGB3 and TAX-4 channels. These studies will enhance our understanding of CNG channel ion permeation, gating and channelopathy and provide guidance for the development of therapeutic strategies to treat degenerative visual disorders.