Molecular physiology and biophysics of cyclic nucleotide-gated channels

环核苷酸门控通道的分子生理学和生物物理学

• 批准号: 10441791
• 负责人: Jian Yang
• 金额: $ 50.15万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10441791
• 关键词: Binding; Biophysics; Brain; Caenorhabditis elegans; Cations; Cells; Charge; Chemicals; Complex; Cone; Cryoelectron Microscopy; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; DNA Sequence Alteration; Disease; Floods; Genes; Human; Ions; Knowledge; Length; Ligands; Light; Link; Missense Mutation; Molecular; Molecular Conformation; Mutate; Mutation; Neurons; Olfactory Receptor Cells; Patients; Photoreceptors; Physiology; Play; Property; Reporting; Resolution; Retinal Cone; Retinitis Pigmentosa; Role; Sensory; Signal Transduction; Smell Perception; Sodium Chloride; Structure; Testing; Vestibule; Vision; Vision Disorders; achromatopsia; cyclic-nucleotide gated ion channels; dimer; gain of function mutation; loss of function; mutant; retinal rods; therapeutic development

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.

项目摘要 环核苷酸门控（CNG）通道在光感受器中将化学信号转换为电信号 和嗅觉受体细胞，对视觉和嗅觉至关重要。CNG通道基因突变导致 视网膜色素变性和色盲。CNG通道由细胞内cGMP或cGMP受体协同激活。 营虽然从CNG通道的广泛功能研究中获得了丰富的知识， CNG通道特性和机制的结构基础是有限的。最近，关闭-和开放- 态的同聚体C.线虫TAX-4 CNG通道和人CNGA 1通道具有 被举报。这些结构揭示了许多有趣的和独特的功能，解释了一些CNG通道 特性.然而，这些结构并不忠实地代表天然哺乳动物CNG通道，天然哺乳动物CNG通道是天然的。 由视杆细胞中CNGA 1和CNGB 1以及CNGA 3和CNGB 3形成的异三聚体复合物 在视锥细胞感光器中。目前还不清楚疾病相关突变（DAMs）如何改变CNG通道 结构和功能。我们最近获得了一个3.5厘米分辨率的冷冻电镜结构的全长人类 CNGA 3/CNGB 3通道处于apo关闭状态。结构显示CNGA 3/CNGB 3通道是 由3个CNGA 3和1个CNGB 3组成，总体结构与TAX-4和CNGA 1相似，但 值得注意的是，CNGB 3的R403（位于孔螺旋中）和R442（位于S6的胞质末端）突出 进入毛孔。我们还发现R410 W（CNGA 3中的DAM）在没有环鸟苷酸的情况下打开了通道。 在这些进展的基础上，我们提出了三个具体的目标，以阐明的分子机制， CNGA 3/CNGB 3和TAX-4通道的变构性、协同性和通道病变。(1)我们将获得高- CNGA 3/CNGB 3通道在封闭和开放状态下的分辨率结构，在不存在和存在下， Ca2+。这些结构可能揭示CNGB 3的独特作用和CNGB 3的基本问题。 为什么天然锥CNG通道具有CNGB 3亚基。(2)我们将阐明 CNGA 3/CNGB 3和TAX-4通道通过功能和结构的变构cGMP活化 部分配体通道的表征。将通过混合CNGA 3或CNGA 4来实现部分cGMP占用。 CNGB 3与不能结合cGMP的突变CNGB 3或CNGA 3亚基，通过串联CNGA 3和/或 CNGB 3串联成含有不能结合cGMP的亚基的串联二聚体，并通过将TAX-4串联成 包含不同数量的WT TAX-4和突变体TAX-4_EA的串联二聚体和串联四聚体 不能结合cGMP的亚单位。(3)我们将研究CNG通道病的分子机制， 重点系统地研究了S4和孔螺旋中的DMA对结构和功能的影响， CNGA 3/CNGB 3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，门控和通道病，并提供指导的治疗策略， 治疗退化性视觉障碍。