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.

项目概要 环核苷酸门控 (CNG) 通道将化学信号转换为光感受器中的电信号 CNG 通道基因突变对视觉和嗅觉至关重要。 视网膜色素变性和色盲症通道由细胞内 cGMP 或 CGMP 协同激活。 尽管从 CNG 通道的广泛功能研究中获得了丰富的知识， 最近，CNG 通道特性和机制的结构基础受到限制。 状态同聚线虫 TAX-4 CNG 通道和人 CNGA1 通道的冷冻电镜结构 这些结构揭示了许多有趣且独特的特征，可以解释一些 CNG 通道。 然而，这些结构并不能忠实地代表天然哺乳动物 CNG 通道。 视杆光感受器中 CNGA1 和 CNGB1 以及 CNGA3 和 CNGB3 形成的异源复合物 目前还不清楚疾病相关突变 (DAM) 如何改变 CNG 通道。 我们最近获得了全长人体的 3.5 Å 分辨率冷冻电镜结构。 CNGA3/CNGB3通道处于apo关闭状态 结构显示CNGA3/CNGB3通道处于apo关闭状态。 由3个CNGA3和1个CNGB3组成，整体结构与TAX-4和CNGA1类似，但是 引人注目的是，CNGB3的R403（位于孔螺旋中）和R442（位于S6的细胞质末端）突出 我们还发现 CNGA3 中的 DAM R410W 在没有 cGMP 的情况下打开通道。 基于这些进展，我们提出了三个具体目标来阐明其分子机制 CNGA3/CNGB3 和 TAX-4 通道的变构、协同性和通道病 (1) 我们将获得高-。 CNGA3/CNGB3 通道在关闭和打开状态下的分辨率结构，在不存在和存在的情况下 Ca2+。这些结构可能揭示 CNGB3 的独特作用和基本问题。 (2) 阐明其分子机制 CNGA3/CNGB3 和 TAX-4 通道通过功能和结构的变构 cGMP 激活 部分配体通道的表征将通过混合 CNGA3 或来实现。 CNGB3 具有无法结合 cGMP 的突变 CNGB3 或 CNGA3 亚基，通​​过连接 CNGA3 和/或 CNGB3 形成串联二聚体，其中含有无法结合 cGMP 的亚基，并将 TAX-4 连接成 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 (3)我们将研究CNG通道病变的分子机制， 重点系统研究 S4 和孔螺旋中 DMA 对结构和功能的影响 CNGA3/CNGB3 和 TAX-4 通道的研究将增强我们对 CNG 通道离子的理解。 渗透、门控和通道病，并为治疗策略的制定提供指导 治疗退行性视觉障碍。