Structural basis of the gating mechanism of connexin 26 and related hearing loss

摘要 作为缝隙连接通道的连接蛋白26直接调节细胞间离子交换， 代谢物和第二信使。连接蛋白26的突变会导致严重的听力损失。 然而，连接蛋白26的结构基础上的分子门控机制仍不清楚， 尤其是缝隙连接蛋白26突变的闭合状态和功能障碍。我们已经开发了 用于高分辨率单粒子低温电子显微镜的天然细胞膜纳米粒子(NCMN)系统 细胞内膜蛋白的结构测定及其生理功能研究 条件。我们最近解决了连接蛋白26在没有或存在的情况下的低温EM结构 钙离子在2.3？和2.9？时处于开放状态，与大约36个脂质分子通过 NCMN系统。然而，连接蛋白26高分辨率的封闭状态仍未被发现。 我们假设我们的NCMN系统在Cx26和Cx26结构测定中的应用 Cx26-R75W在有无钙和钙调蛋白的情况下可以提供独特的 结构信息。目的1：研究浇口机构的闭合构象 Cx26。目的2：阐明Cx26基因R75W单点突变的分子基础 会导致严重的听力损失。拟议的实验结果将为 详细的结构信息和生化分析，以了解门控机制， 还提供了在此听证会中检测到的异常通道门控功能的机械细节 无序。结构信息可能导致一些新的策略来调整故障 CX26-R75W用于听力损失。

SUMMARY Connexin 26, as a gap junction channel, directly regulates the cell-to-cell exchange of ion, metabolites, and second messengers. Mutations of connexin 26 can cause severe hearing loss. However, the molecular gating mechanism on the structural basis of connexin 26 is still unclear, especially the closed state and malfunction of connexin 26 mutations. We have developed the native cell membrane nanoparticle (NCMN) system for high-resolution single-particle cryo-EM structure determination and functional studies of membrane proteins within their physiological conditions. We recently solved the cryo-EM structures of connexin 26 in the absence or presence of calcium ions in an open state at 2.3 Å and 2.9 Å associated with about 36 lipid molecules via the NCMN system. However, the close state of connexin 26 with high resolution is still uncovered. We hypothesize that our NCMN system's application to structure determination of Cx26 and Cx26-R75W in the presence and absence of calcium and calmodulin can provide unique structural information. Aim 1: To investigate the CLOSED conformation of the gating mechanism of Cx26. Aim 2: To elucidate the molecular basis of how the single point mutation R75W in Cx26 leads to profound hearing loss. The proposed experimental results will provide insights into detailed structural information and biochemical analysis in understanding the gating mechanism, also provide mechanistic details on abnormal channel gating function detected in this hearing disorder. The structural information may lead to some novel strategy to modulate the malfunction CX26-R75W for hearing loss.