School of Life Sciences, University of Warwick, Coventry, United Kingdom.
Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.
Elife. 2024 Jun 3;13:RP93686. doi: 10.7554/eLife.93686.
Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.
连接蛋白通过在相邻细胞之间形成间隙连接通道 (GJCs) 来实现细胞间通讯。连接蛋白 26 (Cx26) 可以被 CO 直接调节。这被认为是通过 K125 的氨甲酰化来介导的。我们表明，将 K125 突变为模拟氨甲酰化负电荷的谷氨酸会导致 Cx26 GJCs 持续关闭。通过冷冻电镜，我们观察到 K125E 突变会推动构象平衡向具有狭窄孔入口的通道移动，类似于提高 CO 分压所产生的影响。在先前的连接蛋白结构中，细胞质环是调节的重要部分，K125 位于此处，其处于无序状态。通过进一步的冷冻电镜研究，我们捕获了 Cx26 的不同状态，并观察到细胞质环的密度。这个环的位置、跨膜螺旋的构象以及控制孔道孔径的 N 端螺旋的位置之间的相互作用提供了一种调节机制。
