PDF(Sci-hub)
Abstract：
Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a \'ball-and-chain\' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.
摘要：
失活是离子通道在开放刺激仍然存在时终止通过其孔的离子通量的过程1。在神经元中，钠和钾通道的失活对于动作电位的产生和放电频率的调节至关重要。通道或辅助亚基的细胞质结构域被认为通过“球链”机制m3-7堵塞开放孔以使通道失活。在这里，我们使用低温电子显微镜来鉴定钙激活钾通道中的分子门控机制，方法是在脂质环境中从热自养甲烷杆菌中获得MthK通道的结构-一种纯粹的钙门控和失活通道。在没有Ca2+的情况下，我们在封闭状态下获得了一个单一的结构，原子模拟表明，在环境温度下，脂质双层具有很高的灵活性，门控环的大摇摆运动和孔衬螺旋的弯曲。在Ca2+束缚条件下，我们得到了几个结构，包括多个开放失活的构象，进一步表明高度动态的蛋白质。这些不同的通道构象通过门控环相对于跨膜区的摇摆来区分。指示整个通道的对称破坏。此外，在所有显示开放通道孔的构象中，通道四聚体的一个亚基的N末端粘入毛孔并堵塞它，自由能模拟表明这是一种强烈的相互作用。该N末端的缺失导致功能上的非失活通道和开放状态的结构，而没有孔塞，这表明该先前未解析的N-末端肽负责球链失活机制。
