Simplified bacterial "pore" channel provides insight into the assembly, stability, and structure of sodium channels.
Simplified bacterial "pore" channel provides insight into the assembly, stability, and structure of sodium channels.
复制标题
DOI：
10.1074/jbc.c111.228122
复制
发表时间：
2011-05-06
期刊：
影响因子：
--
通讯作者：
Wallace BA
中科院分区：
文献类型：
--
作者：
McCusker EC;D'Avanzo N;Nichols CG;Wallace BA
真核细胞钠通道是重要的膜蛋白，参与离子渗透、动态平衡和电信号传导。它们是长的、多结构域的蛋白，在异源系统中不能很好地表达，因此，对纯化的钠通道蛋白的结构/功能和生化研究一直是有限的。细菌产生更小的同源四聚体单域通道，专用于钠离子的电导。它们由N端电压传感器和C端孔子亚域组成。我们设计了一个仅有孔的功能通道，由海洋细菌硅杆菌Pomeroyi钠通道的最后两个跨膜螺旋、相互作用的P区和膜外C端区域组成。这种钠“孔”通道形成一个四聚体的折叠结构，能够支持磷脂小泡中的钠流量。只有孔的通道比其全长通道更热稳定，这表明电压传感器亚域可能会破坏全长通道的稳定性。孔子结构域可以独立于电压传感器进行组装、折叠和功能，并表现出与完整通道相似的配体阻断特性。这种简单的孔道结构的可用性应该能够为潜在的新配体的测试提供高水平的表达，并加强我们对支配钠选择性和渗透性的结构特征的理解。
Eukaryotic sodium channels are important membrane proteins involved in ion permeation, homeostasis, and electrical signaling. They are long, multidomain proteins that do not express well in heterologous systems, and hence, structure/function and biochemical studies on purified sodium channel proteins have been limited. Bacteria produce smaller, homologous tetrameric single domain channels specific for the conductance of sodium ions. They consist of N-terminal voltage sensor and C-terminal pore subdomains. We designed a functional pore-only channel consisting of the final two transmembrane helices, the intervening P-region, and the C-terminal extramembranous region of the sodium channel from the marine bacterium Silicibacter pomeroyi. This sodium “pore” channel forms a tetrameric, folded structure that is capable of supporting sodium flux in phospholipid vesicles. The pore-only channel is more thermally stable than its full-length counterpart, suggesting that the voltage sensor subdomain may destabilize the full-length channel. The pore subdomains can assemble, fold, and function independently from the voltage sensor and exhibit similar ligand-blocking characteristics as the intact channel. The availability of this simple pore-only construct should enable high-level expression for the testing of potential new ligands and enhance our understanding of the structural features that govern sodium selectivity and permeability.