Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Department of Nephrology, Hunan Key Laboratory of Kidney Disease and Blood Purification, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China; The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.
Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
J Biol Chem. 2023 Mar;299(3):102914. doi: 10.1016/j.jbc.2023.102914. Epub 2023 Jan 14.
Epithelial Na channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.
上皮钠通道 (ENaC) 和相关通道具有大的细胞外结构域，其中特定的因素相互作用并诱导构象变化，导致通道活性改变。然而，与 ENaC 活性变化相关的细胞外结构转变尚不清楚。我们使用 Xenopus 卵母细胞中的交联和双电极电压钳技术，确定了几个功能亚基间接触对，其中通过在引入的半胱氨酸残基之间诱导或打破二硫键来调节小鼠 ENaC 的活性。具体来说，交联β-亚基手掌域中的 E499C 和 α-亚基手掌域中的 N510C 激活 ENaC，而交联β-E499C 与 α-亚基拇指域中的 Q441C 抑制 ENaC。我们确定了通过不同的机制桥接β-E499C 与α-N510C 或α-Q441C 改变了 Na 自抑制反应。与桥接β-E499C 和α-Q441C 相似，我们发现交联手掌域α-E557C 与拇指域γ-Q398C 强烈抑制 ENaC 活性。总之，我们提出在特定的亚基界面的某些残基形成微开关，在 ENaC 门控及其调节过程中传递构象波。
