Extracellular intersubunit interactions modulate epithelial Na plus channel gating

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 beta-subunit palm domain and N510C in the alpha-subunit palm domain activated ENaC, whereas crosslinking beta E499C with alpha Q441C in the alpha-subunit thumb domain inhibited ENaC. We determined that bridging beta E499C to alpha N510C or alpha Q441C altered the Na+ selfinhibition response via distinct mechanisms. Similar to bridging beta E499C and alpha Q441C, we found that crosslinking palm domain alpha E557C with thumb domain gamma Q398C 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.

Automated summary

Using crosslinking and voltage clamp techniques, we identified specific intersubunit contacts in ENaC that modulate channel activity. Crosslinking between specific residues in the palm and thumb domains of different subunits activated or inhibited ENaC activity. These findings suggest the presence of microswitches at specific subunit interfaces that play a role in ENaC gating and regulation.