Functional Study Of The Palm Domain Of The Epithelial Sodium Channel In Kidney

Background:Epithelial sodium channel（ENaC）,composed ofα,βandγsubunit,is a member of the ENaC/degenerin family.In kidney,ENaCs are expressed in the aldosterone-sensitive distal nephron where hormones like aldosterone regulate the reabsorption of sodium and water via modulating ENaC activity.It has been reported that ENaC is related to the pathogenesis of diseases including Liddle’s syndrome,pseudohypoaldosteronism type 1,salt-sensitive hypertension,nephrotic syndrome,diabetic kidney disease and polycystic kidney disease.At present,the detail mechanism of ENaC channel gating remains unclear.However,human ENaC structure suggests that the palm domain of ENaC resides in an important position.Palm domain connects the peripheral extracellular domain（ECD）to the transmembrane domain（TM）.Therefore,it is likely to play an important role in ENaC channel gating.In addition,although the palm domain sequence is highly conservative,functional ENaC subunit dependence suggests that the palm domain functions of theα,β,andγsubunits may differ.Objectives:In this study,we aim to explore the function of palm domain during ENaC gating.Method:（1）In order to explore whether palm domain is involved in ENaC gating,we made a mouse ENaC structure model via homology modeling.Based on the relative position,the longβ10 strand was chosen as the representative strand of the palm domain.13 residues of theαsubunitβ10,4 selected residues of theβsubunitβ10 and 4 selected residues of theγsubunitβ10 were mutated into Cys,individually.Wild type（WT）and mutant ENaCs were expressed in Xenopus oocytes.Change of current and sodium self-inhibition response due to MTSES,MTSET and Cd²⁺were recorded by two-electrode voltage clamp.Molecular simulation was conducted to predict aqueous tunnels,using specific sites inβ10 as the starting points.（2）In order to explore how palm domain participates in ENaC gating,specific sites at the inter-subunit interfaces were chosen for crosslinking scanning based on the mouse ENaC model.Selected residues were mutated to Cys,individually.ENaC with zero,one or two substituted Cys were expressed in Xenopus oocytes.Oxidizing reagent,reducing reagents and metal ion were used to identify possible inter-subunit interaction.Change of ENaC activity was also assessed.Result:（1）Among the thirteen sites with Cys substitution of the α subunitβ10,only those in the proximal region of β10 could be modified by MTS reagents.Cys introduced at the selected proximal sites of the β and γ subunitβ10 strands could also interact with MTS reagents.Additionally,multiple introduced Cys in the proximal region ofβ10 were modified by low concentrations of thiophilic Cd²⁺.By assessing Na⁺ self-inhibition response,we identified four α ,twoβand twoγsubunitβ10strand mutations that changed the Na⁺self-inhibition response.Results above suggest that the proximal regions of theβ10 strand are accessible to aqueous compounds and Cd²⁺.The proximal regions of the β 10 strand have a role in ENaC gating.These results are consistent with the presence of the central vestibule in mouse ENaC structural model and the results of the aqueous tunnel prediction.（2）Using crosslinking scanning,we identified four pairs of functional inter-subunit interactions involving ENaC palm domains（αpalm-βpalm,αthumb-βpalm,αpalm-γthumb andβpalm-γ-palm）.Notably,crosslinking E499C in theβsubunit palm domain and N510C in theαsubunit palm domain activated ENaC,whereas cross-linkingβE499C with Q441C in theαsubunit thumb domain inhibited ENaC.These inter-subunit interactions propose that residues at the specific subunit interfaces could serve as microswitches that covey the conformational wave during ENaC gating.Conclusion:Extracellular palm domain is involved in inter-subunit interactions and modulates ENaC gating.