Mukherjee Anindit, Wang Zhijian, Kinlough Carol L, Poland Paul A, Marciszyn Allison L, Montalbetti Nicolas, Carattino Marcelo D, Butterworth Michael B, Kleyman Thomas R, Hughey Rebecca P
From the Departments of Medicine.
Cell Biology, and.
J Biol Chem. 2017 Mar 10;292(10):4152-4163. doi: 10.1074/jbc.M117.776146. Epub 2017 Jan 30.
The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, β, and γ). We previously reported that cytoplasmic residues on the β (βCys-43 and βCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over β subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking β subunit palmitoylation sites. β subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking β and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCD cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCD cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.
上皮钠通道（ENaC）在调节细胞外液容量和血压以及气道表面液体容量和黏液纤毛清除方面发挥着重要作用。ENaC是由三个同源亚基（α、β和γ）组成的三聚体。我们之前报道过，β亚基（βCys - 43和βCys - 557）和γ亚基（γCys - 33和γCys - 41）上的细胞质残基发生了棕榈酰化。阻断ENaC棕榈酰化的半胱氨酸突变也降低了通道开放概率。此外，在调节ENaC方面，γ亚基棕榈酰化比β亚基棕榈酰化起主导作用。为了确定哪些棕榈酰转移酶（称为DHHCs）调节该通道，将小鼠ENaC与23种小鼠DHHCs中的每一种在卵母细胞中共同表达。DHHCs 1、2、3、7和14显著增加了ENaC活性。当γ亚基棕榈酰化位点发生突变时，DHHCs对ENaC的激活作用丧失，而DHHCs 1、2和14仍能激活缺乏β亚基棕榈酰化位点的ENaC。与DHHC 7共同表达ENaC可增加β亚基棕榈酰化。野生型ENaC以及缺乏β和γ棕榈酰化位点的通道都与五种激活型DHHCs进行了共免疫沉淀，这表明ENaC与多种DHHCs形成了复合物。逆转录聚合酶链反应（RT-PCR）显示，五种激活型DHHCs的转录本存在于培养的小鼠皮质集合管（mCCD）细胞中，并且DHHC 3在小鼠醛固酮敏感远端肾单位的水通道蛋白2阳性主细胞中表达，而ENaC也定位于此。用棕榈酰化的通用抑制剂处理极化的mCCD细胞，几分钟内就降低了ENaC介导的钠电流。我们的结果表明，特定的DHHCs在调节ENaC中发挥作用。
