Regulation of intrinsic activity of the Epithelial Na+ Channel

描述（由申请方提供）：上皮Na+通道或ENaC对肾上皮的钠吸收具有速率限制作用。通道活性决定最终尿钠浓度，并部分决定钠排泄。通道活动影响身体钠和水的平衡，从而影响血压。该通道主要存在于质膜上的电惰性池中。最近，它被证明，这种无活性池可以激活的丝氨酸蛋白酶的两个通道亚基的切割。因此，这种通道调节模式为表达ENaC的肾上皮细胞提供了快速响应钠负荷、肾滤过和身体钠平衡变化的巨大能力。蛋白酶对沉默通道的激活被认为涉及从通道的α和γ亚基中去除短的抑制结构域。然而，我们的数据清楚地表明，未切割的通道可以是活性的，而一些切割的通道亚基不形成活性通道。我们表明，PO可以调节多种机制，包括通道加工，膜脂刚性，通道分配到脂筏，和ENaC亚基的抑制结构域之间的相互作用。我们建议ENaC亚基之间的多重相互作用决定通道Po。通道亚基的切割提供了去除或改变这些相互作用中的一些的手段，并导致通道活化。我们还提出，通道亚基相互作用影响亚基加工，膜分配和稳定性。统一的假设是，未加工的通道在膜上是稳定的，但本质上是无活性的，除非存在减少亚基-亚基抑制的条件，而且，蛋白水解通过永久去除这种亚基间的相互作用来激活或引发通道的激活。蛋白质水解则倾向于通道亚基的内化，除非加工的亚基在筏膜结构域中受到保护。为了解决控制通道活性的机制，我们开发了新的工具，这些工具利用1）工程化的切割位点2）最近鉴定的组成型ENaC亚基（ENaC）3）通道与ASIC 1晶体结构的同源性映射和4）通道分配到膜结构域的分析。我们的数据将为理解波道调节和进一步修改波道的每个下游过程铺平道路。 公共卫生相关性：我们研究了控制肾上皮钠通道自发活动的机制。这种蛋白质的活性在确定肾脏盐排泄以及因此由身体和血容量和血压的盐保留方面是重要的。了解这种蛋白质的自发活动将有助于了解血压盐敏感性的基础，并且在设计高血压患者的治疗方法时非常重要。

DESCRIPTION (provided by applicant): The Epithelial Na+ Channel or ENaC is rate limiting to sodium absorption in renal epithelia. Channel activity sets the final urinary sodium concentration and determines in part sodium excretion. Channel activity affects body sodium and water balance and therefore blood pressure. This channel exists at the plasma membrane largely in an electrically inactive pool. Recently, it was demonstrated that this inactive pool can be activated by cleavage of two of the channel subunits by serine proteases. Thus, this mode of channel regulation affords ENaC expressing renal epithelia an enormous capacity to rapidly respond to changes of sodium load, renal filtration, and body sodium balance. Activation of silent channels by proteases is proposed to involve the removal of short inhibitory domains from the channel's alpha and gamma subunits. However, our data clearly demonstrate that non-cleaved channels can be active, while some cleaved channel subunits do not form active channels. We demonstrate that Po can be regulated by multiple mechanisms that include channel processing, membrane lipid rigidity, channel partitioning into lipid rafts, and interaction between inhibitory domains of the ENaC subunits. We propose that multiple interactions between the ENaC subunits determine channel Po. Cleavage of the channel subunits provides a mean of removing or altering some of these interactions and leads to channel activation. We also propose that channel subunit interactions affect subunit processing, membrane partitioning and stability. The unifying hypothesis is that unprocessed channels are stable at the membrane but inherently inactive unless conditions exist to reduce subunit-subunit inhibition, and that moreover, proteolysis activates or primes the channels for activation by permanently removing such inter-subunit interactions. Proteolysis then predisposes the channel subunits for internalization unless processed subunits are protected in raft membrane domains. To address the mechanism of control of channel activity, we develop new tools that utilize 1) engineered cleavage sites 2) a recently identified constitutively ENaC subunit (epsilon) 3) homology mapping of the channel to the crystal structure of ASIC1 and 4) analysis of channel partitioning into membrane domains. Our data will pave the way for understanding channel Po regulation and every downstream process that further modify this Po. PUBLIC HEALTH RELEVANCE: We examine the mechanisms that control the spontaneous activity of the renal epithelial sodium channel. The activity of this protein is important in determining kidney salt excretion and consequently salt retention by the body and blood volume and pressure. Understanding the spontaneous activity of this protein would help understand the basis of salt sensitivity of blood pressure and is important in designing therapy for individuals with high blood pressure.