Regulation of intrinsic activity of the Epithelial Na+ Channel

描述（由申请人提供）：上皮Na+通道或ENaC限制了肾上皮的钠吸收。通道活性决定最终尿钠浓度并部分决定钠排泄。通道活动影响体内钠和水的平衡，从而影响血压。该通道主要存在于质膜上的非电性池中。最近，研究表明，丝氨酸蛋白酶可以通过切割两个通道亚基来激活这个无活性的通道池。因此，这种通道调节模式使表达ENaC的肾上皮具有快速响应钠负荷、肾滤过和机体钠平衡变化的巨大能力。蛋白酶激活沉默通道涉及从通道的α和γ亚基中去除短抑制域。然而，我们的数据清楚地表明，非劈裂通道可以是活跃的，而一些劈裂通道亚基不能形成活跃的通道。我们证明，Po可以通过多种机制进行调节，包括通道加工、膜脂刚性、通道划分为脂筏以及ENaC亚基抑制域之间的相互作用。我们提出ENaC亚基之间的多重相互作用决定了通道Po。通道亚基的切割提供了一种消除或改变这些相互作用的方法，并导致通道激活。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，未经加工的通道在膜上是稳定的，但本质上是不活跃的，除非存在减少亚基-亚基抑制的条件，而且，蛋白质水解通过永久消除这种亚基间的相互作用来激活或启动通道的激活。蛋白水解使通道亚基易于内化，除非加工过的亚基在筏膜结构域中受到保护。为了解决通道活性的控制机制，我们开发了新的工具，利用1)工程切割位点2)最近鉴定的ENaC亚基（epsilon） 3)通道与ASIC1晶体结构的同源映射4)通道划分到膜域的分析。我们的数据将为理解通道Po调节和进一步修改该Po的每个下游过程铺平道路。

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.