Regulation of intrinsic activity of the Epithelial Na+ Channel

上皮钠通道内在活性的调节

• 批准号: 8134440
• 负责人: MOUHAMED S. AWAYDA
• 金额: $ 33.79万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8134440
• 关键词: Address; Affect; Amino Acids; Blood Pressure; Blood Volume; Cell membrane; Cells; Cholesterol; Cleaved cell; Coupling; Data; Duct (organ) structure; Engineering; Epithelial; Equilibrium; Event; Excision; Excretory function; Exhibits; Filtration; Fingers; Health; Homeostasis; Human; Hypertension; Image; Individual; Ion Channel; Kidney; Lead; Length; Maps; Mediating; Membrane; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Modeling; Modification; Molecular Conformation; Mutate; Mutation; N-terminal; Nephrons; Peptide Hydrolases; Probability; Process; Proteins; Proteolysis; Proteolytic Processing; Regulation; Regulation of Proteolysis; Role; Secondary to; Serine Protease; Site; Sodium; Sodium Channel; Sodium Chloride; Stretching; Structure; Subtilisins; Techniques; Temperature; Testing; Thick; Transmembrane Domain; Ubiquitination; Water; X-Ray Crystallography; absorption; base; beta pleated sheet; body volume; epithelial Na+ channel; extracellular; gamma ENaC; membrane activity; mutant; novel strategies; patch clamp; pressure; public health relevance; renal epithelium; research study; therapy design; tool; urinary

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.

描述（由申请人提供）：上皮 Na+ 通道或 ENaC 对肾上皮细胞中的钠吸收进行速率限制。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，研究表明，丝氨酸蛋白酶切割两个通道亚基可以激活该非活性池。因此，这种通道调节模式为表达 ENaC 的肾上皮提供了快速响应钠负荷、肾滤过和体内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亚基中去除短抑制域。然而，我们的数据清楚地表明，非裂解通道可以是活跃的，而一些裂解通道亚基不会形成活跃通道。我们证明 Po 可以通过多种机制调节，包括通道加工、膜脂刚性、通道划分为脂筏以及 ENaC 亚基的抑制域之间的相互作用。我们认为 ENaC 亚基之间的多重相互作用决定了通道 Po。通道亚基的裂解提供了去除或改变其中一些相互作用的方法，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，未经处理的通道在膜上是稳定的，但本质上是不活跃的，除非存在减少亚基间抑制的条件，而且，蛋白水解通过永久消除这种亚基间相互作用来激活或启动通道的激活。然后，蛋白水解使通道亚基易于内化，除非加工的亚基在筏膜域中受到保护。为了解决通道活性的控制机制，我们开发了新工具，利用 1) 工程切割位点 2) 最近鉴定的组成型 ENaC 亚基 (epsilon) 3) 通道与 ASIC1 晶体结构的同源映射和 4) 通道划分为膜域的分析。我们的数据将为理解通道 Po 调节以及进一步修改该 Po 的每个下游流程铺平道路。 公共健康相关性：我们研究了控制肾上皮钠通道自发活动的机制。这种蛋白质的活性对于确定肾脏盐排泄以及身体盐潴留以及血容量和压力很重要。了解这种蛋白质的自发活性将有助于了解血压盐敏感性的基础，并且对于设计高血压患者的治疗方法非常重要。