REGULATION OF THE SQUID AXON NA, K, C1 COTRANSPORTER

鱿鱼轴突 NA、K、C1 协同转运蛋白的调节

• 批准号: 6394386
• 负责人: JOHN M RUSSELL
• 金额: $ 22.28万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6394386
• 关键词: Xenopus oocyte; actins; axon; chloride ion; ion transport; membrane transport proteins; neuronal transport; phosphorylation; potassium ion; protein isoforms; protein structure function; sodium ion; squid; tissue /cell culture

DESCRIPTION: (Applicant's Abstract) It is currently believed that much of the regulation and modulation of the Na,K,Cl cotransporter (NKCC) involves phosphorylation of the NKCC protein. Using the internally dialyzed squid giant axon, we have recently made two novel phosphorylation-related observations about the NKCC. In the nominal absence of ATP and ADP, arginine phosphate (Arg-P) supports NKCC fluxes across the squid axolemma. We will pursue this unexpected finding by testing whether Arg-P will support phosphorylation of the NKCC using a squid-specific antibody (which we will develop) to perform immunoprecipitation studies. We will test for whether the effect of arginine phosphate may be due to the replenishment of a compartment of ATP which is poorly accessible to dialysis, but readily accessible to the NKCC. We will perform a series of functional studies to determine whether the Arg-P activated NKCC behaves qualitatively different from the ATP activated NKCC. A second unexpected finding involves the ability of two inhibitors (A3 and DRB) of protein kinase CK2 (aka casein kinase 2) to completely block NKCC-mediated ion fluxes. This is particularly interesting given that the squid NKCC and those from mammalian sources have numerous CK2 consensus phosphorylation sites. To date, we have not identified any other types of protein kinase inhibitors capable of inhibiting the squid NKCC. We will take advantage of the fact that CK2 uses GTP almost as effectively as ATP to test whether GTP can support NKCC fluxes. We will again use the squid-specific antibody to test for whether treatment with A3 or DRB reduces the phosphorylation of the NKCC. Axonal shrinkage stimulates the squid NKCC by shifting the intracellular Cl- inhibition curve toward higher [Cl-]I values. We will test for roles for CK2 and for actin polymerization in NKCC activation by shrinkage. Axonal shrinkage leads to an increase of axoplasmic ionic strength which is known to facilitate the polymerization of actin. A series of studies examining the effects of several known modulators of F-actin on NKCC-mediated flux will be performed to test for a role for actin polymerization in activation of the NKCC. Another series of studies will be directed toward obtaining the functional expression of a clone of the squid NKCC using both mammalian cell and Xenopus oocyte expression systems. We will also develop a cut-open oocyte preparation for the characterization of the cloned squid NKCC. It will be used to functionally compare the squid NKCC with that from mammalian sources. In addition, the cut-open oocyte model will permit the year-round study of squid NKCC in a preparation that gives reasonably good access to intracellular regulatory sites on the NKCC protein.

描述：(申请人摘要) 目前人们认为，对生物多样性的大部分调控 钠、钾、氯共转运体(NKCC)参与NKCC蛋白的磷酸化。 利用内透析的鱿鱼巨型轴突，我们最近制作了两部新的 NKCC的磷酸化相关观察。在名义上没有……的情况下 ATP和ADP，精氨酸磷酸(Arg-P)支持NKCC通过鱿鱼的通量 轴膜。我们将通过测试Arg-P是否会 使用鱿鱼特异性抗体支持NKCC的磷酸化 将会发展)来进行免疫沉淀研究。我们将测试是否 精氨酸磷酸的作用可能是由于补充了 三磷酸腺苷隔室，很难透析，但很容易 可由NKCC访问。我们将进行一系列功能研究，以 确定Arg-P激活的NKCC的行为是否与 三磷酸腺苷激活了NKCC。第二个意想不到的发现涉及两个人的能力 蛋白激酶CK2(又名酪蛋白激酶2)的抑制剂(A3和DRB) 完全阻断NKCC介导的离子通量。这一点特别有趣 鉴于鱿鱼NKCC和哺乳动物来源的鱿鱼有许多CK2 一致的磷酸化位点。到目前为止，我们还没有发现任何其他 能够抑制鱿鱼NKCC的蛋白激酶抑制剂的类型。我们 将利用CK2使用GTP几乎与ATP同样有效的事实 测试GTP是否支持NKCC通量。我们将再次使用 用于测试A3或DRB治疗是否减少的鱿鱼特异性抗体 NKCC的磷酸化。轴突收缩通过以下方式刺激鱿鱼NKCC 使细胞内Cl-抑制曲线向较高的[Cl-]i值移动。我们 将测试CK2和肌动蛋白聚合在NKCC激活中的作用 缩水。轴突收缩导致轴浆离子强度增加 已知其有助于肌动蛋白的聚合。一系列研究 检测几种已知的F-肌动蛋白调节剂对NKCC介导的影响 将进行助熔剂以测试肌动蛋白聚合在 NKCC的激活。另一系列研究将针对 利用两者获得鱿鱼NKCC克隆的功能表达 哺乳动物细胞和非洲爪哇卵母细胞表达系统。我们还将开发一种 切开卵母细胞制备法用于克隆鱿鱼NKCC的鉴定。 它将被用于对鱿鱼NKCC和哺乳动物的NKCC进行功能比较 消息来源。此外，切开的卵母细胞模型将允许全年 对鱿鱼NKCC制剂的研究，该制剂可以很好地获得 NKCC蛋白上的细胞内调控位点。