REGULATION OF THE SQUID AXON NA, K, C1 COTRANSPORTER

描述：（申请人摘要） 目前认为，大部分的调节和调节 Na、K、Cl 协同转运蛋白 (NKCC) 涉及 NKCC 蛋白的磷酸化。 利用内部透析的鱿鱼巨轴突，我们最近制作了两个新颖的 NKCC 磷酸化相关观察。在名义上不存在的情况下 ATP 和 ADP、磷酸精氨酸 (Arg-P) 支持鱿鱼中的 NKCC 通量 轴突。我们将通过测试 Arg-P 是否会影响这一意外发现 使用鱿鱼特异性抗体（我们使用该抗体）支持 NKCC 的磷酸化 将开发）进行免疫沉淀研究。我们将测试是否 磷酸精氨酸的作用可能是由于补充了 ATP 隔室很难进行透析，但很容易 NKCC 可以访问。我们将进行一系列的功能研究 确定 Arg-P 激活的 NKCC 的行为是否与 ATP 激活 NKCC。第二个意外发现涉及两个人的能力 蛋白激酶 CK2（又名酪蛋白激酶 2）抑制剂（A3 和 DRB） 完全阻断 NKCC 介导的离子流。这一点特别有趣 鉴于鱿鱼 NKCC 和来自哺乳动物的鱿鱼具有大量 CK2 共有磷酸化位点。迄今为止，我们还没有发现任何其他 能够抑制鱿鱼 NKCC 的蛋白激酶抑制剂类型。我们 将利用 CK2 使用 GTP 几乎与 ATP 一样有效的事实 测试GTP是否可以支持NKCC助焊剂。我们将再次使用 鱿鱼特异性抗体，用于测试 A3 或 DRB 治疗是否会减少 NKCC 的磷酸化。轴突收缩通过以下方式刺激鱿鱼 NKCC 将细胞内 Cl- 抑制曲线向更高的 [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 收缩。轴突收缩导致轴浆离子强度增加 已知它可以促进肌动蛋白的聚合。一系列研究 检查几种已知的 F-肌动蛋白调节剂对 NKCC 介导的影响 将进行通量以测试肌动蛋白聚合的作用 NKCC 的激活。另一系列研究将针对 使用两者获得鱿鱼 NKCC 克隆的功能表达 哺乳动物细胞和非洲爪蟾卵母细胞表达系统。我们还将开发一个 用于表征克隆鱿鱼 NKCC 的切开卵母细胞制备。 它将用于对鱿鱼 NKCC 与哺乳动物的 NKCC 进行功能比较 来源。此外，切开的卵母细胞模型将允许全年 对鱿鱼 NKCC 的研究，其制备方法可以很好地获取 NKCC 蛋白上的细胞内调节位点。

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.