REGULATION OF THE SQUID AXON NA, K, C1 COTRANSPORTER

描述：（申请人摘要） 目前人们认为，大部分的监管和调节 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的 关于NKCC的磷酸化相关观察。在名义上缺席的情况下 ATP和ADP，精氨酸磷酸盐（Arg-P）支持鱿鱼的NKCC通量 轴膜我们将通过测试Arg-P是否会 支持使用鱿鱼特异性抗体磷酸化NKCC（我们 将开发）进行免疫沉淀研究。我们将测试 精氨酸磷酸盐的作用可能是由于补充了 ATP隔室，透析难以进入，但容易进入 可访问NKCC。我们将进行一系列功能研究， 确定Arg-P激活的NKCC的行为是否与 ATP激活了NKCC第二个意想不到的发现涉及到两个 蛋白激酶CK 2（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。这是特别有趣 鉴于鱿鱼NKCC和来自哺乳动物来源的那些具有许多CK 2， 共有磷酸化位点。到目前为止，我们还没有发现任何其他 类蛋白激酶抑制剂能够抑制鱿鱼NKCC。我们 将利用CK 2几乎与ATP一样有效地利用GTP的事实 以测试GTP是否能够支持NKCC通量。我们将再次使用 鱿鱼特异性抗体，以测试是否与A3或DRB治疗减少 NKCC的磷酸化。轴突收缩刺激鱿鱼NKCC， 使细胞内Cl-抑制曲线向更高的[Cl-]I值移动。我们 将测试CK 2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 已知其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 将进行通量测试肌动蛋白聚合在 激活NKCC。另一系列研究将针对 使用两种方法获得鱿鱼NKCC克隆的功能性表达， 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 切开的卵母细胞制备用于克隆鱿鱼NKCC的表征。 这将用于鱿鱼NKCC与哺乳动物NKCC的功能比较 源此外，切开的卵母细胞模型将允许全年 研究鱿鱼NKCC的准备，使合理的良好访问， 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.