BASOLATERAL CL CHANNELS IN THICK ASCENDING LIMBS

上肢粗的基底外侧 CL 通道

• 批准号: 6094175
• 负责人: Thomas E Andreoli
• 金额: $ 25.11万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6094175
• 关键词: Xenopus oocyte; adenosine triphosphate; basolateral membrane; chimeric proteins; chloride channels; cofactor; complementary DNA; gene expression; injection /infusion; laboratory mouse; laboratory rabbit; messenger RNA; pharmacokinetics; phosphorylation; protein kinase A; renal cortex; renal medulla; renal tubular transport; tissue /cell culture

Our goals are to characterize basolateral Cl-channels from rabbit and mouse medullary thick limbs (MTAL) and from mouse cortical thick limbs (CTAL). ADH enhances net NaCl absorption in microperfused MTAL but not CTAL. In the MTAL, ADH may increase apical salt admittance, and the rise in cell Cl- may increase basolateral Cl- conductance secondary. We previously studied Cl-channels from basolateral vesicles of rabbit outer medulla and expressed a Cl- conductance in Xenopus oocytes using mRNA from rabbit outer medulla. Current progress included: A) Characterizing Cl-channels from rabbit outer medulla and from cultured mouse MTAL and CTAL cells; B) Identifying a putative Cl-channel cDNA, rbCIC-Ka, from rabbit outer medulla; C) Providing evidence that rbCIC-Ka, the 75 kDa gene product of rbCIC-Ka, mediates salt absorption in the MTAL; D) Comparing the properties of Cl-channels in cultured mouse MTAL and cultured mouse CTAL cells; E) Studying the kinetics of Cl-flux through MTAL Cl- channels. Our aims are: (i) To express rbCIC-Ka. (ii) To understand the molecular physiology of basolateral Cl- channels in mouse MTAL and CTAL. (iii) To show that the Cl-conductance increase produced when Xenopus oocytes are infected with 1.8-3.2 kb mRNA from rabbit outer medulla is due to rbCIC-Ka. (iv) We have isolated two highly homologous cDNAs, mmCIC-Ka and mmCIC-Ka, from mouse medulla and cortex, respectively. We wish to establish whether mmCIC-Ka and mmCIC-Ka encode Cl-channels mediating net Cl-absorption in mouse MTAL and CTAL, respectively; and to express the gene products of mmCIC-Ka and mmCIC-Ka. (v) To evaluate which proposed phosphorylation sites on the cytosolic surfaces of rbCIC-Ka, mmCIC-Ka and mmCIC-Ka (the proposed proteins encoded by rbCIC-Ka, mmCIC-Ka and mmCIC-Ka, respectively) are phosphorylated by (ATP + PKA). (vi) To evaluate why anti-rbCIC-Ka, prepared using the cytoplasmic C-terminus of rbCIC-Ka as antigen, blocks rbCIC-Ka channels at extracellular faces. (viii) To evaluate the kinetics of Cl-permeation in Cl-channels from mouse CTAL. These data may help provide a molecular basis for the functional differences between basolateral MTAL and CTAL Cl-channels. The results may help explain the molecular basis for some salt-acquisitive or salt-wasting disorders., and allow the rational design of drugs which ameliorate such derangements.

我们的目标是表征来自兔子和髓质厚的肢体（MTAL）的基底外侧CL通道以及小鼠皮质厚的肢体（CTAL）。 ADH增强了微填充的MTAL中的NaCl净吸收，但不能增强CTAL。在MTAL中，ADH可能会增加根尖盐的入口，并且细胞cl-可能会增加基底外侧cl电导率。我们以前先是研究了兔外髓质基底外侧囊泡的Cl通道，并使用兔外髓质的mRNA在爪蟾卵母细胞中表达了cl频道。当前的进展包括：a）表征来自兔外髓质以及培养的小鼠MTAL和CTAL细胞的CL通道； b）从兔子外髓质中识别出假定的Cl通道cDNA RBCIC-KA； c）提供证据表明，rbcic-ka是rbcic-ka的75 kDa基因产物，可介导mtal中的盐吸收。 d）比较培养的小鼠MTAL和培养的小鼠CTAL细胞中Cl通道的特性； e）通过MTAL CL-通道研究Cl-Flux的动力学。我们的目标是：（i）表达RBCIC-KA。 （ii）了解小鼠MTAL和CTAL中基底外侧CL通道的分子生理。 （iii）表明，当爪蟾卵母细胞被兔外髓质的1.8-3.2 kb mRNA感染时，产生的CL传导增加是由于rbcic-ka引起的。 （iv）我们分别从小鼠髓质和皮层分离出了两个高度同源的cDNA，mmcic-ka和mmcic-ka。我们希望确定MMCIC-KA和MMCIC-KA是否分别编码小鼠MTAL和CTAL中介导净CL-吸附的CL通道；并表达mmcic-ka和mmcic-ka的基因产物。 （v）评估哪些提出了在RBCIC-KA，MMCIC-KA和MMCIC-KA（由RBCIC-KA编码的拟议蛋白），MMCIC-KA和MMCIC-KA编码的蛋白质（分别通过（分别）通过（分别为Atp + PKA）的磷酸化位点。 （vi）评估为什么使用rbcic-ka的胞质C末端作为抗原制备的抗rbcic-ka为何将抗抗原的C-末端阻止rbcic-ka通道处于细胞外面部。 （viii）评估小鼠CTAL的Cl通道中Cl-Permeation的动力学。这些数据可能有助于为基底外侧MTAL和CTAL CL通道之间的功能差异提供分子基础。结果可能有助于解释某些盐积极或浪费盐的疾病的分子基础。