ROMK-CFTR INTERACTIONS IN THE DISTAL NEPHRON

Kir1.1(ROMK)形成小电导(SK)ATP敏感性钾通道(KATP)，介导哺乳动物肾脏粗大升支(TAL)顶端钾循环和主细胞钾分泌。在Kcnj1(Kir1.1)基因缺失的小鼠中，SK通道活性缺失，这些ROMK缺陷小鼠具有与人类ROMK Bartter综合征一致的盐耗表型。因此，ROMK在肾脏钠钾转运中起着至关重要的作用。所有KATP频道都是 被认为是由四个形成孔的亚基(Kir6.x或Kir1.1)结合四个三磷酸腺苷结合盒(ABC)蛋白质形成的。相关的ABC蛋白介导对磺脲类药物(如格列本脲)的敏感性，并被认为在核苷酸的代谢感应中发挥关键作用。在乳管圆线虫卵母细胞共表达的研究表明，在TAL和主细胞的顶端边界表达的CFTR可能是ROMK的ABC伙伴。然而，在肾脏TAL和主细胞中，几乎不知道CFTR在SK通道活性和代谢调节中的作用，CFTR可能作为心尖C1通道的作用以及这种阴离子通道活性对阳离子转运的功能影响，或者ROMK对CFTRCI通道活性的影响。我们假设 这表明：(1)cftr介导格列本脲敏感性，并参与cAMP(CAMP)低时SK通道的核苷酸感知(ATP抑制和ADP激活)；(2)cAMP高时，cftr与ROMK(或cAMP上的ROMK)失去功能相互作用，从而丧失对格列本脲的敏感性，降低ATP抑制SK的能力；(3)顶端cftr氯通道在低cAMP条件下保持沉默，但在高cAMP条件下激活；以及(4)cFtr氯电流部分分流顶端K分泌电流，允许钾分泌电流从主细胞的钠吸收中电离离。这些cAMP介导的CFTR-ROMK相互作用和与细胞高cAMP功能的改变可以在远端肾小管血流较低时促进主干和粗大升肢细胞的K分泌。在表达CFTR-ROMK的莱氏血吸虫卵母细胞中进行的生化和膜片钳研究将被用来评估通道相互作用的性质和cAMP的作用。大鼠TAL和主细胞顶膜的膜片钳技术将被用来评估SK和CFTRCl通道的活性、调节和相互作用。体内和体外微灌流将被用来研究ROMK-CFTR相互作用在大鼠远端肾单位节段的功能后果。在ROMK和/或CFTR缺陷小鼠中进行的类似研究将被用来剖析CFTR对 ROMK函数和ROMK对CFTR函数的影响。

Kir1.1(ROMK) forms the small conductance (SK) ATP-sensitive K channel (KATP) mediating apical K recycling in thick ascending limb (TAL) and K secretion in principal cells of the mammalian kidney. SK channel activity is absent in mice with deletion of the Kcnj 1 (Kir1.1) gene and these ROMK-deficient mice have a salt wasting phenotype consistent with human ROMK Bartter's syndrome. Thus, ROMK is crucial to renal Na and K handling. All KATP channels are thought to be formed by four pore-forming subunits (Kir6.x or Kir1.1) in association four ATP-Binding Cassette (ABC) proteins. The associated ABC proteins mediate sensitivity to sulfonylureas (e.g., glibenclamide) and are believed play crucial roles in metabolic sensing of nucleotides. CFTR, which is expressed at apical borders of TAL and principal cells, has been suggested to be the ABC partner of ROMK based on co-expression studies in X. laevis oocytes. Yet, virtually nothing is known in renal TAL and principal cells about the roles of CFTR in SK channel activity and metabolic regulation, the circumstances where CFTR may function as an apical C1 channel and the functional consequences of this anion channel activity on cation transport, or the effects that ROMK has on CFTR CI channel activity. We hypothesize that: (1) CFTR mediates glibenclamide sensitivity and is involved in nucleotide sensing (ATP inhibition and ADP activation) by SK channels when cell cyclic AMP (cAMP) is low; (2) the functional interaction between CFTR and ROMK (or ROMK on CFTR) is lost when cyclic AMP is high with consequent loss of glibenclamide sensitivity and reduced ability of ATP to inhibit SK; (3) apical CFTR Cl channels are silent during low, but activated during high, cAMP conditions; and (4) CFTR Cl current partially shunts the apical K secretory current allowing for electrical dissociation of K secretion from Na absorption in principal cells. These cAMP-mediated changes in CFTR-ROMK interactions and function with cell high cAMP could enhance K secretion in principal and thick ascending limb cells when distal tubular flow is low. Biochemical and patch clamp studies in X. laevis oocytes expressing CFTR-ROMK will be used to assess the nature of channel interactions and the effects of cAMP. Patch clamping of apical membranes of rat TAL and principal cells will be used to assess SK and CFTR Cl channel activity, regulation and interactions. In vivo and in vitro microperfusion will be used to study the functional consequences of ROMK-CFTR interactions in rat distal nephron segments. Similar studies in ROMK and/or CFTR deficient mice will be used to dissect out the effects of CFTR on ROMK function and of ROMK on CFTR function.