Maturation of K Transport in the Distal Nephron

描述(申请人提供)：大脑皮层集合管中的流动诱导钾分泌(FIKS)是由IBTX敏感的钙/拉伸激活的BK通道介导的，由造孔1和辅助2亚基组成。该通道存在于钠吸收主细胞(PC)和酸碱转运插层细胞(ICs)中。我们假设BK通道定位于一个大分子复合体中，该复合体由机械敏感的顶端钙通道和多种激酶/磷酸酶以及其他信号分子组成，锚定在细胞骨架上，并且管状液流速的增加导致IC和PC特异性反应，在很大程度上由其中BK通道的细胞特异性组成决定。这一假设将在两个特定的目标(SA)中进行检验。SA1建议确定NKCC1在CCDFIKS中的作用。由于IC中BK通道的密度超过PC中的BK通道密度，IC将是调节FIKs的合理候选者。然而，这种作用需要ICs有一个强大的机制来维持高稳态[K]i。我们认为ICs的K摄取是由基侧Na-K-2CI共转运体介导的 (NKCC1)。为了测试这一点，我们将首先(A)检测基侧布美他尼(NKCC抑制剂)和鲁米那IBX是否在体外微灌流的CCDs中抑制相同的转运途径；(B)使用荧光功能探针测试功能性NKCC是否存在于IC和/或PC的基侧膜上；以及(C)检测靶向缺失NKCC1 for Fiks的小鼠CD的能力。SA2将测试PC和IC中的BK通道是否由于独特的细胞特异性BK1变体和2个异构体的表达而受到机械激活的信号通路的差异调节。为此，我们将(A)确定单个IC和PC中BK通道的分子组成，并检查饮食K摄入量和流体切应力(FSS)是否调节BK通道的异构体和异构体表达，然后研究(B)NO/cGMP/PKG、(C)MAPK、(D)棕榈酰化和(E)FSS诱导的磷酸化在调节CCDBK通道中的作用。我们预计，拟议的研究将揭示尿钾排出障碍的发生/维持机制，并确定治疗钾失衡的潜在靶点。 公共卫生相关性：拟议的研究旨在研究肾脏远端肾单位BK钾通道调节的分子机制。这些研究将确定这些通道在低尿流率条件下如何被抑制，并在尿流率增加时如何被激活以将钾分泌到尿液中。这项工作有可能揭示钾排泄障碍发生和/或维持的机制，并确定治疗钾平衡失衡的新疗法的潜在靶点。

DESCRIPTION (provided by applicant): Flow-induced K secretion (FIKS) in the cortical collecting duct (CCD) is mediated by the iberiotoxin (IbTX)-sensitive Ca2+/stretch-activated BK channel, comprised of pore-forming 1 and accessory 2 subunits. The channel is detected in both Na absorbing principal cells (PC) and acid-base transporting intercalated cells (ICs). We hypothesize that the BK channel is localized in a macromolecular complex, comprised of mechanosensitive apical Ca2+ channels and a variety of kinases/phosphatases as well as other signaling molecules, anchored to the cytoskeleton, and that an increase in tubular fluid flow rate leads to IC- and PC-specific responses determined, in large part, by the cell-specific composition of the BK channels therein. This hypothesis will be tested in two Specific Aims (SAs). SA1 proposes to identify the role of NKCC1 in FIKS in the CCD. As the density of BK channels in IC exceeds that in PC, the IC would be the logical candidate to mediate FIKS. However, this role would require that ICs have a robust mechanism to sustain a high steady-state [K]i. We propose that K uptake in ICs is mediated by a basolateral Na-K-2CI cotransporter (NKCC1). To test this, we will first (A) examine whether basolateral bumetanide (NKCC inhibitor) and luminal IBX inhibit the same transport pathway in in vitro microperfused CCDs; (B) test whether functional NKCC is present along the basolateral membranes of IC and/or PC using fluorescent functional probes; and (C) examine the capacity of CDs from mice with targeted deletion of NKCC1 for FIKS. SA2 will test whether BK channels in PC and IC are differentially regulated by mechano-activated signaling pathways due to the expression of unique cell-specific BK1 variants and 2 isoforms. To this end, we will (A) determine the molecular composition of BK channels in individual IC and PC, and examine whether variant and isoform expression is regulated by dietary K intake and fluid shear stress (FSS), and then examine the roles of (B) NO/cGMP/PKG, (C) MAPK, (D) palmitoylation and (E) FSS induced phosphorylation in the regulation of BK channels in the CCD. We anticipate that the proposed studies will uncover mechanisms involved in the development/maintenance of disorders of urinary K excretion and identify potential targets for therapies to treat K imbalances. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to examine the molecular mechanisms underlying regulation of BK potassium channels in the distal nephron of the kidney. The studies will define how these channels are suppressed under conditions of low urinary flow rates, and activated to secrete potassium into the urine when urinary flow rates increase. This work has the potential to uncover mechanisms involved in the development and/or maintenance of disorders of potassium excretion and identify potential targets for novel therapies to treat imbalances in potassium balance.