Maturation of K Transport in the Distal Nephron

描述（由申请人提供）：皮质集合管（CCD）中的流动诱导K分泌（FIKS）由伊比利亚毒素（IbTX）敏感性Ca 2 +/牵张激活BK通道介导，该通道由孔形成1和辅助2亚基组成。该通道在钠吸收主细胞（PC）和酸碱转运插入细胞（IC）中都有发现。我们假设BK通道位于一个大分子复合物中，由机械敏感性顶端Ca 2+通道和各种激酶/磷酸酶以及其他信号分子组成，锚定在细胞骨架上，并且小管流体流速的增加导致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目标（SA）中进行检验。SA 1建议在《防治荒漠化公约》中确定NKCC 1在FIKS中的作用。由于IC中BK通道的密度超过PC，IC将是介导FIKS的逻辑候选者。然而，这种作用将要求IC具有稳健的机制来维持高稳态[K]i。我们认为IC的钾吸收是由基底外侧的Na-K-2CI协同转运蛋白介导的 （NKCC1）。为了测试这一点，我们将首先（A）检查基底外侧布美他尼（NKCC抑制剂）和管腔IBX是否抑制体外微灌注CCD中的相同转运途径;（B）使用荧光功能探针测试功能性NKCC是否沿着IC和/或PC的基底外侧膜存在;和（C）检查来自NKCC 1靶向缺失的小鼠的CD用于FIKS的能力。SA 2将测试PC和IC中的BK通道是否由于独特的细胞特异性BK 1变体和2种亚型的表达而受到机械激活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，并检查变体和同种型表达是否受膳食K摄入量和流体剪切应力（FSS）的调节，然后检查（B）NO/cGMP/PKG，（C）MAPK，（D）棕榈酰化和（E）FSS诱导的磷酸化在CCD中BK通道调节中的作用。我们预计，拟议的研究将揭示参与尿钾排泄障碍的发展/维持机制，并确定治疗钾失衡的潜在靶点。

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.