Maturation of K transport in the distal nephron

描述（由申请人提供）：全身钾含量取决于摄入和输出之间的平衡，后者主要由连接管（CNT）和集合管中的肾钾分泌调节。这些节段中钾分泌的大小由电化学梯度和顶端膜对钾的渗透性决定。在CNT和皮质集合管（CCD）中已经鉴定了两个顶端K选择性通道：低电导SK和高电导Ca/牵张激活BK（或maxi-K）通道。而SK通道仅限于Na和K转运主细胞，BK通道的密度在酸碱转运的插入细胞中最高。我们实验室最近发表的和初步的研究提供了令人信服的证据，SK通道介导的基线K分泌和BK通道参与流量刺激的钙依赖性K分泌。此外，我们已经报道，流量刺激的K分泌和BK通道在CCD的表达水平的速率在出生后的发展和饮食中的K摄入量的慢性变化的反应进行调节。基于这些数据，我们假设，流量刺激的净钾分泌在远端肾单位介导的BK通道在闰细胞中的钙依赖性的方式，并在发展过程中的通道活性的调节和表观遗传因素的BK α亚基剪接变体和β亚基的差异表达决定。将使用综合方法（包括功能（体外微灌注、荧光功能成像、电生理学）、生物化学和分子方法，通常应用于哺乳动物CCD）对该假设进行检验。在SA 1中，我们将定义瞬时流动诱导的细胞Ca浓度增加和持续刺激K分泌之间的关系，专注于导致响应的Ca的时间和来源，以及Ca/钙调蛋白激酶在信号转导途径中的作用。在SA 2中，我们将研究正常出生后发育过程中通道变体和亚型表达的分子调控，以及对膳食钾摄入量和代谢性酸碱紊乱的急性变化的反应;这些研究将在观察到的对流量刺激的钾分泌的影响的背景下进行解释。这项研究的结果应该为健康和疾病中肾K适应的分子生理学提供新的见解（例如，Bartters综合征），并扩展我们对上皮细胞功能的生物力学调节的新认识。简单总结：我们最近已经确定了一个“BK”通道（孔）在肾小管激活高尿流率，这使细胞剪切，拉伸和增加细胞钙浓度。本申请的目的是增强我们对健康和疾病条件下哺乳动物肾脏中BK通道的表达、个体发育、定位和调控的理解。

DESCRIPTION (provided by applicant): Total body K content depends on the balance between intake and output, the latter regulated primarily by renal K secretion in the connecting (CNT) and collecting ducts. The magnitude of K secretion in these segments is determined by the electrochemical gradient and the permeability of the apical membrane to K. Two apical K-selective channels have been identified in the CNT and cortical collecting duct (CCD): a low- conductance SK and a high-conductance Ca/stretch-activated BK (or maxi-K) channel. Whereas the SK channel is restricted to Na and K transporting principal cells, the density of BK channels is highest in acid- base transporting intercalated cells. Recent published and preliminary studies by our lab have provided compelling evidence that the SK channel mediates baseline K secretion and that the BK channel participates in flow-stimulated Ca-dependent K secretion. Furthermore, we have reported that the rates of flow-stimulated K secretion and levels of BK channel expression in the CCD are regulated during postnatal development and in response to chronic changes in dietary K intake. Based on this data, we hypothesize that flow-stimulated net K secretion in the distal nephron is mediated by BK channels in intercalated cells in a Ca-dependent manner, and that the regulation of channel activity during development and in response to epigenetic factors is determined by the differential expression of BK alpha subunit splice variants and beta subunits. This hypothesis will be tested using an integrated approach including functional (in vitro microperfusion, fluorescence functional imaging, electrophysiology), biochemical, and molecular approaches, generally applied to mammalian CCDs. In SA1, we will define the relationship between the transient flow-induced increase in cell Ca concentration and sustained stimulation of K secretion, focusing on the, timing and source of the Ca leading to the response, and the role of Ca/calmodulin kinase in the signal transduction pathway. In SA2, we will examine the molecular regulation of channel variant and isoform expression during normal postnatal development, and in response to acute changes in dietary K intake and metabolic acid-base disturbances; these studies will be interpreted in the context of observed effects on flow-stimulated K secretion. The results of this investigation should provide new insight into the molecular physiology underlying renal K adaptation in health and disease (e.g., Bartters syndrome), and extend our emerging understanding of the biomechanical regulation of epithelial cell function. Lay summary: We have recently identified a "BK" channel (pore) in kidney tubules that is activated at high urinary flow rates, which subject the cells to shear, stretch and an increased cell calcium concentration. The goal of this application is to enhance our understanding about the expression, ontogeny, localization, and regulation of BK channels in the mammalian kidney under conditions of health and disease.